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Principles of Fluorescence Spectroscopy

Chemistry Department

XMU

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Chapter Three

Fluorophores

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Fluorophores

3.1 Organic Fluorescent Molecular

3.2 Metal-ligand compounds3.3 Probes

3.4 Reference

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Probes

3.3.1 Protein probes 3.3.2 Membrane probes3.3.3 DNA probes3.3.4 pH probes3.3.5 Ion probes3.3.6 Potentialmetric prob

es

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Reference

Molecular probes , Handbook of Fluorescent Probes and Research Chemicals

---------- Molecular Probes

Molecular probes , Handbook of Fluorescent Probes and Research Chemicals

---------- Molecular Probes

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Molecular probes

www.probes.com

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homepage

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Protein fluorescence

H2N CH C

CH2

OH

O

H2N CH C

CH2

OH

O

H2N CH C

CH2

OH

O

HN

H2N CH C

CH2

OH

O

HN

H2N CH C

CH2

OH

O

OH

H2N CH C

CH2

OH

O

OH

TRP PHE TYR

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Protein-labeling Probes

DNS – Cl dansyl chloride 丹磺酰氯

Absorption and fluorescence emission spectra of dansyl cadaverine （ 1 ， 5 – 戊二胺） in methanol.

= 10 -15 ns

= 0.1 - 0.3

340 nm540 nm

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Protein-labeling ProbesFICT fluorescein 5-isothiocyanate, 异硫氰酸荧光素

Absorption and fluorescence emission spectra of goat anti-mouse IgG labeled with fluorescein-5-isothio-cyanate in pH 8.0 buffer.

= 4.5 ns

= 0.3 – 0.85

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photo

Two proteobacterial symbionts localized with phylotype-specific 16S rRNA–directed oligonucleotide probes labeled with either fluorescein-5-isothiocyanate or Texas Red sulfonyl chloride. The filamentous bacteria are attached to a hair-like structure secreted from a pore on the dorsal surface of the deep-sea hydrothermal vent polychaete Alvinella pompejana.

用异硫氰酸荧光素或 Texas 红标记的位于种系型特异性 16s- 核蛋白体核糖核酸的两种蛋白细菌共生体 -- 定向寡聚核苷酸探针

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TRITC Tetramethylrhodamine 5- (and 6) isothiocyanate

Effect of protein conjugation on tetramethylrhodamine‘s absorption spectrum. The absorption spectrum of tetramethylrhodamine conjugated to goat anti–mouse IgG (TMR-GAM) shows an additional peak at about 520 nm when compared to the spectrum of the same concentration of free dye (TMR). Partial unfolding of the protein in the presence of 4.8 M guanidine hydrochloride ( 盐酸胍） (TMR-GAM + GuHCl) results in a spectrum more similar to that of the free dye.

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RG

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Oregon Green 514 carboxylic acid

Absorption and fluorescence emission spectra of Oregon Green 514 goat anti-mouse IgG in pH 8.0 buffer.

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Staining

immunofluorescent staining by Oregon Green 514 goat anti–mouse IgG immunofluorescent staining by Oregon Green 514 goat anti–mouse IgG

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6-acryloyl-2-dimethylaminonaphthalene (acrylodan)6 - 烯丙酰 - 2 - 二甲胺基萘（ highly sensitive to polarity)

Normalized emission spectra of prodan in 1) cyclohexane, 2) dimethylformamide, 3) ethanol, and 4) water.

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Texas Red

Absorption and fluorescence emission spectra of Texas Red-X goat anti-mouse IgG in pH 7.2 buffer Absorption and fluorescence emission spectra of Texas Red-X goat anti-mouse IgG in pH 7.2 buffer

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Comparison of protein labeling

Comparison of relative fluorescence as a function of the number of fluorophores attached per protein for goat anti–mouse IgG con

jugates prepared using Oregon Green 514 succinimidyl ester, Oregon Green 488 succinimidyl ester , fluorescein-5-EX succinimidyl ester , and fluorescein isothio

cyanate (FITC, ). Conjugate fluorescence is determined by measuring the fluorescence quantum

yield of the conjugated dye relative to that of the free dye and mul

tiplying by the number of fluorophores per protein.

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Reaction

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Noncovalent protein-labeling probe

0

用于表征蛋白质的微环境

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Membrane probes N CH3

CH3

H3CCH2

CH2

OP OOOCH2

CHH2CO O

C OCO

+

-

卵磷脂

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Ion channel

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Ion channel

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CH

CH

CH

CH

CH

CH

DPH

2,6-ANS

Partitioning probe

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Membrane probes

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Membrane probes

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Membrane probes

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Membrane probes

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Partition

0 10 20 30 40 50 600

2

4

6

41 oC

RuDPPG vesicle containingcholesterolEX=463nm, EM=650nm,

0% 20% 33% 50%

Em

issi

on I

nte

nsi

ty

T oC

0 10 20 30 40 50 600

2

4

6

41 oC

RuDPPG vesicle containingcholesterolEX=463nm, EM=650nm,

0% 20% 33% 50%

Em

issi

on I

nte

nsi

ty

T oC

Emission intensities of DPPG vesicles labeled with of [Ru(bpy)2(dppz)]2+ at various cholesterol concentrations, measured as a f

unction of increasing temperature towards the lipid phase transiti

on temperature

N

N

N

N

Ru

N

N

N

N

2+

[Ru(bpy)2(dppz)]2+

N

N

N

N

Ru

N

N

N

N

2+

[Ru(bpy)2(dppz)]2+

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Absorption and fluorescence emission spectra of ethidium bromde bound to DNA

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DNA stainingAcridine orange

Absorption and fluorescence emission spectra of acridine orange bound to DNA

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DNA stainingHoechst 33258 (bis-benzimide)

Absorption and fluorescence emission spectra of Hoechst 33258 bound to DNA.

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Normal and camptothecin-treated bovine pulmonary artery endothelial cells (BPAEC) were stained with C4-BODIPY 500/510 C9 (B-3824 and Hoechst 33258 (H-1398, H-3569). BODIPY and Hoechst fluorescence were recorded using longpass filters appropriate for fluorescein and DAPI, respectively. An overlay of the images indicates that a red shift in BODIPY fluorescence is significant in normal cells (left panel) compared to that in apoptotic cells (right panel, as indicated by the fragmented nuclear staining of the Hoechst dye). Image contributed by Wan-Nan Uang, Molecular Probes, Inc.

正常细胞 编程性死亡细胞

Hoechst 33258 staining

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DNA staining Viable Madin-Darby canine kidney (MDCK) cells sequentially stained with BODIPY FL C5-ceramide (D-3521), LysoTracker Red DND-99 (L-7528) and Hoechst 33258 (H-1398, H-3569). Green-fluorescent BODIPY FL C5-ceramide

localized to the Golgi apparatus, red-fluorescent LysoTracker Re

d stain accumulated in the lysosomes and blue-fluorescent Hoechst 33258 dye stained the nuclei. The multiple-exposure image was acquired with bandpass filters appropriate for fluorescein,

Texas Red dye and DAPI. Image contributed by Chii-Shiarng Che

n, Molecular Probes, Inc.

Hoechst 33258 stain N 胞核

BODIPY FL C5 stain G 高尔基体

Lysotracker stain L 溶酶体

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DNA and RNA determination

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DNA and RNA determination

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pH indicator5-sulfofluorescein diacetate, sodium salt (SFDA)

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Ionization equilibria of fluorescein

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Ca2+ indicator

Absorption and fluorescence emission spectra of Ca2+-saturated fluo-3 (F-1240) in pH 7.2 buffer

fluo-3, pentaammonium salt

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fluo-3

Ca2+-dependent fluorescence emission spectra of fluo-3 (F-1240, F-3715). The spectrum for the Ca2+-free solution is indistinguishable from the baseline.

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Mg2+ indicator

Absorption and fluorescence emission spectra of Magnesium Green (M-3733) in pH 7.05 buffer containing 35 mM free Mg2+.

Magnesium Green , pentapotassium salt

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Magnesium Green

Mg2+-dependent fluorescence emission spectra of Magnesium Green (M-3733).

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Zn2+ indicator

Fluorescence excitation spectra of APTRA-BTC (A-6895) in solutions containing zero to 25 µM Zn2+.

APTRA-BTC, tripotassium salt

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Cd2+ indicator

Fluorescence emission spectra of a mixture of 1 µM BTC-5N (B-6845) and 1 µM TCPP in solutions containing zero to 1.0 µM Cd2+.

BTC-5N, tetrapotassium salt

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Na+ indicatorSBFI, tetraammonium salt

Fluorescence excitation (detected at 505 nm) and emission (excited at 340 nm) spectra of SBFI (S-1262) in pH 7.0 buffer containing 135 mM (A) or zero (B) Na+.

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SBFI

The excitation spectral response of SBFI (S-1262) to Na+: A) in K+-free solution and B) in solutions containing K+ with the combined

Na+ and K+ concentration equal to 135 mM. The scale on the vertical axis is the same for both p

anels.

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K+ indicator

PBFI, tetraammonium salt

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PBFI

The excitation spectral response of PB

FI (P-1265) to K+ : A) in Na+-free solution and B) in solutions containing Na+ with the combined

K+ and Na+ concentration equal to 135 mM. The scale on the vertical axis is the same for both p

anels.

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SBFLPBFL

Comparison

K+ Na+

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Inorganic phosphate indicatorEnzChek Phosphate Assay Kit

Quantitative analysis of inorganic phosphate using the EnzChek Phosphate Assay Kit (E-6646).

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Cl- indicatorN-(ethoxycarbonylmethyl)-6- methoxyquinolinium bromide (MQAE)

Fluorescence emission spectra of MQAE (E-3101) in increasing concentrations of Cl–.

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Potentiometric probes 　电位探针

The plasma membrane （质膜） of a cell typically has a transmembrane potential ( 跨膜电位） of approximately –70 mV (negative inside) as a consequence of K+, Na+ and Cl– concentration gradients that are maintained by active transport processes. Potentiometric probes offer an indirect method of detecting the translocation of these ions, whereas the fluorescent ion indicators can be used to directly measure changes in specific ion concentrations.

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Potentiometric probesdi-4-ANEPPS

Absorption and fluorescence emission spectra of Di-8-ANEPPS (D-3167) bound to phospholipid bilayer membra

the ANEP dyes are essentially nonfluorescent in aqueous solutions and exhibit spectral properties that are strongly dependent on their environment.

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Potentiometric probes

Cationic

Zwitterionic

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Potentiometric probes

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Potentiometric probes

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Potentiometric probes

Fluorescence emission spectra of 0.15 µM and 0.3 µM JC-1 (T-3168) in 50 mM Tris-HCl, pH 8.2, containing 1% DMSO, showing the concentration-dependent increase of long-wavelength J-aggregate emission at 590 nm.

,5',6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1; CBIC2(3))

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Potential-dependent staining Potential-dependent staining of mitochondria in CC

L64 fibroblasts by JC-1 (T-3168). The mitochondria were visualized by epifluorescence microscopy

using a 520 nm longpass optical filter. Regions of high mitochondrial polarization are indicated by red fluorescence due to J-aggregate formation by the

concentrated dye. Depolarized regions are indicated by the green fluorescence of the JC-1 monomers.

线粒体电位成像

超极化区

去极化区

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cell images NIH 3T3 fibroblasts

（成纤维细胞） stained with JC-1 (T-3168) showing the progressive loss of red J-aggregate fluorescence an

d cytoplasmic( 胞质的） diffusion of green monomer fluorescence following exposure to hydrogen peroxide. Images show the

same field of cells viewed before H2O2 treatment, and 5, 10 and 2

0 minutes after treatment.