sted microscopy - lumc€¦ · recommended mounting media are mowiol, ... labeling procedure and...

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


2 color STED images of neurons

courtesy Dr. W. Zuschratter, IfN, Magdeburg


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


Dye Vendor Laser lines 2 color

Alexa 532 Invitrogen 531/750

Chromeo 494 Active Motif 531/750 recommended

Mega 520 Sigma-Aldrich 531/775

19

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

25

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

32

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

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

ing

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

sted microscopy - lumc€¦ · recommended mounting media are mowiol, ... labeling procedure and...

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