The Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling TechnologiesIMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
The Molecular Probes™ Handbook: A Guide to Fluorescent Probes and Labeling Technologies
IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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The Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling TechnologiesIMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
Chapter 23 — Antifades and Other Tools for Fluorescence
The Molecular Probes™ Handbook: A Guide to Fluorescent Probes and Labeling Technologies
IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.thermofisher.com/probes
Chapter 23 — Antifades and Other Tools for Fluorescence
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The Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling TechnologiesIMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
Section 23.1 Fluorescence Microscopy Accessories and Reference Standards
To obtain accurate and reproducible results from �uorescence imaging applications, it is essential to maximize the intensity and sta-bility of the �uorescence signal in the experimental sample. We have developed several e�ective antifade reagents that minimize photo-bleaching of �uorescently labeled specimens. �e Image-iT® FX signal enhancer (I36933) blocks nonspeci�c binding of dye-labeled antibod-ies, resulting in improved signal:background characteristics in images of immunolabeled cells and tissues. Our FluoCells® prepared micro-scope slides provide ready-to-use, multicolor-labeled cell or tissue preparations for educational and commercial �uorescence microscopy demonstrations. In collaboration with Grace Bio-Labs, we also o�er a wide selection of microscopy accessories, including sample chambers, slides and coverslips.
Likewise, accurate and reproducible results depend on optimal per-formance of the optical system. �e spectral compatibility of dyes and probes with excitation light sources (Table 23.1) and emission wave-length �lters must be carefully evaluated. Downloadable reference spectra are available through our online Fluorescence SpectraViewer tool (www.invitrogen.com/handbook/spectraviewer, Using the Fluorescence SpectraViewer—Note 23.1), and guidance in choosing optical �lters can be found in Selecting Optical Filters for Fluorescence Microscopy—Note 23.2. Careful calibration and instrumentation ad-justment are also required for high-precision imaging of �uorescent probes, particularly in multicolor applications that involve multiple exposures, repetitive scans or three-dimensional sectioning. We o�er a variety of microsphere reference standards designed to facilitate adjust-ment and calibration of both conventional �uorescence microscopes and confocal laser-scanning microscopes. In addition, the Reference Dye Sampler Kit (R14782) provides ready-made stock solutions of �ve extensively characterized �uorescence standards for use in spectro�uo-rometers and �uorescence microplate readers. We are also the source of the NIST-traceable �uorescein standard (F36915), which is directly traceable to the �uorescein standard adopted and maintained by the National Institute of Standards and Technology.
Antifade Reagents and Mounting MediaLoss of �uorescence through irreversible photobleaching pro-
cesses can lead to a signi�cant reduction in sensitivity, particularly when target molecules are of low abundance or when excitation light is of high intensity or long duration.1–3 To minimize photobleaching of experimental samples, we have developed the ProLong®, ProLong® Gold, SlowFade® and SlowFade® Gold Antifade Kits and reagents, which have been shown to increase the photostability of many of our �uorophores in �xed cells, �xed tissues and cell-free prepara-tions. �e primary function of any antifade reagent is to sustain dye �uorescence, usually by inhibiting the generation and di�usion of reactive oxygen species. Other strategies for avoiding photobleaching include reducing the excitation light intensity by using high–numeri-cal aperture objectives and low magni�cation as well as hardware control of the excitation light’s spatial 3 and temporal 4 distribu-tion. Loss of �uorescence signal due to attenuated excitation can be compensated to some extent by use of high-quality optical �lters and high-e�ciency photodetectors.
23.1 Fluorescence Microscopy Accessories and Reference Standards
Table 23.1 Fluorescence excitation sources.
Source Principal Lines (nm)
Mercury-arc lamp 366, 405, 436, 546, 578 *
Xenon-arc lamp 250–1000 *
Tungsten–halogen lamp 350–1000 *
Violet diode laser 405
Helium–cadmium laser 325, 442
Argon-ion laser 457, 488, 514
Nd:YAG laser 532 †
Helium–neon laser 543, 594, 633
Yellow diode laser 561
Krypton-ion laser 568, 647
Red diode laser 635
* Continuous white-light source. † Frequency-doubled principal line output.
Figure 23.1.1 A comparison of the performance of Molecular Probes® antifade reagents. Bovine pulmonary arterial endothelial cells were labeled with �uorescein phalloidin and mounted using A) PBS, ProLong® Gold antifade reagent (P36930, P36934) or ProLong® Antifade Kit (P7481); B) PBS, SlowFade® Gold antifade reagent (SS36936, S36937) or SlowFade® Antifade Kit (S2828). Samples were imaged using a 1.3 NA 40× oil immersion lens, Omega® XF100-2 �lter set and frame-capture rate of 1 image/second. Images were acquired with a Hamamatsu Orca ER camera using the same exposure time for all samples. Y-axis values represent averages of the top 10% of the intensity-binned pixel values.
600
200
400
800
0
Time (seconds)
5 15 20 350 10 25 30
1400
1000
1200
Ave
rage
�uo
resc
ence
inte
nsity
PBSSlowFade® Gold
SlowFade®
®
®
ProLong® Gold Antifade ReagentProLong® Gold antifade reagent is an improved version of the
ProLong® antifade reagent, a component of the ProLong® Antifade Kit described below. �e ProLong® Gold antifade reagent causes little or no quenching of the �uorescent signal while protecting the sample from
A
B
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Section 23.1 Fluorescence Microscopy Accessories and Reference Standards
NOTE 23.1
Using the Fluorescence SpectraViewerThe Fluorescence SpectraViewer (www.invitrogen.com/handbook/spectraviewer) is an online tool that allows researchers to assess the spectral
compatibility of dyes and probes in the course of designing experiments that utilize �uorescence detection techniques. This note outlines the functional-ity of the SpectraViewer (Figure 1A) and examples of its utility in the experimental design process (Figure 1B, Figure 1C).
Figure 1B Evaluating �uorophores for multiplex detection experiments: Excitation spectra. This overlay of the �uorescence excitation spectra of Alexa Fluor® 488 and Alexa Fluor® 568 dyes provides a useful initial assessment of their suitability for use in a multiplex detection experi-ment. It also serves to highlight scaling factors that are critical determi-nants of the �nal detected signal levels but that are excluded from the SpectraViewer comparison. The plot indicates that excitation of Alexa Fluor® 568 at 488 nm is relatively ine�cient (~5% of maximum indicat-ed by the cursor). However, this consideration takes no account of the molar absorptivities (extinction coe�cient, EC) of the �uorophores. The maximum EC values of Alexa Fluor® 488 and Alexa Fluor® 568 dyes are actually quite similar (75,000 cm–1 M–1 and 93,000 cm–1 M–1, respectively, as listed in The Molecular Probes® Handbook data tables). An even more signi�cant weighting factor is the relative abundances of the molecular targets of the �uorophores in the detector �eld of view; in general, cel-lular abundances of proteins vary over more orders of magnitude than the extinction coe�cients of �uorophores. In the situation illustrated with excitation limited to 488 nm, the preferred course would be to use Alexa Fluor® 568 to detect the more abundant of the two molecular tar-gets and Alexa Fluor® 488 to detect the less abundant target, thereby o�setting the absorptivity and target abundance factors.
Figure 1A SpectraViewer features: A. Fluorophore selection menu. Up to 5 �uorophore data sets may be displayed simultaneously. Open the drop-down menu (.) and select from the list of available excitation/emis-sion data sets. The number of available data sets (as of July 2010) is 300, encompassing organic dyes, �uorescent proteins and Qdot® nanocrys-tals. Note that data sets may be added, deleted or modi�ed at our edi-torial discretion and without notice, although in practice we aim to keep such changes to a minimum to maintain stability of the database. These data sets may be downloaded from our website as 4-column text �les for importing into other plotting and calculation utilities. For each selected �uorophore data set, a legend containing sample context information is displayed on the panel to the right of the plot. B. For each �uorophore data set, excitation (ex) and emission (em) data may be displayed or hid-den by checking or unchecking the respective boxes. C. Y-axis scaling for excitation and emission spectra is in terms of percentage of peak inten-sity value. For Qdot® nanocrystals, which exhibit quasi-continuous exci-tation pro�les, the 100% intensity value has been arbitrarily de�ned as that at 300 nm. X-axis values on all plots are wavelengths in nanometers (nm). D. Excitation source spectral characteristics may be superimposed on the plot in the form of laser lines selected from a drop-down menu or �lter characteristics input as numeric center wavelength (CWL) and bandpass (BP) values in nm in the boxes provided. In this example, laser excitation at 488 nm is indicated. E. Emission �lter spectral characteris-tics may be superimposed on the plot in the form of numeric CWL and BP values in nm entered in the boxes provided. In this example, a typical FITC emission �lter with CWL = 535 nm and BP = 50 nm is indicated. The transmission window of the �lter is shown on the plot as a green-shaded rectangle. F. Mouse-controlled X,Y cursor. The crosshairs may be moved to any user-selected location within the plot window and are coupled to a numeric display of the corresponding (X,Y) values.
Figure 1C Evaluating �uorophores for multiplex detection experiments: Emission spectra. The �uorescence emission spectra of Alexa Fluor® 488 and Alexa Fluor® 568 dyes are shown with the spectral characteristics of typical emission �lters superimposed. In this case, the main practical concern is the extent of overspill of Alexa Fluor® 488 �uorescence into the Alexa Fluor® 568 detection channel (CWL = 645 nm, BP = 75 nm), which can lead to false indications of molecular target colocalization in imaging applications. As in the excitation spectra comparison, this peak-normalized overlay provides an initial assessment of the suitability of �u-orophore combinations for multiplex detection, but weighting of �uores-cence signals by the relative abundances of molecular targets and other factors will heavily in�uence the �nal experimentally observed outcome.
A
B
C
The Molecular Probes™ Handbook: A Guide to Fluorescent Probes and Labeling Technologies
IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.thermofisher.com/probes
Chapter 23 — Antifades and Other Tools for Fluorescence
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Section 23.1 Fluorescence Microscopy Accessories and Reference Standards
photobleaching (Figure 23.1.1, Figure 23.1.2). Furthermore, unlike the reagents in the ProLong® Antifade Kit, the ProLong® Gold antifade reagent is premixed and ready to use—just add a drop to the preparation and mount. As with our original ProLong® antifade reagent, ProLong® Gold reagent cures within 24 hours and the sample can be saved for months a�er mounting. ProLong® Gold reagent o�ers excellent compatibility with a multitude of dyes and dye complexes, making it an especially valuable tool for multicolor applications 5–7 (Figure 23.1.3). �e ProLong® Gold antifade reagent is available in a single 10 mL bottle (P36930), as well as in a set of �ve 2 mL bottles (P36934).
As an added convenience, we also o�er ProLong® Gold antifade reagent containing DAPI, the popular nuclear and chromosome stain that emits blue �uorescence upon binding to DNA. �e addition of DAPI in the mounting media eliminates the need for a separate counter-staining step. ProLong® Gold antifade reagent with DAPI is available in a single 10 mL bottle (P36931), as well as in a set of �ve 2 mL bottles (P36935).
reagent, which has proven to e�ectively enhance the resistance of many di�erent �uorophores to photobleaching. Furthermore, specimens mounted using the ProLong® Antifade Kit exhibit little or no quenching of the �uorescent signal of most dyes.
Figure 23.1.3 HeLa cell transfected with pShooter pCMV/myc/mito/GFP, then �xed and permeabilized. Green Fluorescent Protein (GFP) localized in the mitochondria was labeled with anti-GFP mouse IgG2a (A11120) and detected with orange-�uo-rescent Alexa Fluor® 555 goat anti–mouse IgG (A21422), which colocalized with the dim GFP �uorescence. F-actin was labeled with green-�uorescent Alexa Fluor® 488 phalloidin (A12379), and the nucleus was stained with blue-�uorescent DAPI (D1306, D3571, D21490). The sample was mounted using ProLong® Gold antifade reagent (P36930). Some GFP �uorescence is retained in the mitochondria after �xation (left), but immunolabeling and detection greatly improve visualization (right).
Figure 23.1.2 A 20-second time series showing enhanced resistance to photobleaching a�orded by ProLong® Gold antifade reagent. Fixed bovine pulmonary artery endothelial cells were labeled with anti–α-tubulin (A11126) and visualized with �uo-rescein goat anti–mouse IgG (F2761). The samples were mounted in ProLong® Gold antifade reagent (P36930; top) or phos-phate-bu�ered saline (bottom). Images were acquired at 5-second intervals using a 40x/1.3 NA oil immersion objective with continuous illumination from a standard 100 watt mercury-arc lamp.
Figure 23.1.4 The relative photobleaching rates of �uo-rescein and Texas Red® �uorophores with bu�er alone (right series) or after treatment with the ProLong® antifade reagent (left series). Bovine pulmonary artery endothe-lial cells were �xed, permeabilized and labeled with Texas Red®-X phalloidin (T7471), which labels F-actin, and with a mouse monoclonal anti–ß-tubulin antibody and �uorescein goat anti–mouse IgG antibody (F2761), which label micro-tubules. Images were acquired at appropriate wavelengths using a cooled CCD camera. The numbers on the left of each pair of frames represent the duration of continuous excita-tion in seconds.
0
125
250
375
500
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Section 23.1 Fluorescence Microscopy Accessories and Reference Standards
Figure 1 Functions of �uorescence microscope �lter set components. The desired excita-tion wavelength (λ2) is selected from the spectral output of the lamp by the excitation �lter (EX) and directed to the sample via the dichroic beamsplitter (DB). The beamsplitter separates emitted �uorescence (– – –) from scattered excitation light (—). The emission �lter (EM) selectively transmits a portion of the sample’s �uorescence emission (λ4) for de-tection and blocks other emission components (λ5).
LAMP
SAMPLE
EX
EM
DB
DETECTOR
λ1
λ3
λ4
λ5
λ2
λ2
NOTE 23.2
Selecting Optical Filters for Fluorescence Microscopy
Figure 2 Potential-dependent staining of mitochondria in CCL64 �broblasts by JC-1 (T3168). The mitochondria were visualized by epi�uorescence microscopy using a 520 nm longpass optical �lter. Regions of high mitochondrial polarization are indicated by red �uorescence due to J-aggregate formation by the concentrated dye. Depolarized regions are indicated by the green �uorescence of the JC-1 monomers. The image was contributed by Lan Bo Chen, Dana Farber Cancer Institute, Harvard Medical School.
Sensitive and versatile fluorescence detection techniques are of ever-increasing importance and popularity in biological research micros-copy. In the now-standard epi-illuminated microscope con�guration, the optical �lter set performs a critical function in separating the �uorescence emission photons that will form the �nal image from the more-intense excitation light �eld. For practical purposes, it is necessary to reduce the excitation light intensity in the detection path by a factor of 106–107. This design objective has to be achieved in parallel with capturing as many of the available �uorescence photons as possible. High capture e�ciency allows compensating reductions in overall excitation light levels, with ac-companying reductions in dye photobleaching and cellular phototoxicity.
This technical note provides some basic �uorescence microscopy information, including discussions of:
• The Optical Filter Set• The Trade-O� in Optical Filter Design• Selecting an Optical Filter Set
The Fluorescence SpectraViewer (www.invitrogen.com/handbook/spectraviewer) is an online tool that allows researchers to assess the spectral compatibility of dyes, probes and optical �lters in the course of designing experiments (Using the Fluorescence SpectraViewer—Note 23.1).
The Optical Filter SetA set of optical �lters for selective excitation and detection of �uo-
rescence typically consists of a minimum of three components: an exci-tation �lter, a dichroic beamsplitter (“dichroic mirror”) and an emission �lter (“barrier �lter”) (Figure 1). The excitation �lter selectively transmits a portion of the spectral output from the light source (Table 23.1). The dichroic beamsplitter then re�ects the selected light, directing it to the sample. Fluorescence emission photons traveling from the sample towards the detector are transmitted by the dichroic beamsplitter, while excita-tion light re�ected back from the sample is diverted out of the detection light path. The emission �lter blocks unwanted spectral components of the emitted �uorescence (e.g., sample auto�uorescence) as well as any residual excitation light. An interactive Java tutorial demonstrating these functions is available online at the Molecular Expressions website of Florida State University (http://micro.magnet.fsu.edu/primer/java/�uorescence/�ltersetpro�les/index.html).
The Trade-O� in Optical Filter Set DesignFor optimal �uorescence detection when using a single dye, the excita-
tion and emission �lters should be centered on the dye’s absorption and emission peaks. To maximize the signal, one can choose excitation and emission �lters with wide bandwidths. However, this strategy may result in unacceptable overlap of the emission signal with the excitation signal, resulting in poor resolution. To minimize spectral overlap, one can instead choose excitation and emission �lters that are narrow in bandwidth and are spectrally well separated to increase signal isolation. This approach will reduce optical noise but may also reduce the signal strength to unac-ceptable levels. When overlapping signals from multiple �uorophores in the same sample are being di�erentiated, both the spectra of the dyes and their expected intensities must be considered before choosing an
The Molecular Probes™ Handbook: A Guide to Fluorescent Probes and Labeling Technologies
IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.thermofisher.com/probes
Chapter 23 — Antifades and Other Tools for Fluorescence
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Section 23.1 Fluorescence Microscopy Accessories and Reference Standards
optical �lter. Complete spectral data for Molecular Probes® �uorophores can be found at www.invitrogen.com/handbook/spectraviewer using our interactive Fluorescence SpectraViewer utility (Using the Fluorescence SpectraViewer—Note 23.1). An interactive Java tutorial illustrating the trade-o� among these parameters is available online at the Molecular Expressions website of Florida State University (http://micro.magnet.fsu.edu/primer/java/�uorescence/�uorocubes/index.html).
Selecting an Optical Filter SetFilter set selection may involve a straightforward recommendation or
a complex analysis of the spectral relationships of dyes and optical �lters.1 Emission �lters are available with either longpass or bandpass wavelength transmission pro�les. A typical longpass emission �lter might transmit all wavelengths ≥530 nm, whereas a typical bandpass �lter might transmit only wavelengths between 515 and 545 nm. Longpass �lters should be used when the application requires maximum emission collection and when spectral discrimination is not desirable or necessary, which is generally the case for probes that generate a single emitting species in specimens with relatively low levels of background auto�uorescence. Longpass �lters are also useful for simultaneous detection of spectrally distinct dual emissions such as the monomer and J-aggregate forms of JC-1 (T3168, Section 12.2, Figure 2) and the monomer and excimer forms of BODIPY® FL C5-ceramide (D3521, B22650; Section 12.4; Figure 3).
Bandpass �lters are designed to maximize the signal-to-noise ratio for applications where discrimination of signal components is more important than overall image brightness. The spectral sensitivity of the detection system should also be considered in order to achieve optimum detec-tor signal-to-noise or accurate color rendition. Some applications, such as confocal laser-scanning microscopy, may require the use of sensitive photomultiplier (PMT) detectors. Alternatively, an electron-multiplying charge-coupled device (EMCCD) may be employed for quantitative imaging or microspectro�uorometry. Dual-, triple- and quadruple-band �lter sets enable microscopists to excite and detect two, three or four �uorophores simultaneously instead of performing sequential image acquisitions with intervening �lter changes (Figure 4).
Selecting optimal �lter sets for �uorescence microscopy applications requires matching optical �lter speci�cations to the spectral characteristics of dyes. Comparisons should be made with care because some dyes have signi�cantly di�erent spectral properties in a particular application than those reported for the dye in solution. For example, the spectral characteris-tics of many nucleic acid stains depend on whether the dyes are in aqueous solution or bound to DNA or RNA. Similarly, styryl dyes such as FM® 1-43 (T3163, T35356; Section 14.4, Section 16.1) and di-4-ANEPPS (D1199, Section 22.2) have emission maxima that depend on whether they are dissolved in solvent or associated with membranes. To provide selection guidelines, we have compiled excitation and emission spectra for many of the most widely used probes in �uorescence microscopy in an online tool, the Fluorescence SpectraViewer (www.invitrogen.com/handbook/spectraviewer).
Technical SupportWe invite customers to call our Technical Assistance Department for
help in selecting the correct optical �lter for a speci�c application. When calling, please be prepared to describe the dye(s), instrumentation and method of detection being used. A technical support scientist will then o�er advice on the most e�ective �lter con�guration for the speci�ed purposes. Alternatively, we recommend contacting Chroma Technology Corp., Omega Optical, Inc. or Semrock, Inc. or the microscope manufacturer for this information. Chroma Technology, Omega Optical and Semrock provide complete transmission curves and information on their specialty �lters for ratio imaging, uncaging, multiphoton and other applications at their respective web sites (www.semrock.com, www.omega�lters.com, www.chroma.com).
1. Biophotonics Int (1999) 6:54.
Figure 4 Optical transmission characteristics of a triple-band �lter set (XF63, Omega Optical Inc.) designed for simultaneous imaging of DAPI, �uorescein and Texas Red® dyes. Transmission curves for the individual �lter set components are shown in blue (ex-citation �lter), black (dichroic beamsplitter) and red (emission �lter). Graphic supplied by and used with permission of Omega Optical Inc., Brattleboro, VT.
100
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Wavelength (nm)
% T
rans
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Figure 3 Selective staining of the Golgi apparatus using the green-�uorescent BODIPY® FL C5-ceramide (D3521) (right panel). At high concentrations, the BODIPY® FL �uorophore forms excimers that can be visualized using a red longpass optical �lter (left panel). The BODIPY® FL C5-ceramide accumulation in the trans-Golgi is su�cient for excimer formation (J Cell Biol (1991) 113:1267). Images contributed by Richard Pagano, Mayo Foundation.
The Molecular Probes™ Handbook: A Guide to Fluorescent Probes and Labeling Technologies
IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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The Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling TechnologiesIMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
Section 23.1 Fluorescence Microscopy Accessories and Reference Standards
Bovine pulmonary arterial epithelial cells (BPAEC) labeled with �uorescein phalloidin (F432) photobleached to about 12% of the ini-tial value in 30 seconds in PBS, while staying at the initial value under the same illumination conditions when mounted using the ProLong® Antifade Kit (Figure 23.1.5). As shown in Figure 23.1.6, the ProLong® Antifade Kit provides more �uorescence output than a popular p-phenylenediamine–containing antifade reagent 8 when used to mount �uorescein-stained HEp-2 cells. �e ProLong® antifade reagent also inhibits the fading of tetramethylrhodamine, as well as the fading of DNA-bound nucleic acid stains such as DAPI, propidium iodide and YOYO®-1 (Section 8.1), again without signi�cantly quenching the �uo-rescence of these dyes.9 �e compatibility of the ProLong® antifade re-agent with a multitude of dyes and dye complexes makes it an especially valuable tool for multicolor analysis procedures such as multiplexed �uorescence in situ hybridization.10
Figure 23.1.5 Bovine pulmonary artery endothelial cells were labeled with �uorescein phalloi-din (F432), which labels �lamentous actin, and placed under constant illumination on the micro-scope with a FITC �lter set using a 60× objective. Images were acquired at 1-second intervals for 30 seconds. Under these illumination conditions, �uorescein photobleached to about 12% of its initial value in 30 seconds in PBS (left), but stayed at the initial value under the same illumination conditions when mounted using the reagents in the ProLong® Antifade Kit (right, P7481).
SlowFade® Antifade KitOur original SlowFade® antifade formulation (S2828) was de-
signed to reduce the fading rate of �uorescein to almost zero. Because it provides nearly constant emission intensity from �uorescein, this SlowFade® antifade reagent is especially useful for quantitative mea-surements and applications that employ confocal laser-scanning mi-croscopy.11 However, this original SlowFade® formulation substan-tially quenches the �uorescence of �uorescein and almost completely quenches that of the Alexa Fluor® 350, Alexa Fluor® 405 and Cascade Blue® �uorophores.
Each SlowFade® Antifade Kit contains:
• SlowFade® antifade reagent in 50% (v/v) glycerol, ready to use and su�cient for at least 200 coverslip-size experiments
• Concentrated SlowFade® antifade reagent solution, provided for those applications in which glycerol may not be compatible
• Equilibration bu�er, which raises the pH of the sample, increasing the protection a�orded by the SlowFade® antifade formulation
• Detailed protocols for mounting samples
SlowFade® Gold Antifade ReagentTo overcome the limitations of the original SlowFade® antifade
reagent, especially with blue �uorophores, we have developed the SlowFade® Gold antifade reagent. �e SlowFade® Gold antifade formu-lation slows �uorescein’s fading rate by about �vefold without signi�-cantly reducing its initial �uorescence intensity, thereby dramatically increasing the signal-to-noise ratio in photomicro scopy. Moreover, quenching of the Alexa Fluor® 350, Alexa Fluor® 405, Cascade Blue®, tetramethylrhodamine and Texas Red® dyes is minimal. In fact, the SlowFade® Gold antifade reagent reduces the fading rate of the Cascade Blue® �uorophore to almost zero, while decreasing its emission intensity by only about 30%.
�e SlowFade® Gold antifade reagent is available in a single 10 mL bottle (S36936), as well as in a set of �ve 2 mL bottles (S36937). As with the ProLong® Gold antifade reagents, we also o�er SlowFade® Gold antifade reagent containing the blue-�uorescent nuclear coun-terstain DAPI. SlowFade® Gold antifade reagent with DAPI is available
Figure 23.1.6 Bleaching pro�les of A) �uorescein and B) Texas Red® dye conjugates in cell sam-ples. In these photobleaching experiments, human epithelial (HEp-2) cells were probed with human anti-nuclear antibodies and then developed for visualization with �uorophore-labeled secondary re-agents. Identical samples were mounted in ProLong® antifade reagent (n), Product X (+) or medium containing no antifade reagent (s). Although these data were normalized, we observed little or no quenching of samples mounted with the ProLong® mounting medium.
Nor
mal
ized
�uo
resc
ene
emis
sion
ProLong®
Product X
No antifade
Illumination time (sec)Illumination time (sec)
Nor
mal
ized
�uo
resc
ene
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A B
Figure 23.1.7 Human carcinoma (HeLa) cell labeled with Qdot® nanocrystals and mounted with Qmount® media. Mitochondria were detected with anti–OxPhos complex V inhibi-tor protein IgG (A21355) and labeled with Qdot® 625 goat F(ab’)2 anti–mouse IgG antibody (A10195, red �uorescence); the Golgi apparatus was detected with rabbit anti-giantin anti-body and labeled with Qdot® 585 goat F(ab’)2 anti–rabbit IgG antibody (Q11411MP, yellow �uorescence); tubulin was detected with rat anti-tubulin antibody and labeled with DSB-X™ biotin goat anti–rat IgG antibody (D20697) and Qdot® 525 streptavidin (Q10141MP, green �uorescence). The nucleus was labeled with Qnuclear™ Deep Red Stain (Q10363, purple �uo-rescence), and the slide was mounted with Qmount® Qdot® mounting media (Q10336).
The Molecular Probes™ Handbook: A Guide to Fluorescent Probes and Labeling Technologies
IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.thermofisher.com/probes
Chapter 23 — Antifades and Other Tools for Fluorescence
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The Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling TechnologiesIMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
Section 23.1 Fluorescence Microscopy Accessories and Reference Standards
in a single 10 mL bottle (S36938), as well as in a set of �ve 2 mL bottles (S36939). �ese reagents permit simultaneous nuclear staining and pro-tection of the stained sample from photobleaching.12
Unlike the ProLong® and ProLong® Gold antifade reagents, the SlowFade® and SlowFade® Gold antifade reagents do not cure over time so samples can be viewed immediately; however, SlowFade® Gold re-agents are intended for short-term use (3–4 weeks) only and mounted samples may degrade over longer time periods. Secondary sealing of coverslips with wax or nail polish is recommended when working with high magni�cation objectives or preparing specimens for storage and subsequent imaging.
6.6) make them largely incompatible with ProLong® Gold, SlowFade® Gold and other mounting media designed primarily for use with or-ganic dyes. Qmount® Qdot® mounting media (Q10336) is a specialized mountant that preserves the �uorescence signal of Qdot® nanocrystals with little to no quenching of the signal’s initial intensity. �e for-mulation cures within 12 hours and is provided in a convenient and easy-to-use dropper bottle. �is mounting medium o�ers excellent compatibility with all eight Qdot® nanocrystal spectral types (Section 6.6) and their conjugates, as well as the nuclear counterstain Qnuclear™ Deep Red stain (Q10363, Section 12.5), making it an especially valu-able tool for multicolor Qdot® nanocrystal imaging applications (Figure 23.1.7). Qmount® Qdot® mounting media is not recommended for use with Alexa Fluor® dyes or �uorescent proteins.
Figure 23.1.8 Reduced background staining a�orded by Image-iT® FX signal enhanc-er. Mouse brain cryosections were permeabilized and antigen retrieval was carried out. The sections were then treated for 30 minutes with Image-iT® FX signal enhancer (I36933, left) or left untreated (right). Sections were labeled with the neural cell body selective antibody anti–Hu C/D (A21271) and visualized using TSA™ Kit #2 (T20912) with the HRP conjugate of goat anti–mouse IgG and Alexa Fluor® 488 tyramide. Sections were mounted using the reagents in the ProLong® Antifade Kit (P7481).
Image-iT® FX Signal EnhancerBy e�ciently blocking nonspeci�c electrostatic interactions of
anionic �uorescent dyes with cationic cell and tissue constituents, the Image-iT® FX signal enhancer (I36933) dramatically improves the signal-to-noise ratio of immunolabeled cells and tissues,13–15 allowing clear visualization of targets that would normally be indistinguishable due to background �uorescence (Figure 23.1.8, Figure 23.1.9, Figure 23.1.10). Background staining seen with �uorescent conjugates of strep-tavidin, goat anti–mouse IgG antibody or goat anti–rabbit IgG antibody is largely eliminated when Image-iT® FX signal enhancer is applied to �xed and permeabilized cells prior to staining.
FluoCells® Prepared Microscope SlidesIdeal for educators and instrument manufacturers, our popular
FluoCells® prepared microscope slides contain multilabeled cell prepa-rations for observation by epi�uorescence or confocal laser-scanning microscopy. �e multicolor staining in these cell and tissue prepara-tions can deliver publication-quality images and lasts through repeated viewings. �ese slides are especially useful for setting up microscopes and camera systems and for assessing the capabilities of optical �lter sets.16–18 When stored properly, these permanently mounted specimens will retain their bright and speci�c staining patterns for at least six months from the date of purchase. We currently o�er �ve di�erent FluoCells® prepared microscope slides:
Figure 23.1.9 Increased label speci�city and resolution provided by Image-iT® FX signal en-hancer. Fixed and permeabilized bovine pulmonary artery endothelial cells were treated with Image-iT® FX signal enhancer (I36933, left) or left untreated (right) and then labeled with tetra methylrhodamine streptavidin (S870).
Figure 23.1.10 Golgi complexes in �xed and permeabilized HeLa cells labeled with anti–golgin-97 anti body (A21270) and visualized with green-�uorescent Alexa Fluor® 488 goat anti–mouse IgG antibody (A11001). Actin was stained with red-�uorescent Alexa Fluor® 594 phalloidin (A12381); nuclei were stained with blue-�uorescent DAPI (D1306, D3571, D21490). Treatment with Image-iT® FX signal enhancer (I36933) largely eliminates nonspeci�c dye binding (bottom panel) as compared with untreated slide (top panel).
The Molecular Probes™ Handbook: A Guide to Fluorescent Probes and Labeling Technologies
IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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Chapter 23 — Antifades and Other Tools for Fluorescence
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The Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling TechnologiesIMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
Section 23.1 Fluorescence Microscopy Accessories and Reference Standards
Figure 23.1.12 FluoCells® prepared slide #3 (F24630) contains a mouse kidney section stained with a combina-tion of �uorescent dyes. Alexa Fluor® 488 wheat germ ag-glutinin (W11261) is a green-�uorescent lectin that was used to label elements of the glomeruli and convoluted tubules. The �lamentous actin prevalent in glomeruli and the brush border were stained with red-orange–�uorescent Alexa Fluor® 568 phalloidin (A12380). Finally, the nuclei were stained with the blue-�uorescent DNA stain DAPI (D1306, D3571, D21490). This pseudocolored image was acquired on a Zeiss® confocal microscope located at the Institute of Neuroscience, University of Oregon.
Figure 23.1.13 FluoCells® prepared slide #3 (F24630) con-tains a section of mouse kidney stained with a combination of �uorescent dyes. Alexa Fluor® 488 wheat germ aggluti-nin (W11261), a green-�uorescent lectin, was used to label elements of the glomeruli and convoluted tubules. The �la-mentous actin prevalent in glomeruli and the brush border were stained with red-�uorescent Alexa Fluor® 568 phal-loidin (A12380). Finally, the nuclei were counterstained with the blue-�uorescent DNA stain DAPI (D1306, D3571, D21490). This image is a composite of three micrographs acquired using �lter sets appropriate for �uorescein, tetra-methylrhodamine and DAPI.
Figure 23.1.14 FluoCells® prepared slide #3 (F24630) con-taining a 16 µm cryostat section of mouse kidney stained with green-�uorescent Alexa Fluor® 488 wheat germ ag-glutinin (W11261), red-orange–�uorescent Alexa Fluor® 568 phalloidin (A12380) and blue-�uorescent DAPI (D1306, D3571, D21490). The image represents an optical section obtained by simultaneous two-photon excitation of all three dyes at 797 nm using a Bio-Rad® Radiance 2100 multi-photon microscope system. The image was acquired at the 2001 3D Microscopy of Living Cells Course, University of British Columbia, Vancouver, Canada, by John Jordan, Bio-Rad® Laboratories; and Iain Johnson, Life Technologies.
• FluoCells® prepared slide #1 (F36924) shows bovine pulmonary artery endothelial cells (BPAEC) stained with MitoTracker® Red CMXRos for labeling mitochondria, Alexa Fluor® 488 phalloidin for labeling F-actin and DAPI for labeling the nucleus.
• FluoCells® prepared slide #219 (F14781, Figure 23.1.11) again contains BPAEC, but this time stained with Texas Red®-X phalloidin for labeling F-actin, mouse monoclonal anti–α-tubulin antibody in conjunction with BODIPY® FL goat anti–mouse IgG antibody for labeling mi-crotubules and DAPI for labeling the nucleus.
• FluoCells® prepared slide #3 (F24630; Figure 23.1.12, Figure 23.1.13, Figure 23.1.14) contains a 16 µm cryostat section of a mouse kidney. Green-�uorescent Alexa Fluor® 488 wheat germ agglutinin stains the glomeruli and convoluted tubules; red-�uorescent Alexa Fluor® 568 phalloidin labels actin, which is especially prevalent in glomeruli and the brush border of proximal convoluted tubules; �nally, DAPI stains the nuclei with blue �uorescence.
• FluoCells® prepared slide #4 (F24631, Figure 23.1.15) contains a 16 µm cryostat section of a mouse intestine. Alexa Fluor® 350 wheat germ agglutinin labels the mucus of goblet cells with blue �uorescence; the red-�uorescent Alexa Fluor® 568 phalloidin labels actin �laments, which are especially prevalent in the brush border of the intestinal mucosa; and SYTOX® Green nucleic acid stain labels nuclei with green �uorescence.
• FluoCells® prepared slide #6 (F36925, Figure 23.1.16) contains muntjac skin �broblast cells stained with a combination of �uorescent stains. Green-�uorescent Alexa Fluor® 488 phalloi-din labels the prominent �lamentous actin in these cells; a mouse monoclonal anti–OxPhos Complex V inhibitor protein antibody in conjunction with the orange-�uorescent Alexa Fluor® 555 goat anti–mouse IgG antibody labels mitochondria; far-red–�uorescent TO-PRO®-3 nucle-ic acid stain labels nuclei. Because it contains no blue-�uorescent dyes, this slide is ideal for use with confocal laser-scanning microscopes that rely on non–UV laser light sources.
FocalCheck™ �uorescent microspheres are speci�cally designed for examining the alignment, sensitivity and stability of confocal laser-scanning microscopes.17,20 �ey are particularly useful for con�rming the optical sectioning thickness (Z-resolution) in three-dimensional imaging applica-tions. �ese polystyrene beads—available in 6 µm and 15 µm diameters—have been treated using a proprietary method in which a �uorescent dye is allowed to penetrate to only a limited depth within the microsphere. �e resulting beads have a well-de�ned dye layer that, when viewed in
Figure 23.1.11 FluoCells® prepared slide #2 (F14781), which shows bovine pulmonary artery endothelial cells (BPAEC) that have been stained with an anti–ß-tubulin mouse mono-clonal antibody in conjunction with BODIPY® FL goat anti–mouse IgG (B2752) for labeling microtubules, Texas Red®-X phalloidin (T7471) for labeling F-actin and DAPI (D1306, D3571, D21490) for labeling nuclei. This multiple-exposure image was acquired using bandpass optical �lter sets appro-priate for DAPI, �uorescein and Texas Red® dye.
The Molecular Probes™ Handbook: A Guide to Fluorescent Probes and Labeling Technologies
IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.thermofisher.com/probes
Chapter 23 — Antifades and Other Tools for Fluorescence
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The Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling TechnologiesIMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
Section 23.1 Fluorescence Microscopy Accessories and Reference Standards
Figure 23.1.16 FluoCells® prepared slide #6 (F36925) showing a �xed, permeabilized and labeled muntjac skin �broblast. Mitochondria were labeled with mouse anti–OxPhos Complex V inhibitor protein antibody and visual-ized using orange-�uorescent Alexa Fluor® 555 goat anti–mouse IgG antibody (A21422). F-actin was labeled with green-�uorescent Alexa Fluor® 488 phalloidin (A12379), and the nucleus was stained with TO-PRO®-3 iodide (T3605, pseudocolored magenta).
Figure 23.1.18 Images from confocal laser-scanning mi-croscope optical cross-sectioning of our 15 µm FocalCheck™ microspheres that have a dark red–�uorescent ring stain with a green-�uorescent stain throughout the bead (F7239). The left panel provides a clear visual representation of poor instrument alignment. Correct image registration has been achieved in the right panel, where the dark red ring is aligned with the green disk. The image was contrib-uted by Paulette Brunner, Keck Imaging Center, University of Washington, and Yu-Zhong Zhang, Life Technologies.
Figure 23.1.15 FluoCells® prepared slide #4 (F24631) con-tains a section of mouse intestine stained with a combination of �uorescent stains. Alexa Fluor® 350 wheat germ agglutinin (W11263) is a blue-�uorescent lectin that was used to stain the mucus of goblet cells. The �lamentous actin prevalent in the brush border was stained with red-orange–�uorescent Alexa Fluor® 568 phalloidin (A12380). Finally, the nuclei were stained with SYTOX® Green nucleic acid stain (S7020). This image is a composite of three digitized images obtained with �lter sets appropriate for �uorescein, DAPI and tetramethylrhodamine.
cross section in the confocal laser-scanning microscope, appears as a �uorescent ring of varying di-mensions depending on the focal plane (Figure 23.1.17, Figure 23.1.18). We refer to this proprietary staining procedure as ring staining to di�erentiate it from routine staining throughout the bead.
FocalCheck™ microspheres are available in a variety of color con�gurations provided by �ve di�erent �uorescent stains:
• Blue (365/430 nm)• Green (505/515 nm)• Orange (560/580 nm)• Red (575/600 nm)• Dark red (660/680 nm)
�e excitation/emission maxima of the di�erent stains are well matched to the laser sources and optical �lters commonly used in confocal laser-scanning microscopy and are especially useful in testing and aligning confocal laser-scanning microscopes with multiple laser lines and detection channels (Figure 23.1.19). Moreover, because the dyes are localized within the bead and therefore protected from environmental factors, the FocalCheck™ microspheres are brighter and much more photostable than conventional surface-stained beads.
Figure 23.1.17 Confocal laser-scanning microscope optical cross-sectioning and alignment with FocalCheck™ micro-spheres. A) Serial optical sectioning from top to bottom along the z-axis of ring-stained microspheres reveals a continuous pattern of disc-to-ring-to-disc images. B) The diameter of the �uorescent ring (or disc) seen is dependent on the depth of the optical focal plane. C) In the confocal laser-scanning mi-croscope, separate light paths exist for UV and visible wave-lengths. Also, emitted �uorescence is detected by di�erent photomultipliers. Proper optical alignment may be obtained with either of two types of FocalCheck™ microspheres. For example, the microspheres with an orange ring stain that are blue-�uorescent throughout the bead allow UV/visible wave-length alignment in three dimensions upon aligning the or-ange ring with the blue disc. Focal alignment is also possible simultaneously in three colors by aligning the green, orange and dark red rings of the FocalCheck™ microspheres contain-ing �uorescent green/orange/dark red ring stains.
C Blue
Orange
Red
GreenOrange
Alignment
Alignment
A
B 1
2
3
21 3
A
C
B
Figure 23.1.19 Normalized excitation spectra of the dyes contained in the FocalCheck™ microspheres. Emission lines of several commonly used laser excitation sources are superimposed on the dyes’ excitation spectra to illustrate the wide range of usage of these beads as calibration references. Ar = Argon-ion laser. Kr-Ar = Krypton-argon laser. He-Ne = Helium-neon laser.
Ar351
Ar488
Ar514
He-Ne543
Kr-Ar568
He-He633
Kr-Ar647
Wavelength (nm)
300 400 500 600 700
The Molecular Probes™ Handbook: A Guide to Fluorescent Probes and Labeling Technologies
IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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Chapter 23 — Antifades and Other Tools for Fluorescence
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Section 23.1 Fluorescence Microscopy Accessories and Reference Standards
FocalCheck™ products are available in various di�erent color con-�gurations, including three suspensions that contain microspheres ex-hibiting ring stains of two or three di�erent �uorescent colors:
• Blue-�uorescent and orange-�uorescent ring stains (15 µm, F7234)• Green-�uorescent and dark red–�uorescent ring stains (15 µm,
F7240)• Green-�uorescent, orange-�uorescent and dark red–�uorescent
ring stains (6 µm, F14806; 15 µm, F7235)
We also supply four suspensions that contain microspheres exhib-iting a ring stain of one �uorescent color combined with a stain of a second �uorescent color throughout the bead:
• Green-�uorescent ring stain with blue-�uorescent stain through-out (6 µm, F14808; 15 µm, F7237)
• Green-�uorescent ring stain with dark red–�uorescent stain throughout (15 µm, F7238)
• Orange-�uorescent ring stain with blue-�uorescent stain through-out (15 µm, F7236)
• Dark red–�uorescent ring stain with green-�uorescent stain throughout (6 µm, F14807; 15 µm, F7239, Figure 23.1.18)
�e sharp ring stains exhibited by the FocalCheck™ microspheres produce a striking visual representation of instrument misalignment or other aberrations, making them ideal as reference standards for confocal laser-scanning microscopy. Correct image registration is in-dicated when the multiple ring images of the ring-stained FocalCheck™ beads (or the ring and disk images of the combination ring-stained and stained-throughout FocalCheck™ beads) are perfectly coincident in all dimensions (Figure 23.1.17).
contains smaller-dia meter microspheres that have spectral and physi-cal features similar to those of our 6 µm and 15 µm FocalCheck™ micro-spheres. Because we prepare these 1.0 µm beads using �uorescent stains that are restricted to the surface only, they exhibit sharper and thinner
Figure 23.1.20 A double-labeled microsphere from the FocalCheck™ DoubleGreen Fluorescent Microsphere Kit (F36905). The bead was imaged as a z-series using a Carl Zeiss® LSM 510 META system. The two green-�uorescent dyes were separated by spectral unmixing, and one of the dyes was pseudocolored red. In this composite image, the complete z-series is shown prior to software rendering. Rendering �lls in the missing information between the slices by interpolation to create a solid object.
�uorescent ring patterns when viewed in cross section with a confocal laser-scanning microscope. �e FocalCheck™ �in-Ring Fluorescent Microspheres Kit contains three 200 µL suspensions of 1.0 µm beads. Each suspension contains beads with a di�erent color con�guration:
• Green-�uorescent (495/515 nm) and red-�uorescent (575/600 nm) ring stains
• Green-�uorescent (495/515 nm) ring stain with dark red–�uores-cent (660/680 nm) stain throughout the bead
• Red-�uorescent (575/600 nm) ring stain with blue-�uorescent (365/430 nm) stain throughout the bead
FocalCheck™ Microspheres Pre-Mounted on Microscope Slides
In addition to the bead suspensions described above, we o�er FocalCheck™ microspheres pre-mounted on microscope slides. �e FocalCheck™ Fluorescent Microsphere Kits feature mounted samples of three di�erent color con�gurations, in either the 6 µm (F24633) or the 15 µm (F24634) bead size:
• FocalCheck™ beads with green-�uorescent, orange-�uorescent and dark red–�uorescent ring stains
• FocalCheck™ beads with green-�uorescent ring stain and blue-�uorescent stain throughout the bead
• FocalCheck™ beads with dark red–�uorescent ring stain and green-�uorescent stain throughout the bead
FocalCheck™ Fluorescence Microscope Test SlidesFocalCheck™ �uorescence microscope test slides #1, #2 and #3 are
speci�cally designed for calibrating �uorescence microscope systems and evaluating system and �lter performance:
• FocalCheck™ �uorescence microscope test slide #1 (F36909) is ideal for routine checking and calibration of confocal and wide�eld �uo-rescence microscopes 21
• FocalCheck™ �uorescence microscope test slide #2 (F36913) pro-vides a robust, reproducible method of evaluating the performance of spectral imaging systems,22 as well as the ability to discriminate closely overlapping spectra. �e slide consists of 6 µm–diameter microspheres labeled with a series of spectrally overlapping dyes
• FocalCheck™ �uorescence microscope test slide #3 (F36914) is use-ful for basic evaluation of �lter performance and as a general prac-tice slide for �uorescence microscopy and digital imaging
�e slides each contain 10 sample areas arranged in two rows coated with proprietary �uorescent microspheres designed speci�cally for microscopy applications. �e microspheres are mounted in opti-cal cement (refractive index ~1.52) for maximal stability. �e optical thickness of the mounted bead specimens may result in spherical aber-ration or inability to focus when using certain types of objectives. Users who encounter these problems are encouraged to contact our Technical Assistance Department.
FocalCheck™ Fluorescent Microspheres for Spectral Unmixing
We have prepared two FocalCheck™ Fluorescent Microspheres Kits for testing spectral separation on spectral imaging systems.23 �ese mi-crospheres are stained with two di�erent �uorescent dyes that appear
The Molecular Probes™ Handbook: A Guide to Fluorescent Probes and Labeling Technologies
IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.thermofisher.com/probes
Chapter 23 — Antifades and Other Tools for Fluorescence
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The Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling TechnologiesIMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
Section 23.1 Fluorescence Microscopy Accessories and Reference Standards
similar in color by eye but are su�ciently di�erent to be resolved by linear-unmixing tech-niques. When linear-unmixing data-processing algorithms are applied, the dyes are shown to be spectrally distinct and spatially separated—one appears only within the outer ring and the other appears throughout the microsphere (Figure 23.1.20). �ese 6 µm, dual-stained microspheres are provided mounted on a microscope slide in each of the following kits:
• FocalCheck™ DoubleGreen Fluorescent Microspheres Kit (F36905), with a green-�uorescent (500/512 nm) ring dye and a slightly longer-wavelength green-�uorescent (512/525 nm) core dye
• FocalCheck™ DoubleOrange Fluorescent Microspheres Kit (F36906), with an orange-�uores-cent (532/552 nm) ring dye and a slightly longer-wavelength orange-�uorescent (545/565 nm) core dye
For generating reference spectra, these kits also contain two additional slides con-taining micro spheres stained uniformly with each of the individual dyes. Downloadable reference spectra are also available through our online Fluorescence SpectraViewer tool (www.invitrogen.com/handbook/spectraviewer; Using the Fluorescence SpectraViewer—Note 23.1).
MultiSpeck™, TetraSpeck™ and Constellation™ Fluorescent Microspheres
Our MultiSpeck™ and TetraSpeck™ �uorescent microspheres greatly facilitate the adjustment and calibration of conventional �uorescence microscopes, confocal laser-scanning microscopes and associated image-processing equipment for multicolor applications. �ese uniform, multiply stained microspheres are useful for colocalizing and focusing di�erent wavelengths of light in the same optical plane, as well as for checking multicolor image resolution, magni�cation and sensitivity.
Microscopy Standards Kit (M7901) exhibit three relatively distinct emission bands—blue, green and red—throughout every particle (Figure 23.1.21). �e spectral characteristics (excitation/emission peaks at 365/405 nm, 520/525 nm and 580/600 nm) are compatible with optical �lter sets designed for commonly used blue, green and red �uorophores (e.g., DAPI, �uorescein and rhodamine or Texas Red® dyes or their Alexa Fluor® dye counterparts). �e MultiSpeck™ beads can be used as external references for assessing image registration in two and three dimensions, allowing the researcher to accurately determine the spatial relationships of di�erent labels in multiparameter experiments.
• Suspension of MultiSpeck™ multispectral microspheres• Mixed suspension of separately stained blue-, green- and red-�uorescent microspheres,
which exhibit the same three excitation/emission bands as the multispectral microspheres but in separate beads
• Mounting medium• Slide-mounting protocol
Both suspensions are provided at a ready-to-use density and can be mounted on slides or incorporated into an experimental sample. Each kit supplies a su�cient amount of material for ~50 slide preparations using either of the two bead suspensions provided.
TetraSpeck™ Fluorescent Microspheres and Sampler KitsOur TetraSpeck™ �uorescent microspheres 17,19,20 expand the multispectral strategy intro-
duced with the MultiSpeck™ beads in two important ways. First, the TetraSpeck™ beads have been stained throughout with a mixture of four di�erent �uorescent dyes, yielding four well-separated excitation and emission peaks (Figure 23.1.22). �e excitation/emission maxima of the dyes are
Figure 23.1.21 Multiple exposures of a prepared slide from our MultiSpeck™ Multispectral Fluorescence Microscopy Standards Kit (M7901). This kit provides �uorescent MultiSpeck™ microspheres that exhibit three relatively dis-tinct excitation/emission bands—blue, green and red—all in the same particle. Thus, the same microsphere can appear a di�erent color depending on the optical �lter set used. This photograph was taken through bandpass optical �lter sets appropriate for DAPI, �uorescein and Texas Red® dyes, with the �eld of view shifted slightly between exposures.
Figure 23.1.22 Four separate exposures of three Tetra-Speck™ beads (T7283) photographed using optical �lter sets appropriate for DAPI, �uorescein, tetramethylrhodamine and the Texas Red® dye. The stage was shifted after each exposure. Note that the same beads appear blue, green, or-ange or red, depending on the �lters used.
The Molecular Probes™ Handbook: A Guide to Fluorescent Probes and Labeling Technologies
IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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Chapter 23 — Antifades and Other Tools for Fluorescence
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The Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling TechnologiesIMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
Section 23.1 Fluorescence Microscopy Accessories and Reference Standards
Figure 23.1.24 Constellation™ microspheres (C14837) provide an assortment of sizes and colors for use in �uo-rescence microscopy demonstrations.
365/430 nm (blue), 505/515 nm (green), 560/580 nm (orange) and 660/680 nm (dark red). Second, these microspheres are available in �ve nominal sizes (actual bead diameters are indicated on the product labels), spanning the range from subresolution to nearly cell-size particles:
Figure 23.1.23 Luminescent microsphere products provide an extensive range of sizes and �uorescent colors, illustrated by a sample of our Constellation™ microspheres for imaging (C14837).
Figure 23.1.25 One PS-Speck™ green-�uorescent micro-sphere (P7220) used for point-spread function analysis. Shown are individual images, in pseudocolor, taken in x-y planes with 0.25 µm increments in the z-axis between planes. The image was contributed by Regina Armstrong, Uniformed Services University of the Health Sciences, Bethesda, Maryland.
• 0.1 µm (T7279)• 0.2 µm (T7280)• 0.5 µm (T7281)
• 1.0 µm (T7282)• 4.0 µm (T7283)
Each of these products provides a 0.5 mL suspension sample of TetraSpeck™ microspheres that is su�cient for about 100 slides. We o�er the TetraSpeck™ Fluorescent Microspheres Sampler Kit (T7284) and the TetraSpeck™ Fluorescent Microspheres Size Kit (T14792). �e TetraSpeck™ Fluorescent Microspheres Sampler Kit consists of separate suspension samples of our 0.1 µm, 0.5 µm and 4.0 µm TetraSpeck™ beads, each su�cient for preparing about 20 slides. �e TetraSpeck™ Fluorescent Microspheres Size Kit contains six microscope slides; �ve slides with a mounted sample of the 0.1 µm, 0.2 µm, 0.5 µm, 1.0 µm or 4.0 µm diameter TetraSpeck™ micro-spheres, and a sixth slide with a mixture of all �ve sizes. TetraSpeck™ microspheres have been used to calibrate the spatial distribution of illumination for high-content screening (HCS),24,25 and as reference markers for image alignment in high-resolution immuno�uorescence colocal-ization analysis.24,26 In addition, various FluoSpheres® and TetraSpeck™ beads have been used as reference standards for two-photon excitation microscopy, and in particular for the in situ determination of the two-photon excitation point-spread function (PSF).27–29
Constellation™ Microspheres for ImagingConstellation™ microspheres for imaging (C14837; Figure 23.1.23, Figure 23.1.24) are 3 mL
suspensions of assorted �uorescent microspheres with a variety of sizes and colors. Designed for use in laboratory tutorials and customer training sessions, they provide inexpensive and robust test samples for demonstrating �lter switching, focus adjustment and other functional capabilities of �uorescence microscopes. �e Constellation™ microspheres can be stored at room temperature, protected from light.
PS-Speck™ Microscope Point Source Kit�e �uorescent microspheres in the PS-Speck™ Microscope Point Source Kit (P7220) have
a diameter of 0.175 ± 0.005 µm, making them ideal subresolution �uorescent sources for cali-brating instrument optics. �ey are particularly useful for measuring the point-spread function (PSF) required for computational image deconvolution procedures 17,30–32 (Figure 23.1.25, Figure 23.1.26). �is kit’s four ready-to-use suspensions contain bright monodisperse particles in the following �uorescent colors (and excitation/emission peaks):
• Blue (360/440 nm)• Green (505/515 nm)
• Orange (540/560 nm)• Deep red (633/660 nm)
�e kit also includes mounting medium and a mounting protocol for the user’s conve-nience. Each suspension provides su�cient material to mount about 100 slides. PS-Speck™ microspheres are too large to represent point source objects for microscopes equipped with high numerical aperture (e.g., NA 1.4) oil immersion objectives. In such cases, we recommend our 0.1 µm TetraSpeck™ �uorescent microspheres (T7279) or 0.1 µm and 0.04 µm FluoSpheres® carboxylate-modi�ed microspheres (Section 6.5) for PSF determinations.
InSpeck™ Microscopy Image Intensity Calibration KitsInSpeck™ Microscope Image Intensity Calibration Kits provide microsphere standards that
generate a series of well-de�ned �uorescence intensity levels (Figure 23.1.27) for constructing cali-bration curves and evaluating sample brightness.6,25,33 InSpeck™ microspheres have been used to estimate the global background and signal response for high-content screening (HCS).25
The Molecular Probes™ Handbook: A Guide to Fluorescent Probes and Labeling Technologies
IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.thermofisher.com/probes
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Section 23.1 Fluorescence Microscopy Accessories and Reference Standards
Most of the kits are o�ered in a choice of two di�erent microsphere sizes (2.5 µm or 6 µm) and �ve di�erent �uorescent colors:
Each kit includes six separate suspensions of InSpeck™ �uorescent microspheres with relative �uorescence intensities of 100%, 30%, 10%, 3%, 1% and 0.3% (Figure 23.1.27), covering the range of intensities commonly encountered in microscopy applications. Unstained control beads and mounting medium are also supplied. �e aqueous suspensions of microspheres may be applied directly to the sample for calibrating �uorescence intensities or mounted separately in an adjacent well or on another slide. Each suspension provides su�cient material to prepare about 100 slides.
Fluorescence Reference StandardsFluorescein NIST-Traceable Standard
�e National Institute of Standards and Technology (NIST) chose high-grade Molecular Probes® �uorescein to create Standard Reference Material 1932 (SRM® 1932), a certi�ed �uores-cein solution. We now o�er a NIST-traceable �uorescein standard (F36915) that not only meets the stringent criteria established by NIST, but is also directly traceable to SRM® 1932. We supply our NIST-traceable �uorescein standard as a calibrated 50 µM solution of �uorescein in 100 mM sodium borate bu�er, pH 9.5; under these conditions, �uorescein is completely ionized 34 and is therefore in its most �uorescent form (Figure 23.1.28, Figure 23.1.29), exhibiting an extremely high quantum yield of 0.93.
Academic researchers and industry scientists alike can use our NIST-traceable �uorescein standard to assess day-to-day or experiment-to-experiment variation in �uorescence-based in-strumentation, as well as to determine the Molecules of Equivalent Soluble Fluorophore (MESF) value for an experimental solution. �e MESF value is de�ned not as the actual number of dye molecules present, but rather as the number of �uorophores that would yield a �uorescence intensity equivalent to that of the experimental solution when analyzed on the same instrument under the same conditions.35–38 Consequently, the MESF value is an important tool for character-izing the �uorescence intensity of a solution containing spectrally similar dye molecules attached to antibodies, nucleic acids, microspheres or other substrates that might enhance or diminish the �uorescence. When its pH is carefully matched with that of the experimental solution, our NIST-traceable �uorescein standard can be used for accurate MESF determinations of a wide range of green-�uorescent dye solutions and on an assortment of �uorescence-based instruments.
Figure 23.1.26 An orthogonal (x-z) display representing a point-spread function. The microsphere used is a component of the PS-Speck™ Microscope Point Source Kit (P7220). This pseudocolored image was generated electronically from a series of microsphere images taken in x-y planes. The image was contributed by Jennifer Kramer, Scanalytics.
Figure 23.1.27 Flow cytometric analysis of the beads in the 6 µm InSpeck™ Green Microscope Image Intensity Calibration Kit (I14785). The microspheres have nominal relative �uorescence intensities of 100%, 30%, 10%, 3%, 1%, 0.3%. For each lot, actual relative intensities are determined by �ow cytometry and printed on the product labels.
Num
ber
of p
artic
les
Forward scattering
Fluorescence intensity
100
101102
103
104
0
60
120
180
O OHO
CO
O
_
monoanion
O OHO
COH
H
O
+
cation
O OO
CO
O
_
dianion
_
O OHO
COH
O
neutral
lactone
O OHHO
OO
Figure 23.1.28 Ionization equilibria of �uorescein. Figure 23.1.29 The pH-dependent spectra of �uorescein (F1300): A) absorption spectra, B) emission spectra.
8.0
7.0
6.5
6.0
5.0
Ex = 488 nm
300 400 500
650600500 550
pH 9.0
8.0
7.0
6.5
6.0
5.0
pH 9.0
Fluo
resc
ence
em
issi
onA
bso
rptio
n
Wavelength (nm)
A
B
Wavelength (nm)
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Section 23.1 Fluorescence Microscopy Accessories and Reference Standards
Figure 23.1.31 Absorption and �uorescence emission spectra of �uorescein in 0.1 M sodium hydroxide.
Figure 23.1.30 Absorption and �uorescence emission spectra of quinine sulfate, dihydrate in 0.5 M sulfuric acid.
Figure 23.1.33 Absorption and �uorescence emission spectra of sulforhodamine 101 in ethanol.
Figure 23.1.32 Absorption and �uorescence emission spectra of 5-carboxytetramethylrhodamine (5-TAMRA) in methanol.
Figure 23.1.34 Absorption and �uorescence emission spectra of nile blue in ethanol.
Table 23.2 Spectroscopic data for components of the Reference Dye Sampler Kit.
Component SolventAbs (nm) *
Em (nm) * QY †
Quinine sulfate Water 347 ‡ 455 ‡ 0.55 ‡
Fluorescein Water 490 § 514 § 0.92 §
5-CTMR ** Methanol 542 568 0.68
Sulforhodamine 101
Ethanol 578 605 0.90
Nile blue Ethanol 636 665 0.27
* Approximate absorbance (Abs) and �uorescence emission (Em) maxima. † Fluorescence quantum yield at 22°C. ‡ Standard values in 0.5 M H2SO4. § Standard values in 0.1 M NaOH. ** CTMR = Carboxytetramethylrhodamine.
extensively characterized �uorescence standards with emission spec-tra covering the entire visible wavelength range.39 All �ve �uorescent standards are supplied as 1 mM stock solutions in 1 mL units, su�cient to prepare approximately 500 diluted working samples for spectro�uo-rometry. �e compositions of the stock solutions are as follows:
• Quinine sulfate in 0.1 M sulfuric acid (H2SO4) (Figure 23.1.30)• Fluorescein in dimethylsulfoxide (DMSO) (Figure 23.1.31)• 5-Carboxytetramethylrhodamine in DMSO (Figure 23.1.32)• Sulforhodamine 101 in DMSO (Figure 23.1.33)• Nile blue perchlorate in DMSO (Figure 23.1.34)
Spectroscopic data for the �ve standards are summarized in Table 23.2. Reference spectra for all �ve �uorescent standard solutions are pro-vided through our online Fluorescence SpectraViewer tool (www.invitro-gen.com/handbook/spectraviewer).
Sample Chambers, Slides and CoverslipsIn collaboration with Grace Bio-Labs, we o�er a collection of acces-
sories for imaging and microscopy. �ese accessories make slide prepara-tion easy, facilitate sample perfusion and simplify sample manipulation during in situ hybridization and other procedures that involve multiple wash steps.
CultureWell™ Cell Culture Systems�e CultureWell™ cell culture systems provide an integrated set of
tools for preparing cultured cells for staining and imaging. Each sys-tem uses medical-grade silicone gaskets preassembled with standard optical-quality coverslips into convenient inserts that �t into matching cell culture plates (Figure 23.1.35). �e entire system is provided sterile and ready to use. Cell culture, treatment and staining are performed on the coverslip, which adheres securely to the culture plate via a silicone backing. �e samples can then be imaged with or without the silicone gaskets. Two types of systems are available in several con�gurations to suit a variety of needs (Table 23.3).
�e CultureWell™ multiwell cell culture systems use precut sili-cone gaskets to form convenient no-leak wells on 24 × 50 mm cover-slips. �e wells are spaced for compatibility with micro�uidic handling robots. Low numbers of wells are ideal for titering antibody dilutions or other staining conditions, whereas the higher numbers of wells fa-cilitate high-throughput screening. Each insert includes four cover-slips with gaskets, preassembled into a culture plate. Silicone dividers (C24770, Figure 23.1.36) are also available for separating portions of the coverslip into leak-proof wells, for di�erent treatment and washing conditions.
�e CultureWell™ multislip cell culture systems comprise multiple coverslips arrayed on a sheet of silicone and assembled into a conve-nient insert. �e silicone backing adheres the coverslips to the tissue culture plate, preventing movement during plating, cell culture and washing steps. Each coverslip can be removed separately for individual
The Molecular Probes™ Handbook: A Guide to Fluorescent Probes and Labeling Technologies
IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.thermofisher.com/probes
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Section 23.1 Fluorescence Microscopy Accessories and Reference Standards
staining experiments. �e CultureWell™ cell culture systems are provided in a set of 10 preas-sembled inserts in plates.
CultureWell™ Chambered Coverslips�e CultureWell™ chambered coverslips (Table 23.3, Figure 23.1.37) are the same gasketed
coverslips provided in our CultureWell™ cell culture systems, but they are not preassembled into inserts. �e chambered coverslip can be placed in CultureWell™ plates (C24769) or other cell culture dishes for cell culture and staining. �e silicone gasket can be easily removed and the coverslip placed on a slide for microscopy. �e CultureWell™ chambered coverslips provide maximum versatility for designing smaller scale cell culture applications. �ey are provided in sets of �ve sterile pouches, with four chambered coverslips per pouch.
CultureWell™ Chambered Coverglasses�e CultureWell™ chambered coverglass—provided sterile and ready to use—contains 16
wells that can each hold up to 250 µL, allowing cells to be cultured in a number of di�erent conditions on a single slide (Figure 23.1.38). A silicone gasket forms a leakproof seal between the polystyrene upper structure and the coverglass. When the cells are ready to be imaged, cover-glass removal is made easy by the use of a simple tool that separates the parts without the need for excessive force, eliminating the risk of coverglass breakage (Figure 23.1.39). Removal of the
Figure 23.1.35 CultureWell™ cell culture system.
Figure 23.1.37 CultureWell™ chambered coverslips.
Figure 23.1.36 CultureWell™ silicone dividers (C24770). Figure 23.1.38 The CultureWell™ removable chambered coverglass for cell culture (C37000).
Table 23.3 CultureWell™ cell culture systems and chambered coverslips.
Cat. No.
Size of Coverslip
(mm)
Number of Wells per Coverslip Well Dimensions Depth Volume per Well
Number of Coverslips per Insert
CultureWell™ multiwell cell culture systems *†
C24762 24 × 50 2 15 mm diameter 1 mm 250–400 µL 4
C24763 24 × 50 2 15 mm diameter 2 mm 300–500 µL 4
C24764 24 × 50 3 9.5 mm square 1 mm 300–500 µL 4
C24765 24 × 50 4 9 mm diameter 1 mm 50–100 µL 4
C24766C24767
24 × 50 8 6 mm diameter 1 mm 15–30 µL 4
C24768 24 × 50 50 3 mm diameter 1 mm 3–10 µL 4
CultureWell™ chambered coverslips ‡
C24775 24 × 50 2 15 mm diameter 1 mm 250–400 µL NA
C24776 24 × 50 2 15 mm diameter 2 mm 300–500 µL NA
C24777 24 × 50 3 9.5 mm diameter 1 mm 300–500 µL NA
C24778 24 × 50 4 9 mm diameter 1 mm 50–100 µL NA
C24779 24 × 50 8 6 mm diameter 1 mm 15–30 µL NA
C24780 24 × 50 50 3 mm diameter 1 mm 3–10 µL NA
CultureWell™ multislip cell culture systems *
C24761 18 × 18 NA NA NA NA 8
C24760 12 × 12 NA NA NA NA 15
* Each system is supplied in packages of 10 inserts, each preassembled in an 86 mm × 128 mm plate. † A trial size is also available (C24767) that includes two inserts — four coverslips with eight 6 mm wells each — in two plates. ‡ Chambered coverslips are supplied in sets, in �ve pouches of four coverslips each. Packs of 10 sterile plates are also available separately (C24769). NA = Not applicable.
Figure 23.1.39 Using the CultureWell™ removable cham-bered coverglass for cell culture (C37000). When the cells are ready to be imaged, coverglass removal is made easy by the use of a simple tool (included with the coverglass) that separates the parts without the need for excessive force, eliminating the risk of coverglass breakage.
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Section 23.1 Fluorescence Microscopy Accessories and Reference Standards
silicone gasket leaves no residue. �e components of the chambered coverglass are manufactured and assembled with special orientation features to allow easy location of a speci�c specimen af-ter the coverglass is mounted. Frosted microscope slides are also provided for mounting. �e CultureWell™ removable chambered coverglass has several important features:
• Black silicone gasket reduces light scatter, enhancing �uorescence applications.• Inert, non-cytotoxic silicone permits edge-to-edge growth of cells in the wells.• Wells are spaced to allow the use of multichannel pipettes for fast and easy cell culturing.• Silicone gasket remains attached to the coverglass a�er separation, allowing the wells to be
used as reagent reservoirs.• Gasket design is ideally suited for small-volume incubations, in situ hybridization and
immunostaining.• Chambered coverglass is provided sterile and ready to use.
We o�er the CultureWell™ chambered coverglass in a package containing eight chambered coverglasses and the removal tool (C37000), as well as in a sample size containing a pair of cham-bered coverglasses (C37005).
cally clear plastic cover, making them ideal for light, epi�uorescence and confocal laser-scanning microscopy.40 By simply pressing an imaging chamber gasket to a microscope slide or coverslip, a sealed chamber is formed to contain mounting medium. �e watertight chamber supports and stabilizes thick and free-�oating specimens, permitting resolution of �ne internal structures and analysis of markers without the compression or movement artifacts that a�ect observations made using an ordinary coverslip.
CoverWell™ Perfusion Chamber GasketsCoverWell™ perfusion chamber gaskets (Table 23.4, Figure 23.1.41) are designed for live-
cell imaging and manipulation. With the same silicone gasket technology as the CoverWell™ imaging chamber gaskets, these gaskets form watertight “press-to-seal” chambers with dual-access ports for addition and removal of perfusing media. �e access ports can be covered using
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Section 23.1 Fluorescence Microscopy Accessories and Reference Standards
adhesive seal tabs (A18211), which are available separately. �e heat-resistant gaskets can be sterilized and used for direct culturing of cells and tissues. CoverWell™ perfusion chamber gaskets are available in single- or multiwell con�gurations, allowing multiple experiments to be performed on a single microscope slide or coverslip.
are silicone gaskets with a clear plastic cover that are expressly de-signed for immunocytochemistry and in situ hybridization.41,42 �e gasket is simply pressed onto a wet or dry microscope slide to form a watertight chamber that holds reactants in place and prevents evapora-tion. �e chambers improve the uniformity and sensitivity of staining by enclosing a large sample area while minimizing the reagent volume required. �e incubation chamber gaskets are easily removed and re-applied for multiple-step procedures. �ese chamber gaskets are heat resistant, autoclavable and nuclease free.
Press-to-Seal Silicone Isolators and Secure-Seal™ Adhesive Spacers
For the ultimate in utility and �exibility, Press-to-Seal silicone isola-tors (Table 23.5, Figure 23.1.43) are removable hydrophobic barriers that can be customized to meet speci�c experimental requirements. �ey may be used to isolate cells grown in culture dishes or to separate spec-imens on microscopy slides during staining procedures. �e silicone material can be autoclaved and adheres to any smooth surface. Isolators without adhesive can be easily removed and reapplied for multiple in-cubation steps. Isolators are also available with adhesive on one side for added security or permanent mounting. In addition, we o�er un-cut silicone sheets that can easily be trimmed to prepare customized enclosures.
Similar to the Press-to-Seal silicone isolators, Secure-Seal™ adhe-sive spacers are ultra-thin gaskets with adhesive that can be stacked to any depth desired. For high-resolution microscopy, the spacer and specimen can be sandwiched between two No. 0 glass coverslips. �e spacers are available in several con�gurations (Table 23.5).
Table 23.5 Press-to-Seal gaskets and Secure-Seal™ spacers.
Cat. No.Number of Wells Well Dimensions Depth
Quantity per Package
Press-to-Seal silicone isolators
P18174 1 20 mm diameter 0.5 mm 50
P18175 1 20 mm diameter 1.0 mm 50
P24740 * 1 20 mm diameter 0.5 mm 50
P24741 * 1 20 mm diameter 1.0 mm 50
P24742 * 24 4.5 mm diameter 2.0 mm 25
P24743 * 8 9 mm diameter 0.5 mm 25
P24744 * 8 9 mm diameter 1.0 mm 25
Secure-Seal™ adhesive spacers
S24735 * 1 13 mm diameter 0.12 mm 100
S24736 * 1 20 mm diameter 0.12 mm 100
S24737 * 8 9 mm diameter 0.12 mm 100
Press-to-Seal silicone sheets (13 cm × 18 cm)
P18178 NA NA 0.5 mm 5
P18179 NA NA 1.0 mm 5
P24745 * NA NA 0.5 mm 5
* With adhesive on one side. NA = Not applicable.
Figure 23.1.43 Press-to-Seal silicone isolators.
Figure 23.1.44 HybriSlip™ hybridization covers.
Table 23.6 Tools for hybridization experiments.
Cat. No. Chamber Dimensions DepthUsable Volume
Quantity per
Package
HybriWell™ hybridization sealing system
H24720 13 mm diameter 0.25 mm 30 µL 100
H24721 20 mm diameter 0.15 mm 30 µL 100
H24723 22 mm × 22 mm 0.15 mm 30–50 µL 100
H18210 40 mm × 21 mm 0.15 mm 50–100 µL 100
H24722 40 mm × 22 mm 0.25 mm 180–200 µL 100
Secure-Seal™ hybridization chambers
S24734 22 mm × 22 mm 0.8 mm 250 µL 50
S24730 20 mm diameter 0.8 mm 200 µL 40
S24731 20 mm diameter 1.3 mm 280 µL 40
S24732 9 mm diameter 0.8 mm 20 µL 20
S24733 9 mm diameter 1.3 mm 40 µL 20
HybriSlip™ hybridization covers
H18200 22 mm × 22 mm NA NA 500
H18201 40 mm × 22 mm NA NA 500
H18202 60 mm × 22 mm NA NA 500
Seal tabs
A18211 Adhesive seal tab NA NA 400
NA = Not applicable.
HybriSlip™ Hybridization CoversHybriSlip™ hybridization covers (Table 23.6, Figure 23.1.44) are
nuclease free, ready-to-use and designed speci�cally for in situ hybrid-ization.43 �ese hydrophobic coverslips do not require lengthy prepara-tion or blocking procedures to prevent probe trapping or binding. �ey are heat resistant and do not curl, even at high temperatures, making them ideal for denaturation steps or in situ PCR incubations. HybriSlip™ covers are available in three sizes.
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Section 23.1 Fluorescence Microscopy Accessories and Reference Standards
Figure 23.1.48 ProPlate™ multi-array system (P37004). Indi-vidual modules (P37001), covered with seal strips (P37002), �t into a tray (P37003), producing a modular plate with a standard microplate footprint and well spacing.
HybriWell™ Hybridization Sealing SystemsHybriWell™ hybridization sealing systems (Table 23.6, Figure 23.1.45) are coverslip–seal
combinations that attach to microscope slides to form microwells optimized for carrying out in situ hybridization procedures. �ese ready-to-use hybridization gaskets have a special adhesive that bonds to glass slides in seconds, creating a water-tight seal that is temperature resistant but can also be removed cleanly and easily a�er hybridization. Solutions are easily added or re-moved through dual-access ports without disrupting the specimen. �e hydrophobic coverslips are nuclease free and will not trap or bind probes, allowing uniform distribution of the reagent over the specimen. �e HybriWell™ sealing systems also include a quick-seal tool to secure the hydrophobic cover to the microscope slide, as well as nuclease-free adhesive seal tabs to cover the access ports. Adhesive seal tabs (A18211) are also available separately in sets of 400.
(Table 23.6, Figure 23.1.46) are designed to isolate single or multiple specimens on a slide dur-ing in situ hybridization procedures. Access ports in the chamber surface allow for the addi-tion or removal of solutions and are easily sealed using adhesive seal tabs (A18211), available separately. Because they are deeper than the HybriWell™ chambers, the Secure-Seal™ chambers provide optimum surface-to-volume �uid dynamics, which facilitate more uniform hybridiza-tion. However, the shallower chambers created by the HybriWell™ sealing systems hold a smaller reagent volume, minimizing the amount of probe required.
ONCYTE® MultiWells with Slide and Matching Gasket�e versatile ONCYTE® MultiWells (Figure 23.1.47) consist of a two-piece set that includes
a slide printed with nitrocellulose circles and a matching removable gasket to enclose and iso-late each sample. �e nitrocellulose coating on the slide is specially formulated for �uorescence imaging. �is ultra-thin microporous coating ensures uniform binding of tissue prints, cells or macromolecules and becomes transparent in a variety of mounting media. �e matching press-to-seal silicone gaskets adhere easily to the surface of the slide to isolate specimens and reagents and prevent cross contamination. A coverslip can be added to create enclosed chambers for long incubations. Gaskets can be removed and cleaned simply by peeling them o�. ONCYTE® MultiWells are available in two con�gurations: a set of 20 slides and gaskets, each with 12 wells, 5 mm in diameter (O24750), or a set of 20 slides and gaskets, each with a single well, 13 mm in diameter (O24751).
ProPlate™ Multi-Array System�e ProPlate™ multi-array system (P37004) from Grace Bio-Labs allows integration of mi-
croscope array technology with automated microplate processing (Figure 23.1.48). Individual modules (P37001), covered with seal strips (P37002), �t into a tray (P37003), producing a modu-lar plate with a standard microplate footprint and well spacing. �e modular design allows loading of 1–4 slides per tray for plate washing and reading. Individual ProPlate™ modules may also be processed by hand without the use of the tray. �e large well volumes and isolation of 16 individual arrays (2 × 8) on a single microscope slide is particularly well suited for proteomics applications, including protein expression analysis, protein-protein interactions, antibody pro-�ling and high-throughput automated analysis of multiple proteins. �e ProPlate™ multi-array system can also be used to process cDNA or oligonucleotide arrays.44
The Molecular Probes™ Handbook: A Guide to Fluorescent Probes and Labeling Technologies
IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.thermofisher.com/probes
Chapter 23 — Antifades and Other Tools for Fluorescence
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The Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling TechnologiesIMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
Section 23.1 Fluorescence Microscopy Accessories and Reference Standards
Atto�uor Cell Chamber�e Atto�uor cell chamber (A7816, Figure 23.1.49) is a durable and practical coverslip holder
designed for viewing live-cell specimens on upright or inverted microscopes; spare O-rings for the Atto�uor cell chamber are available in sets of 10 (O14804). Features of the Atto�uor cell chamber include:
• Surgical stainless steel construction• Autoclavable, allowing cells to be grown directly in the chamber• O-ring seal design that prevents sample contamination by oil and leakage of media from
the coverslip• Accepts 25 mm–diameter round coverslips and mounts in a standard 35 mm–diameter
stage holder• �in 0.5 mm base, allowing clearance for the objective when focusing
Coverslip Mini-Rack and Coverslip Maxi-RackOur unique coverslip mini-rack (C14784, Figure 23.1.50) is a miniature support designed
to vertically hold eight standard round or square coverslips. �e mini-rack �ts easily into a standard 50 mL beaker and can accommodate a small stir bar beneath the rack. Use of the mini-rack eliminates the necessity for repeatedly moving coverslips between solutions with for-ceps. Because it is constructed of Te�on®, the mini-rack does not adsorb biopolymers, withstands strong acids and bases, is not damaged by heat and may be sterilized by a variety of methods such as autoclaving, organic solvent treatment or ethylene oxide exposure. �e mini-rack is easily dis-assembled for cleaning and storage. �e mini-rack is particularly useful in immunocytochemi-cal and in situ hybridization procedures involving sequential wash steps where thorough and consistent removal staining, �xation, permeabilization or blocking reagents from the coverslips is critical. �e coverslip maxi-rack (C24784, Figure 23.1.51) provides e�cient support for the simultaneous staining and washing of up to 50 samples on 18 mm square or circular coverslips in a self-contained covered container. �e maxi-rack includes a convenient handle to remove the rack from the staining solution.
The Molecular Probes™ Handbook: A Guide to Fluorescent Probes and Labeling Technologies
IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
thermofisher.com/probes
Chapter 23 — Antifades and Other Tools for Fluorescence
960www.invitrogen.com/probes
The Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling TechnologiesIMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
Section 23.1 Fluorescence Microscopy Accessories and Reference Standards
PRODUCT LIST 23.1 FLUORESCENCE MICROSCOPY ACCESSORIES AND REFERENCE STANDARDS Cat. No. Product QuantityA18211 Adhesive seal-tab, for HybriWell™ hybridization sealing system *set of 400* 1 setA7816 Atto�uor cell chamber *for microscopy* eachC14837 Constellation™ microspheres for imaging *mixture of assorted sizes and colors* 3 mLC24784 coverslip maxi-rack *for 50 coverslips* eachC14784 coverslip mini-rack *for 8 coverslips* eachC18160 CoverWell™ imaging chamber gasket, one chamber, 20 mm diameter, 0.5 mm deep *set of 40* 1 setC18161 CoverWell™ imaging chamber gasket, one chamber, 20 mm diameter, 1.0 mm deep *set of 40* 1 setC24726 CoverWell™ imaging chamber gasket with adhesive, one chamber, 20 mm diameter, 0.5 mm deep *set of 40* 1 setC24727 CoverWell™ imaging chamber gasket with adhesive, one chamber, 20 mm diameter, 1.0 mm deep *set of 40* 1 setC18155 CoverWell™ incubation chamber gasket, one chamber, 13 mm diameter, 0.2 mm deep *set of 25* 1 setC18156 CoverWell™ incubation chamber gasket, one chamber, 13 mm diameter, 0.5 mm deep *set of 50* 1 setC18150 CoverWell™ incubation chamber gasket, one chamber, 40 mm x 22 mm, 0.2 mm deep *set of 25* 1 setC18151 CoverWell™ incubation chamber gasket, one chamber, 40 mm x 22 mm, 0.5 mm deep *set of 50* 1 setC18139 CoverWell™ perfusion chamber gasket, eight chambers, 9 mm diameter, 0.5 mm deep *set of 20* 1 setC18140 CoverWell™ perfusion chamber gasket, eight chambers, 9 mm diameter, 1.0 mm deep *set of 20* 1 setC18141 CoverWell™ perfusion chamber gasket, eight chambers, 9 mm diameter, 2.0 mm deep *set of 20* 1 setC18142 CoverWell™ perfusion chamber gasket, eight chambers, 9 mm diameter, 2.5 mm deep *set of 20* 1 setC18128 CoverWell™ perfusion chamber gasket, four chambers, 19 mm x 6 mm, 0.5 mm deep *set of 40* 1 setC18136 CoverWell™ perfusion chamber gasket, one chamber, 20 mm diameter, 1.0 mm deep *set of 40* 1 setC18120 CoverWell™ perfusion chamber gasket, one chamber, 32 mm x 19 mm, 0.5 mm deep *set of 40* 1 setC18121 CoverWell™ perfusion chamber gasket, one chamber, 32 mm x 19 mm, 1.0 mm deep *set of 40* 1 setC24769 CultureWell™ cell culture plate *set of 10* 1 setC37005 CultureWell™ chambered coverglass for cell culture *sixteen wells per coverglass* *set of 2* 1 packC37000 CultureWell™ chambered coverglass for cell culture *sixteen wells per coverglass* *set of 8* 1 setC24770 CultureWell™ coverslip divider *set of 4* 1 setC24760 CultureWell™ multislip cell culture system MSI-12 *plate and insert, �fteen 12 mm coverslips per insert* *set of 10* 1 setC24761 CultureWell™ multislip cell culture system MSI-18 *plate and insert, eight 18 mm coverslips per insert* *set of 10* 1 setC24762 CultureWell™ multiwell cell culture system CWI 2R-1.0 *plate and insert, four 24 mm x 50 mm coverslips per insert, two 1 mm-deep wells per coverslip* *set of 10* 1 setC24763 CultureWell™ multiwell cell culture system CWI 2R-2.0 *plate and insert, four 24 mm x 50 mm coverslips per insert, two 2 mm-deep wells per coverslip* *set of 10* 1 setC24764 CultureWell™ multiwell cell culture system CWI 3S-1.0 *plate and insert, four 24 mm x 50 mm coverslips per insert, three 1 mm-deep wells per coverslip* *set of 10* 1 setC24765 CultureWell™ multiwell cell culture system CWI 4R-1.0 *plate and insert, four 24 mm x 50 mm coverslips per insert, four 1 mm-deep wells per coverslip* *set of 10* 1 setC24766 CultureWell™ multiwell cell culture system CWI 8R-1.0 *plate and insert, four 24 mm x 50 mm coverslips per insert, eight 1 mm-deep wells per coverslip* *set of 10* 1 setC24767 CultureWell™ multiwell cell culture system CWI 8R-1.0 TS *plate and insert, four 24 mm x 50 mm coverslips per insert, eight 1 mm-deep wells per coverslip* *set of 2* 1 setC24768 CultureWell™ multiwell cell culture system CWI 50R-1.0 *plate and insert, four 24 mm x 50 mm coverslips per insert, �fty 1 mm-deep wells per coverslip* *set of 10* 1 setC24775 CultureWell™ multiwell chambered coverslip CWCS 2R-1.0 *24 mm x 50 mm coverslips, two 1 mm-deep wells per coverslip* *set of 20* 1 setC24776 CultureWell™ multiwell chambered coverslip CWCS 2R-2.0 *24 mm x 50 mm coverslips, two 2 mm-deep wells per coverslip* *set of 20* 1 setC24777 CultureWell™ multiwell chambered coverslip CWCS 3S-1.0 *24 mm x 50 mm coverslips, three 1 mm-deep wells per coverslip* *set of 20* 1 setC24778 CultureWell™ multiwell chambered coverslip CWCS 4R-1.0 *24 mm x 50 mm coverslips, four 1 mm-deep wells per coverslip* *set of 20* 1 setC24779 CultureWell™ multiwell chambered coverslip CWCS 8R-1.0 *24 mm x 50 mm coverslips, eight 1 mm-deep wells per coverslip* *set of 20* 1 setC24780 CultureWell™ multiwell chambered coverslip CWCS 50R-1.0 *24 mm x 50 mm coverslips, �fty 1 mm-deep wells per coverslip* *set of 20* 1 setF36924 FluoCells® prepared slide #1 *BPAE cells with MitoTracker® Red CMXRos, Alexa Fluor® 488 phalloidin, DAPI* eachF14781 FluoCells® prepared slide #2 *BPAE cells with mouse anti-α-tubulin, BODIPY® FL goat anti-mouse IgG, Texas Red®-X phalloidin, DAPI* eachF24630 FluoCells® prepared slide #3 *mouse kidney section with Alexa Fluor® 488 WGA, Alexa Fluor® 568 phalloidin, DAPI* eachF24631 FluoCells® prepared slide #4 *mouse intestine section with Alexa Fluor® 350 WGA, Alexa Fluor® 568 phalloidin, SYTOX® Green* eachF36925 FluoCells® prepared slide #6 *muntjac cells with mouse anti-OxPhos Complex V inhibitor protein, Alexa Fluor® 555 goat anti-mouse IgG, Alexa Fluor® 488
phalloidin, TO-PRO®-3*each
F36915 �uorescein *NIST-traceable standard* *nominal concentration 50 µM* *special packaging* 5 x 1 mLF36905 FocalCheck™ DoubleGreen Fluorescent Microspheres Kit, 6 µm *mounted on slides* 1 kitF36906 FocalCheck™ DoubleOrange Fluorescent Microspheres Kit, 6 µm *mounted on slides* 1 kitF36909 FocalCheck™ �uorescence microscope test slide #1 *for alignment, intensity, and calibration* eachF36913 FocalCheck™ �uorescence microscope test slide #2 *for spectral imaging systems* eachF36914 FocalCheck™ �uorescence microscope test slide #3 *5 colors, high and low intensities* eachF24633 FocalCheck™ Fluorescent Microspheres Kit, 6 µm *mounted on slides* 1 kitF24634 FocalCheck™ Fluorescent Microspheres Kit, 15 µm *mounted on slides* 1 kitF14807 FocalCheck™ microspheres, 6 µm, �uorescent dark-red ring stain/green throughout 0.5 mLF14808 FocalCheck™ microspheres, 6 µm, �uorescent green ring stain/blue throughout 0.5 mLF14806 FocalCheck™ microspheres, 6 µm, �uorescent green/orange/dark-red ring stains 0.5 mLF7234 FocalCheck™ microspheres, 15 µm, �uorescent blue/orange ring stains 0.5 mLF7239 FocalCheck™ microspheres, 15 µm, �uorescent dark-red ring stain/green throughout 0.5 mL
The Molecular Probes™ Handbook: A Guide to Fluorescent Probes and Labeling Technologies
IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.thermofisher.com/probes
Chapter 23 — Antifades and Other Tools for Fluorescence
961www.invitrogen.com/probes
The Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling TechnologiesIMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
Section 23.1 Fluorescence Microscopy Accessories and Reference Standards
Cat. No. Product QuantityF7237 FocalCheck™ microspheres, 15 µm, �uorescent green ring stain/blue throughout 0.5 mLF7238 FocalCheck™ microspheres, 15 µm, �uorescent green ring stain/dark red throughout 0.5 mLF7240 FocalCheck™ microspheres, 15 µm, �uorescent green/dark-red ring stains 0.5 mLF7235 FocalCheck™ microspheres, 15 µm, �uorescent green/orange/dark-red ring stains 0.5 mLF7236 FocalCheck™ microspheres, 15 µm, �uorescent orange ring stain/blue throughout 0.5 mLF14791 FocalCheck™ Thin-Ring Fluorescent Microspheres Kit, 1.0 µm *three suspensions* 1 kitH18200 HybriSlip™ hybridization cover, 22 mm x 22 mm *RNase free* *set of 500* 1 setH18201 HybriSlip™ hybridization cover, 40 mm x 22 mm *RNase free* *set of 500* 1 setH18202 HybriSlip™ hybridization cover, 60 mm x 22 mm *RNase free* *set of 500* 1 setH24720 HybriWell™ hybridization sealing system, 13 mm diameter chamber, 0.25 mm deep *set of 100* 1 setH24721 HybriWell™ hybridization sealing system, 20 mm diameter chamber, 0.15 mm deep *set of 100* 1 setH24723 HybriWell™ hybridization sealing system, 22 mm x 22 mm chamber, 0.15 mm deep *set of 100* 1 setH18210 HybriWell™ hybridization sealing system, 40 mm x 21 mm chamber, 0.15 mm deep *set of 100* 1 setH24722 HybriWell™ hybridization sealing system, 40 mm x 22 mm chamber, 0.25 mm deep *set of 100* 1 setI36933 Image-iT® FX signal enhancer 10 mLI7221 InSpeck™ Blue (350/440) Microscope Image Intensity Calibration Kit, 2.5 µm 1 kitI7225 InSpeck™ Deep Red (633/660) Microscope Image Intensity Calibration Kit, 2.5 µm 1 kitI7219 InSpeck™ Green (505/515) Microscope Image Intensity Calibration Kit, 2.5 µm 1 kitI14785 InSpeck™ Green (505/515) Microscope Image Intensity Calibration Kit, 6 µm 1 kitI7223 InSpeck™ Orange (540/560) Microscope Image Intensity Calibration Kit, 2.5 µm 1 kitI14786 InSpeck™ Orange (540/560) Microscope Image Intensity Calibration Kit, 6 µm 1 kitI7224 InSpeck™ Red (580/605) Microscope Image Intensity Calibration Kit, 2.5 µm 1 kitI14787 InSpeck™ Red (580/605) Microscope Image Intensity Calibration Kit, 6 µm 1 kitM7901 MultiSpeck™ Multispectral Fluorescence Microscopy Standards Kit *in suspension* 1 kitO24751 ONCYTE® MultiWells, one well, 13 mm diameter, with slide and matching gasket *set of 20* 1 setO24750 ONCYTE® MultiWells, 12 wells, 5 mm diameter, with slide and matching gasket *set of 20* 1 setO14804 O-rings for Atto�uor cell chamber, set of 10 eachP18174 Press-to-Seal silicone isolator, one well, 20 mm diameter, 0.5 mm deep *set of 50* 1 setP18175 Press-to-Seal silicone isolator, one well, 20 mm diameter, 1.0 mm deep *set of 50* 1 setP24743 Press-to-Seal silicone isolator with adhesive, eight wells, 9 mm diameter, 0.5 mm deep *set of 25* 1 setP24744 Press-to-Seal silicone isolator with adhesive, eight wells, 9 mm diameter, 1.0 mm deep *set of 25* 1 setP24740 Press-to-Seal silicone isolator with adhesive, one well, 20 mm diameter, 0.5 mm deep *set of 50* 1 setP24741 Press-to-Seal silicone isolator with adhesive, one well, 20 mm diameter, 1.0 mm deep *set of 50* 1 setP24742 Press-to-Seal silicone isolator with adhesive, 24 wells, 2.5 mm diameter, 2.0 mm deep *set of 25* 1 setP24745 Press-to-Seal silicone sheet with adhesive, 13 cm x 18 cm, 0.5 mm thick *set of 5* 1 setP18178 Press-to-Seal silicone sheet, 13 cm x 18 cm, 0.5 mm thick *set of 5* 1 setP18179 Press-to-Seal silicone sheet, 13 cm x 18 cm, 1.0 mm thick *set of 5* 1 setP7481 ProLong® Antifade Kit 1 kitP36930 ProLong® Gold antifade reagent 10 mLP36934 ProLong® Gold antifade reagent *special packaging* 5 x 2 mLP36931 ProLong® Gold antifade reagent with DAPI 10 mLP36935 ProLong® Gold antifade reagent with DAPI *special packaging* 5 x 2 mLP37002 ProPlate™ adhesive seal-strips *set of 50 seal-strips and one applicator* 1 setP37001 ProPlate™ multi-array slide module *set of 2* 1 setP37004 ProPlate™ multi-array system *includes four 16-well slide modules, one tray and cover, ten seal-strips and one applicator* 1 setP37003 ProPlate™ tray and cover *includes one tray and cover* 1 setP7220 PS-Speck™ Microscope Point Source Kit *blue, green, orange and deep red �uorescent beads* 1 kitQ10336 Qmount® Qdot® mounting media 3 x 2 mLR14782 Reference Dye Sampler Kit *�ve 1 mM solutions, 1 mL each* 1 kitS24732 Secure-Seal™ hybridization chamber gasket, eight chambers, 9 mm diameter, 0.8 mm deep *set of 20* 1 setS24733 Secure-Seal™ hybridization chamber gasket, eight chambers, 9 mm diameter, 1.3 mm deep *set of 20* 1 setS24730 Secure-Seal™ hybridization chamber gasket, one chamber, 20 mm diameter, 0.8 mm deep *set of 40* 1 setS24731 Secure-Seal™ hybridization chamber gasket, one chamber, 20 mm diameter, 1.3 mm deep *set of 40* 1 setS24734 Secure-Seal™ hybridization chamber gasket, one chamber, 22 mm x 22 mm, 0.8 mm deep *set of 50* 1 setS24737 Secure-Seal™ spacer, eight wells, 9 mm diameter, 0.12 mm deep *set of 100* 1 set
PRODUCT LIST 23.1 FLUORESCENCE MICROSCOPY ACCESSORIES AND REFERENCE STANDARDS —continued
The Molecular Probes™ Handbook: A Guide to Fluorescent Probes and Labeling Technologies
IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
thermofisher.com/probes
Chapter 23 — Antifades and Other Tools for Fluorescence
962www.invitrogen.com/probes
The Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling TechnologiesIMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
Section 23.2 Flow Cytometry Reference Standards
Figure 23.2.1 AlignFlow™ Plus (A7303) �ow cytometry alignment beads excited at 488 nm by an argon-ion laser and monitored in three emission channels. The broad �uorescence emission is detected in all three channels. Note the exceptionally small variation of �uores-cence intensity of the beads. Contributed by Carleton Stewart, Roswell Park Cancer Institute.
Num
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of p
artic
les
Green Orange Red
102 104103 102 104103 102 104103
Fluorescence intensity
23.2 Flow Cytometry Reference StandardsFlow cytometers are designed to perform quantitative measurements
on individual cells and other particles with speed, accuracy and preci-sion. As with all high-performance instrumentation, �ow cytometers must be calibrated frequently to ensure accuracy and reliability. �e stability, uniformity and reproducibility of our �uorescent microsphere products make them ideal reference standards for �ow cytometry. However, be-cause of the high variability in quantum yields of bound dyes and the heterogeneity of protein labeling, as well as problems with stoichiometry and accessibility in binding to targets, bead standards containing a known number of �uorophores per bead do not necessarily provide accurate in-formation about the number of ligands bound to a cell.1–4
AlignFlow™ and AlignFlow™ Plus Flow Cytometry Alignment Beads
In order to ensure accurate and reproducible quantitative results, �ow cytometers should be checked at least daily for proper perfor-mance. AlignFlow™ and AlignFlow™ Plus �ow cytometry alignment beads permit the calibration of a �ow cytometer’s laser(s), optics and stream �ow without wasting valuable and sensitive experimental material.5–7 �ese �uorescently stained polystyrene microspheres are highly uniform with respect to both size and �uorescence intensity, and they are designed to approximately replicate the size, emission wavelength and intensity of bio-logical samples. Because the dyes are contained inside the microsphere’s matrix instead of on the surface, AlignFlow™ beads have excellent pho-tochemical and physical stability, providing reliable reference signals for aligning, focusing and calibrating �ow cytometers. �e �uorescent dyes have been carefully selected for optimal excitation by laser sources com-monly used in �ow cytometry.
�e 2.5 µm AlignFlow™ �ow cytometry alignment beads are avail-able in four versions: for UV (350–370 nm) excitation (A7304), for 488 nm excitation (A7302), for 633 nm excitation (A7312) and for 630–660 nm excitation (A14835); the 6 µm AlignFlow™ Plus beads are available for the same four excitation-wavelength ranges: for UV (350–370 nm) excitation (A7305), for 488 nm excitation (A7303), for 633 nm excitation (A7313)
and for 630–660 nm excitation (A14836). �e UV light–excitable beads emit from 400 nm to 470 nm, the 488 nm light–excitable beads emit broadly from 515 nm to 660 nm (Figure 23.2.1), the 633 nm light–ex-citable beads emit from 645 nm to 680 nm, and the 630–660 nm light–excitable beads emit from 670 nm to 720 nm. �e AlignFlow™ and AlignFlow™ Plus �ow cytometry alignment beads are supplied as sus-pensions packaged in dropper vials for convenient dispensation.
LinearFlow™ Flow Cytometry Intensity Calibration Kits provide �ow cytometer operators with intensity references for generating cali-bration curves, establishing photomultiplier settings and evaluating sample brightness.8–10 Each kit contains �uorescent microspheres in which the degree of staining has been carefully controlled to provide precisely determined intensity levels when excited in a �ow cytometer (Figure 23.2.2). �e microspheres are supplied as suspensions packaged in dropper vials for convenient dispensation. �e LinearFlow™ Flow Cytometry Intensity Calibration Kits are available in two di�erent bead sizes (2.5 µm or 6 µm) and �ve di�erent �uorescent colors covering the spectral ranges commonly encountered in �ow cytometry:
• Blue (for UV excitation/430 nm emission). Available in 2.5 µm (L14812) and 6 µm (L14813) sizes; both kits contain microspheres stained at 100%, 20%, 4.0% and 0.8% relative �uorescence intensity levels.
• Green (for 488 nm excitation/515 emission). Available in 2.5 µm (L14821) and 6 µm (L14822) sizes; both kits contain microspheres stained at 100%, 10%, 2.0%, 0.4%, 0.1% and 0.02% relative �uo-rescence intensity levels. �e LinearFlow™ Green Flow Cytometry Low-Intensity Calibration Kits contain 2.5 µm (L14823) or 6 µm (L14824) diameter beads stained at 0.1%, 0.02%, 0.004% and 0.001% relative �uorescence intensity levels. �e �uorescence from the beads with the lowest intensity level is approximately half that of the auto�uorescence typically observed from unstained cells.
• Orange (for 488 nm excitation/575 nm emission). Available in 2.5 µm (L14814) and 6 µm (L14815) sizes; both kits contain micro-spheres stained at 100%, 10%, 2.0%, 0.4%, 0.1% and 0.02% relative �uorescence intensity levels.
• Carmine (for 488 nm excitation/620 nm emission). Available in 2.5 µm (L14816) and 6 µm (L14817) sizes; both kits contain micro-spheres stained at 100%, 10%, 2.0%, 0.4%, 0.1% and 0.02% relative �uorescence intensity levels.
• Deep Red (for 633 nm excitation/660 nm emission). Available in 2.5 µm (L14818) and 6 µm (L14819, Figure 23.2.2) sizes; both kits contain microspheres stained at 100%, 20%, 4.0%, 0.8%, 0.2% and 0.04% relative �uorescence intensity levels.
�e 365/430 nm �uorescence excitation/emission maxima of the microspheres in the LinearFlow™ Blue Kits provide a close spectral match to samples stained with DAPI, Hoechst 33258 or Hoechst 33342 nucleic acid stains. �ese kits are ideal for intensity calibration of �ow cytometers equipped with UV laser excitation. �e microspheres in the
The Molecular Probes™ Handbook: A Guide to Fluorescent Probes and Labeling Technologies
IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.thermofisher.com/probes
Chapter 23 — Antifades and Other Tools for Fluorescence
963www.invitrogen.com/probes
The Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling TechnologiesIMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
Section 23.2 Flow Cytometry Reference Standards
Figure 23.2.3 Normalized emission spectra of PeakFlow™ Green �ow cytometry reference beads (P14827, solid line) and �uorescein-labeled cells (dashed line). The narrow emission spectrum of PeakFlow™ beads is approximately centered on the broader emission spectrum of �uorescein.
Figure 23.2.2 Fluorescence intensity histogram of the six di�erent 6 µm polystyrene bead samples supplied in the LinearFlow™ Deep Red Flow Cytometry Intensity Calibration Kit (L14819). Fluorescence measurements were performed with a �ow cytometer using excitation at 633 nm. This histo-gram is a composite of two graphs; the same mixture of micro spheres was sampled and analyzed using two distinct PMT voltage settings in order to cover the full intensity range.
80
40
0
Red �uorescence
Num
ber
of p
artic
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120
101 102 103 104100
0.04%
0.2%
0.8%
4%
20%
100%
Table 23.7 Spectral characteristics of PeakFlow™ �ow cytometry reference beads.
Cat. No. Size (µm) Nominal Color Abs * (nm) Em * (nm) Emission Matches Cells Stained with:
* Approximate absorption and emission maxima for beads in suspension. † Suitable for excitation by the UV (351–364 nm) spectral line of argon-ion lasers. ‡ Suitable for excitation by the 488 nm spectral line of argon-ion lasers. § Suitable for excitation by the 633 nm spectral line of He-Ne lasers.
LinearFlow™ Green Kits are designed for calibrating the green (FL1) detection channel. Although the microspheres actually have an excitation maximum of ~505 nm, they are e�ectively excited by the 488 nm spectral line of the argon-ion laser. �eir emission maximum of ~515 nm closely matches that of samples labeled with �uorescein, Oregon Green® 488 or Alexa Fluor® 488 dyes or with SYTOX® Green nucleic acid stain. Microspheres in the LinearFlow™ Orange Kit are spec-trally similar to phycoerythrin and tetramethylrhodamine conjugates, making this kit useful for calibrating the orange (FL2) channel. Although these microspheres actually have an excitation maximum of ~570 nm, they are e�ectively excited by the 488 nm spectral line of the argon-ion laser. Microspheres in the LinearFlow™ Carmine Kit exhibit excitation and emission spectra similar to the spectra of the propidium iodide complex with DNA or the spectra of Texas Red® or Alexa Fluor® 594 dyes and are suitable for calibrating the red (FL3) channel. �e microspheres in the LinearFlow™ Carmine Kit have an excitation maximum of ~580 nm, but they can also be excited by the 488 nm spectral line of the argon-ion laser. �e microspheres in the LinearFlow™ Deep Red Kit have maximal emission at ~660 nm, closely matching that of Alexa Fluor® 647 dye, Cy®5 dye and allophycocyanin, and they are useful for calibrating �ow cytometers equipped with 633 nm He-Ne laser excitation. Although primarily intended for 633 nm excitation, the LinearFlow™ Deep Red microspheres can be adequately excited at 488 nm and will provide ac-curate relative intensity readings with this excitation.
PeakFlow™ Flow Cytometry Reference BeadsPeakFlow™ �ow cytometry reference beads are stained with �uorescent dyes that have been
carefully selected to produce emission peaks coincident with labeled cells used in typical �ow cytometry applications. �e emission pro�les for these standards are intentionally narrow in comparison to �uorescein-labeled cells (Figure 23.2.3). Consequently, PeakFlow™ beads serve as reference sources with emissions centered upon the expected �uorescence of the experimental sample. Because PeakFlow™ beads are highly uniform with respect to both size and �uores-cence intensity, and because they approximate the size, emission wavelength and intensity of many biological samples, they can be used to calibrate a �ow cytometer’s laser source, optics, stream �ow and cell sorting system without wasting valuable and sensitive experimental mate-rial. Furthermore, due to their narrow emission pro�les, PeakFlow™ beads of two di�erent �uo-rescent colors exhibit minimal spectral overlap, and little or no color compensation is needed when setting up for multicolor experiments.
As with all of our �ow cytometry standard microspheres, PeakFlow™ beads are stained in-ternally rather than on the surface. �e dyes are therefore insulated from environmental interac-tions that could cause variable �uorescence output, resulting in excellent signal stability. �ese �uorescent polystyrene microspheres are supplied as suspensions packaged in dropper vials for convenient dispensation, with a choice of seven �uorescent colors and, for most products, two di�erent sizes (Table 23.7).
The Molecular Probes™ Handbook: A Guide to Fluorescent Probes and Labeling Technologies
IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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Chapter 23 — Antifades and Other Tools for Fluorescence
964www.invitrogen.com/probes
The Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling TechnologiesIMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
Section 23.2 Flow Cytometry Reference Standards
Figure 23.2.4 Compensation using the AbC™ Anti-Mouse Bead Kit (A10344). (A) R-Phycoerythrin (R-PE)–conjugated mouse anti–human CD56 antibodies (MHCD56044) label the AbC™ capture beads for a positive signal, and negative beads provide a negative signal. (B) FITC-conjugated mouse anti–human CD3 antibodies (MHCD03014) label the AbC™ capture beads for a positive signal, and negative beads provide a negative signal. (C) Dual-parameter plot showing gated human lymphocytes labeled with R-PE–conjugated mouse anti–human CD56 and FITC-conjugated mouse anti–human CD3 antibodies using compensation settings obtained with the AbC™ Anti-Mouse Bead Kit.
R-PE �uorescence
Eve
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50
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103 104 105
A
FITC �uorescence
Eve
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102-102 00
50
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B
CD56 (R-PE �uorescence)
CD
3 (F
ITC
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resc
ence
)
102–1020 103 104 105
105
104
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0–102
C
AbC™ and ArC™ Bead Kits for Flow Cytometry CompensationAbC™ Anti-Mouse and AbC™ Anti-Rat/Hamster Bead Kits
�e AbC™ Anti-Mouse Bead Kit (A10344) provides a consistent, accurate and simple-to-use technique for the setting of �ow cytom-etry compensation when using �uorophore-conjugated mouse anti-bodies. �e kit contains two types of specially modi�ed polystyrene microspheres, the AbC™ capture beads, that bind all isotypes of mouse immunoglobulin, and the negative beads that have no antibody bind-ing capacity. A�er incubation with a �uorophore-conjugated mouse antibody, the two bead components provide distinct positive and nega-tive populations of beads that can be used to set compensation (Figure 23.2.4). Because of the consistent nature of bead scatter and high surface antibody–binding capacity, more consistent and accurate compensa-tion settings for any combination of �uorophore-labeled mouse anti-bodies can be achieved. �e AbC™ Anti-Rat/Hamster Bead Kit (A10389) provides parallel functionality for protocols using �uorophore-labeled rat or hamster antibodies. �e AbC™ capture beads and negative beads have a diameter of approximately 6 µm (actual size for each lot is listed on the component vial). �e bead suspensions are supplied in dropper vials for convenient sample application.
vides a consistent, accurate and simple-to-use technique for the set-ting of �ow cytometry compensation when using any of the LIVE/DEAD® �xable dead cell stains. �e LIVE/DEAD® Fixable Dead Cell Stain Kits (Section 15.3) use an amine-reactive dye labeling method to evaluate the viability of mammalian cells by �ow cytometry.11 �e ArC™ Amine-Reactive Compensation Bead Kit includes two types of specially modi�ed polystyrene microspheres to allow easy compen-sation of the LIVE/DEAD® �xable stains: the ArC™ reactive beads (Component A), which bind any of the amine-reactive dyes, and the ArC™ negative beads (Component B), which have no reactivity. A�er incubation with any amine-reactive dye, the two kit components pro-vide distinct positive and negative populations of beads that can be used to set compensation.
�uorescent particle-size calibration standards for use in forward light scattering measurements of cell size by �ow cyto metry.12,13 �is kit con-tains suspensions of six di�erent non�uorescent microspheres pack-aged in convenient dropper vials. �e individual standards contain highly uniform polystyrene microspheres with nominal diameters of 1.0 µm, 2.0 µm, 4.0 µm, 6 µm, 10 µm and 15 µm.
CountBright™ Absolute Counting BeadsFlow cytometry provides a rapid method for quantitating cell charac-
teristics; however, most �ow cytometers cannot directly provide the cell concentration or absolute count of cells in a sample. Absolute cell counts have been widely used in quantitating cell populations and disease pro-gression14–18 and are generally obtained either by combining a separate cell concentration determination from a hematology analyzer with �ow cytometry population data (multiple-platform testing) or by adding an internal microsphere counting standard to the �ow cytometry sample (single-platform testing). �e single-platform method is preferred as it is technically less complicated and more accurate than multiple-platform testing.19 To facilitate this single-platform method, we o�er CountBright™ absolute counting beads (C36950), a calibrated suspension of microspheres that are brightly �uorescent across a wide range of excitation and emis-sion wavelengths and contain a known concentration of microspheres. For absolute counts, a speci�c volume of the CountBright™ microsphere suspension is added to a speci�c volume of sample, such that the ratio of sample volume to microsphere volume is known.20 �e volume of sample analyzed can be calculated from the number of microsphere events and then used with cell events to determine cell concentration. In general, at least 1000 bead events should be acquired to assure a statistically signi�-cant determination of sample volume. Su�cient reagents are provided for 100 �ow cytometry assays, each using 50 µL of counting beads per test.
CountBright™ absolute counting beads are broadly �uorescent and can be used with either a �uorescence or scatter threshold. Fluorescence can be excited by wavelengths from UV to 635 nm; �uorescence
The Molecular Probes™ Handbook: A Guide to Fluorescent Probes and Labeling Technologies
IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.thermofisher.com/probes
Chapter 23 — Antifades and Other Tools for Fluorescence
965www.invitrogen.com/probes
The Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling TechnologiesIMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
emission can be read between 385 nm and 800 nm. �e �uorescence intensity of the micro-spheres has been adjusted to be about 5–50 times brighter than the anticipated intensities of typically stained cells. When using a scatter threshold, the microsphere signal should be above the threshold. �e microspheres can be gated by a single parameter, but a combination of pa-rameters can be used to resolve microspheres from cells and other events.
CountBright™ absolute counting beads can be used with any sample type, including no-wash/lysed whole blood. �e microspheres in the reagents are approximately 7 µm in diameter and have sedimentation properties similar to lymphocytes. �e accuracy of cell counts based on CountBright™ absolute counting beads depends on sample handling and the precise delivery of the volume of beads. �e CountBright™ absolute counting beads must be mixed well to as-sure a uniform suspension of microspheres. A�er vortexing for 30 seconds, the microsphere suspension can be pipetted by standard techniques; however, more viscous solutions such as blood require reverse pipetting for accurate volume delivery. Cell suspensions may be diluted but should be assayed without wash steps. Other sample preparation steps that can lead to cell or microsphere loss should also be avoided. For antibody protocols, CountBright™ absolute count-ing beads should be used with reagents titered for no-wash staining.
P24671 PeakFlow™ Ultra Red �ow cytometry reference beads, 6 µm *695 nm emission* 3 mL
The Molecular Probes™ Handbook: A Guide to Fluorescent Probes and Labeling Technologies
IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
