STED Microscopy Sample preparation Dr. Nathalie Garin Components of a microscope

Objective lens

Cover slip

Immersion

Sample

The last 3 optical elements are usually added by the user ! 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

www.leica-microsystems.com/products/total- histology/consumables/catalog/catalog-us/

http://www.hecht-assistent.de/index-us.html http://www.menzel.de/ APPLICATIONS Protein localization and dual color imaging

STED Microscopy – sample preparation 6 Nuclear proteins labelled with Alexa 488 D. Gruenwald; J. Hopkins, New York

direct immunofluorescence STED CW: Spectral Conditions

Example: OregonGreen 488

No Excitation at

lSTED

Dyes with excitation from 458 nm to 514 nm Depletion at 592 nm STED: Spectral Conditions

Excitation STED

Detection Band

No Excitation at

lSTED

Dyes with excitation at 532 nm or 640 nm Tunable 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

STED Microscopy – Principles and Applications 10 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

STED Microscopy – Principles and Applications 11 2 color STED images of neurons

courtesy Dr. W. Zuschratter, IfN, Magdeburg

STED Microscopy – Principles and Applications 13 2 color STED:

BD Horizon V500 Oregon Green 488

Channel 1 Channel 2 Multicolor Imaging – TCS STED CW

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

Excitation @ 458 nm Multicolor Imaging

Example: Pacific Orange, Alexa488 STED raw data STED color separation

Sample courtesy: MPI Goettingen, NBDX & Alexa488 (postprocessing: spectral separation) 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

STED Microscopy – Principles and Applications 18 Some large Stoke’s shift dyes for pulsed STED

Dye Vendor Laser lines 2 color Alexa 532 Invitrogen 531/750 Chromeo 494 Active Motif 531/750 recommended Mega 520 Sigma-Aldrich 531/775

STED Microscopy – Principles and Applications 19 Configuration examples

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 488 500 – 580 dye) NBDX 458 500 - 580 Alexa488 (or any green 488 500 – 580 dye)

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 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 Gold and 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

Y

primary Ab dye secondary Ab Y 7 – 10nm

Fab fragments: 3.5 – 5.5nm

Y

Y Y Y Y Y

direct IF indirect IF

25 Thickness of sample Refractive Index Match & Mismatch

Oil Oil

Coverslip Coverslip

Oil Water Refractive indMatchex mismatch results in spherical aberrationMismatch

 Matching RIs crucial for confocal microscopy

…and even more so for nanoscopy!

A. Egner and S. W. Hell Doughnut visualized with 80 nm gold beads

Reflection mode, 488 nm & 592 nm, zoom 64 center section yz

xz

Oil Objective PL APO 63x1.3 Sample embedded in Glycerol (80/20%) Thick specimen (100µm): muscle fibers Mounting & Immersion

Refractive indices Immersion / Mounting Media Good results were obtained with:

. Water: 1.33447 . Mowiol . Glycerol 23°C: 1.45100 . Glycerol . Vectashield (100%) 1.46 . ProLongGold . Mowiol 1.46 . 86% glycerol + 4% NPG (N-propyl-gallate) . TDE/Water: 1.33 – 1.52 . 86% glycerol + 2.5% DABCO

. Immersion Oil: 1.518 at pH7.4 . Glas 1.52

Penetration depth To avoid:  10 – 25 µm . Vectashield (Vectorlabs) . Slowfade (invitrogen) . Para-phenylenediamine (PPD) 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. APPLICATIONS STED live-imaging

32 CW STED imaging with fluorescent proteins

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 Potential dyes for STED CW

proteins laser line Cerulean 458 CyPet 458 eCFP 458 Emerald 488 mBanana 514 Topaz 514 YPet 514 ZsYellow 514 STED Microscopy – Principles and Applications 35 More live imaging: (Abp1-eGFP in yeast cells)

Confocal

Courtesy of Marko Kaksonen EMBL, Heidelberg 1 Hz

STED Live Cell STED with Fluorogen Activating Proteins (FAPs) Specific labeling of genetically encoded marker without background problems

Fluorogen

Protein-FAP DNA

live cell

Szent et al. Nature Biotechnol 2008; Fitzpatrick et al. Bioconjugate Chem 2009 Live Cell STED with Fluorgen Activating Proteins (FAPs)

Confocal STED

Fluorogen Activating Protein (FAP) labeled ADRB2 TCS STED in living NIH 3T3 cells Images: James Fitzpatrick/Jochen Sieber STED

Szent et al. Nature Biotechnol 2008; Fitzpatrick et al. Bioconjugate Chem 2009 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. Influence of scan speed

Alexa 488 in  - Tubulin

4

3

2

1 Resonant bleachingexponents[images] Galvoscanner

0 0 200 400 600 800 1000 1200 1400 8000 scan rate [Hz] 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

confocal STED

Thank you for your attention!