Post on 04-Jun-2018
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STED MicroscopySample preparation
Dr. Nathalie Garin
Objective lens
Cover slip
Immersion
Sample
The last 3 optical elements are usually added by the user !
Components of a microscope
Coverslip thickness
Objective lenses designed for use with 170 µm coverslips(i.e. considered in the optical calculation of the lens)
Some lenses are equipped with an adjustable collar correcting for different thickness
Mismatch causes
o spherical and chromatic aberration
o spread of focus spot
o loss in resolution
o intensity decreases
o distortion of structures inside deeper layers of the sample
15 µm cover slip deviation approx. deteriorates ½ of resolution & intensity
Coverslip Thickness
# Thickness [mm]
0 0,08 – 0,12
1 0,13 – 0,16
1,5 0,16 – 0,19
2 0,19 – 0,23
Source: Hecht-Assistent / Menzel
Coverslip Sources
http://www.hecht-assistent.de/index-us.htmlhttp://www.menzel.de/
www.leica-microsystems.com/products/total-histology/consumables/catalog/catalog-us/
APPLICATIONSProtein localization and dual color imaging
6STED Microscopy – sample preparation
Nuclear proteins labelled with Alexa 488D. Gruenwald; J. Hopkins, New York
direct immunofluorescence
Example: OregonGreen 488
STED CW: Spectral Conditions
Dyes with excitation from 458 nm to 514 nmDepletion at 592 nm
No Excitation at lSTED
STED
Detection
Band
Excitation
No Excitation at lSTED
STED: Spectral Conditions
Dyes with excitation at 532 nm or 640 nmTunable depletion from 660 to 780 nm
Some working green dyes for CW STED
Dye Vendor Laser line 2 color
Alexa 488 Invitrogen 488 or 514
Atto 488 ATTO-TEC 488 or 514
Chromeo 488 Active Motif 488 or 514
Chromeo 505 Active Motif 488 or 514 recommended
DyLight 488 Pierce Technology 488 or 514
FITC Invitrogen 488 or 514
FluoProbes 488 Interchim 488 or 514
Oregon Green 488 Invitrogen 488 or 514 recommended
Oregon Green 514 Invitrogen 514
10STED Microscopy – Principles and Applications
Some working dyes for pulsed STED
Dye Vendor Laser lines 2 color
Abberior STAR 635 Abberior 640/750 recommended
Alexa 647 Invitrogen 640/750
Atto 647N ATTO-TEC 640/750 recommended
Atto 655 ATTO-TEC 640/750
Atto 665 ATTO-TEC 640/775
11STED Microscopy – Principles and Applications
2 color STED images of neurons
courtesy Dr. W. Zuschratter, IfN, Magdeburg
13STED Microscopy – Principles and Applications
2 color STED:
Channel 1 Channel 2
BD Horizon V500Oregon Green 488
Fluorophore Exc / nm Em / nm Source Coupling
NBD-X 467 538 Invitrogen Succinimidyl
Pacific Orange 400 556 Invitrogen IgG
Horizon V500 415 500 Becton & Dickinson Streptavidin
Multicolor Imaging – TCS STED CW
Excitation @ 458 nm
Multicolor Imaging
Example: Pacific Orange, Alexa488
Sample courtesy: MPI Goettingen, NBDX & Alexa488 (postprocessing: spectral separation)
STED color separationSTED raw data
Some large Stoke’s shift dyes for CW STED
Dye Vendor Laser line remarks
Abberior STAR 440SX Abberior 458 recommended
Atto 425 Rockland 458
BD Horizon V500 Becton Dickinson 458 recommended
Pacific Orange Invitrogen 458
18STED Microscopy – Principles and Applications
Dye Vendor Laser lines 2 color
Alexa 532 Invitrogen 531/750
Chromeo 494 Active Motif 531/750 recommended
Mega 520 Sigma-Aldrich 531/775
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Some large Stoke’s shift dyes for pulsed STED
STED Microscopy – Principles and Applications
Configuration examples
TCS STED (depletion at 765nm)
Dye1 Dye2
Name Excitation Emission Name Excitation Emission
Chromeo494 531 505 - 605 ATTO647N 635 665 – 705
Mega520 531 665 - 705 ATTO 655 635 665 – 705
TCS STED CW
Dye1 Dye2
Name Excitation Emission Name Excitation Emission
BD Horizon V500 458 465 - 515 OregonGreen488 or 514 514 520 – 580
Pacific orange 458 535 - 580 Alexa488 (or any green
dye)
488 500 – 580
NBDX 458 500 - 580 Alexa488 (or any green
dye)
488 500 – 580
Tips and tricks I
Carry out your stainings similar to regular fluorescent imaging. Good samples in regular fluorescence imaging are also suitable for STED imaging.
Check the mounting medium for compatibility with the fluorescent dyes (especially concerning the large Stoke’s shift dyes) and doesn’t show high levels of auto-fluorescence.
Recommended mounting media are mowiol, 86% glycerol, Prolong Goldand TDE, with or without anti-fade agents.
Vectashield isn’t compatible with large Stoke’s shift dyes.
Use sequential acquisition for 2 color STED imaging.
Labeling procedure and density may influence image quality.
Immunolabeling, classical steps
Fixation (usually 4% PAF)
Washing
Permeabilisation (Triton, saponin)
Washing
Blocking (BSA or NGS)
Incubation of the primary antibody (recommended : overnight at 4°C)
Immunolabeling, classical steps
Fixation (usually 4% PAF)
Washing
Permeabilisation (Triton, saponin)
Washing
Blocking (BSA or NGS)
Incubation of the primary antibody (recommended : overnight at 4°C)
Washing
Incubation with secondary antibody 1 hour at RT
Washing
Postfixation in 4% PFA
washing
Consider the size of the label
YYY YYY
direct IF indirect IF
Y
secondary Ab
Y
primary Ab dye
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7 – 10nmFab fragments: 3.5 – 5.5nm
Thickness of sample
Refractive index mismatch results in spherical aberration
Oil
Coverslip
WaterMismatchMatch
Oil
Coverslip
Oil
Matching RIs crucial for confocal microscopy
…and even more so for nanoscopy!
Refractive Index Match & Mismatch
A. Egner and S. W. Hell
Doughnut visualized with 80 nm gold beads
Reflection mode, 488 nm & 592 nm, zoom 64
Oil Objective PL APO 63x1.3
yz
xz
center section
Sample embedded in Glycerol (80/20%)Thick specimen (100µm): muscle fibers
Mounting & Immersion
Refractive indices Immersion / Mounting Media
Water: 1.33447
Glycerol 23°C: 1.45100
Vectashield (100%) 1.46
Mowiol 1.46
TDE/Water: 1.33 – 1.52
Immersion Oil: 1.518
Glas 1.52
To avoid:
Vectashield (Vectorlabs)
Slowfade (invitrogen)
Para-phenylenediamine (PPD)
Good results were obtained with:
Mowiol
Glycerol
ProLongGold
86% glycerol + 4% NPG (N-propyl-gallate)
86% glycerol + 2.5% DABCO
at pH7.4
Penetration depth
10 – 25 µm
Protocoles
Mowiol embedding media (pH7.4):
•take 6g Glycerol (analytical grade)
•add 2.4 g Mowiol 4-88 (Calbiochem # 475904)
•add 6 ml Aqua dest.
•add 12 ml 0.2 M TRIS Buffer pH74.
•add 2.5% DABCO (=Anti -Bleaching-Reagent 1,4- Diazabicyclo-82.2.29-octan; Fluka #33480)
•stir for 4 hours (magnetic stirrer)
•let rest suspension for 2 hours
•incubate for 10 min at 50°C (water bath)
•centrifuge at 5000 g for 15 min
•take the supernatant and freeze it in aliquots at -20°C
Glycerol – PBS- antifading (pH7.4):
•86%glycerol+4% n-propyl-gallate(NPG)
Thiodiethanol (pH7.4)
•TDE, Sigma, #88559) has been used with excellent results (Staudt T, Lang MC, Medda R, Engelhardt J, Hell SW (2007).
“2,2'-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy.” Microsc Res Tech. 70(1):1-
9.). The TDE concentration must be gradually enhanced to obtain a final refractive index of 1.514, which is reached using a
TDE concentration of 97%. Sequential steps in TDE 50%, 70% (15 minutes at each step), then in 97% + antifade as final
mounting media must be undertaken
•If using TDE, the coverslip must be sealed using invisible nail
Mounting media must have antifade freshly mixed in it.
•N-propyl gallate (NPG, 4%) while not very soluble, is non-toxic and can be used on live cells.
•DABCO (2.5%) is a well-known antifade.
APPLICATIONSSTED live-imaging
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proteins vendor laser line remarks
eGFP Tsien et al 488
eYFP Invitrogen 514 recommended
Citrin Tsien et al 514 recommended
Venus Miyawaki et al 514
CW STED imaging with fluorescent proteins
Potential dyes for STED CW
proteins laser line
Cerulean 458
CyPet 458
eCFP 458
Emerald 488
mBanana 514
Topaz 514
YPet 514
ZsYellow 514
35STED Microscopy – Principles and Applications
More live imaging: (Abp1-eGFP in yeast cells)
Confocal
STED
Courtesy of Marko KaksonenEMBL, Heidelberg1 Hz
DNAProtein-FAP
live cell
Fluorogen
Specific labeling of genetically encoded marker without background problems
Live Cell STED with Fluorogen Activating Proteins (FAPs)
Szent et al. Nature Biotechnol 2008; Fitzpatrick et al. Bioconjugate Chem 2009
Confocal
STED
STED
Fluorogen Activating Protein (FAP) labeled ADRB2 in living NIH 3T3 cells Images: James Fitzpatrick/Jochen Sieber
TCS STED
Szent et al. Nature Biotechnol 2008; Fitzpatrick et al. Bioconjugate Chem 2009
Live Cell STED with Fluorgen Activating Proteins (FAPs)
Tips and tricks II
Bleaching rate of fluorescent proteins and dyes are directly correlated with the pixel dwell time. Fast scan speed with higher number of averaging are more gentle to the fluorophore and thus may produce a higher number of frames before bleaching.
0 200 400 600 800 1000 1200 1400 8000
0
1
2
3
4
ble
ach
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exp
on
en
ts [im
ag
es]
scan rate [Hz]
Galvoscanner
Re
so
na
nt
Alexa 488 in - Tubulin
Influence of scan speed
Tips and tricks II
Bleaching rate of fluorescent proteins and dyes are directly correlated with the pixel dwell time. Fast scan speed with higher number of averaging are more gentle to the fluorophore and thus may produce a higher number of frames before bleaching.
Signal to noise ratio (SNR) is correlated to the pixel size. Bigger pixel give “brighter” images. Oversampling of at least 2 is still recommended
Slightly reduce STED depletion power for better SNR. Compromising the resolution with lower STED intensities may increase SNR.
Change the pinhole size. Increasing the pinhole size may increase the SNR in samples that aren’t too densely labeled.
Consider the use of lower excitation power and accumulation with low noise detectors (HyD and APD at low gains).
Tips and tricks II
Mounting medium:
o Antifadings: vitamin C, A, ….
o No riboflavin or phenol red
Living up to Life
Thank you
for your attention!
confocal STED