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STimulated Emission Depletion (STED)
Living up to Life
Sample Preparation for STED CW 1 and 2-color
ACTIN
MICROTUBULES
Confocal
Confocal
STED
STED
Myriam Gastard, PhD, Application and Tech Support, Exton, PA, 19341.
www.leica-microsystems.com
03/14/2011
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Living up to Life
Resolution Enhancement by STED
confocal
FWHM 65 nm
FWHM < 65nm
FWHM ~260nm
FWHM ~260nm
STED
STED
STED
STED
STED
STED
STED
STED
STED
STED
STED
STED
STED
STED
STED
STED
A threefold improved resolution can make 9 spots out of 1 !
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3/16/2011
Resolution: Reality check by STED
Living up to Life
STED CW resolution (in red) vs confocal resolution (in green): AKTpS473 (Rockland Immuno, Inc, PA)
REMEMBER
Do NOT expect to have a resolution below 100 nm if the protein(s) or structure of interest
are bigger than 100 nm !
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1-color STED CW
Living up to Life
STED: Spectral Conditions
Excitation
STED
Detection Band
Requirements for STED CW:
• Dye excitable with Argon laser
• Still significant emission at λSTEDc
• No Excitation at λSTEDc
350
400
450
500
550
600
650
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2-color STED CW
Living up to Life
What is the challenge?
458
488
592 depletion laser
Shift stock
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1 and 2-color STED CW
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Secondary Antibodies
1-color STED CW
• DyLight 488: gave the highest resolution, bright.
• Oregon green 488: very bright, be careful with precipitates.
• Chromeo 505: Bright and allows better separation.
• Alexa 488 , Alexa 514: not as bright or stable than the previous ones.
2 -color STED CW
in combination with one dye from the single color STED CW list
• Atto 425, antibody or Streptavidin conjugated.
• BD Horizon V500, Streptavidin conjugated.
• Pacific Orange, antibody conjugated.
• NBD-X (Succinimidyl).
Do Not Use: DAPI, QDOTs, or other fluorescent dyes which are not excitable
with the Argon laser.
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Living up to Life
2-color STED CW
Recommended pair for 2-color STED CW
Channel 1
Channel 2
Comment
V500 (Strep)
+
Oregon Green 488/Chromeo
505
Strongly recommended, best
combination so far (V500 staining via
secondary biotinilated antibody biotin
+ streptavidin)
No dye separation required
V500 (Strep)
+
Any green dye, reported to work
for STED CW
No dye separation required
Pacific Orange (Secondary
Antibodies conjugated)
+
DyLight488 / Alexa488
(excluding Oregon Green 488
and Chromeo 505)
Dye separation required
Atto 425 (Anti-mouse)
+
DyLight 488, Chromeo 505,
Oregon Green 488, Alexa 488
To be used with protein or structure
of interest bigger than 100 nm due to
a lower efficiency of the dye in STED
CW.
No dye separation required.
NBD-X
+
Any other green dye reported to
work for STED CW
No commercial conjugates for NBD-X
so far
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Living up to Life
2-color STED CW
Fluorescent Proteins
458 nm excitation
488 nm excitation
514 nm excitation
eCFP
eGFP
eYFP
mCerulean
Emerald
mCitrine, mVenus
NBDx
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Living up to Life
STED CW: “Must Remember”
Cells or tissue?
Coverslip #1.5 or glass bottom
dishes
Fluorescence (1or 2-color)
Coverslip # 1.5
Chosen protocol?
Mounting media?
Sample thickness is < 30µm
• Prolong kit (Not Prolong Gold)
• Moviol + Antifade
Sample thickness is > 30µm
• Prolong kit (Not Prolong gold)
•TDE + Antifade
• Moviol + Antifade
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Living up to Life
STED CW: Thiodiethanol preparation for tissue mounting
(TDE, Sigma, #88559)
6 Well-plate
TDE 25%
TDE 50%
TDE 75%
TDE 97% +
Antifade
• 15-30 minutes incubation at each step (depending on the sections thickness).
• Mix VERY WELL the antifade (pipette in and out) with the TDE 97% prior to mount the tissue
sections (eliminate the bubbles by centrifugation).
• Use nail polish (high quality uncolored nail polish) on the #1.5 coverslip corner to keep it in
place for the night (must be kept flat).
• Seal the coverslip edges the following morning with nail polish.
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Living up to Life
2-color STED CW
Mounting Media
Best
Good
Do Not Use
Prolong kit + Antifade
Mowiol + Antifade
(will polymerize)
(will polymerize)
Slowfade
Thiodiethanol
(TDE, Sigma, #88559). Add
Antifade (will stay
visceous)
Vectashield
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Living up to Life
STED CW: Antifading reagents
• DABCO (2.5%): Not as effective as the PPD, but is less toxic.
• Or you can also use the proprietory antifade from the Prolong kit, without DAPI.
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STED CW: 2-color protocol
Living up to Life
This is just an example and we are encouraging you to stay as close as possible to your usual protocol.
Only adjust the “Must Know” to your protocol.
• Cells grow on # 1.5 coverslip.
• Cells fixed with PAF 4% 10 min., RT, (or methanol, 5-7 min, -20 ºC).
• Rinse 3x in PBS.
• Block with PBS/Triton X 0.2%/Normal goat serum 10%, 30 min in rotation at RT
• Incubate in primary Ab in PBS/Tx/NGS 1 hr, RT.
• Rinse 3x in PBS.
• Block for 30 min.
• Incubate in secondary: Pacific Orange, Atto 425, V500, 1 hours at RT or on rotation at 4 degree C overnight.
PS: For the Horizon V500 labeling: biotinylated antibody for 1 hour at RT followed by Horizon V500 Streptavidin, 1 hr at RT.
Rinse, rinse, rinse!!!! At least 3x in PBS, in rotation at RT.
• Block in PBS/Tx/ normal serum (in accordance with the “second” secondary Ab species.
• Incubate in 2nd primary Ab for 1hr at RT Rinse in PBS.
• Block.
• Incubate in DyLight 488, 1 hr, RT Rinse 3x in PBS.
• Rince once in tap water.
• Mount in Prolong Kit (or TDE + antifade).
• Let cure at least overnight (at best 48 hrs to reach the maximum RI).
• Keep the slide at 4 degree C and protected from the light.
• Enjoy the STED CW imaging!
Myriam Gastard, PhD, personal communication, Leica Microsystems, Inc. USA
www.leica-microsystems.com
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Living up to Life
Examples of optimization of the acquisition for selected dyes pairs,
+
Dye1
Name
Excitation
Emission
Pacific Orange
458
535 - 580
Pacific Orange
458
Horizon V500
Dye2
Name
Excitation
Emission
+
DyLight 488, Alexa 488 / Any 488
(excluding Oregon Green 488)
488
496
500 – 530
505 - 530
500 -580
+
Any 488 (excluding Oregon Green 488)
488
496
514
500 – 580
505 – 580
525 - 580
458
465 – 500
465 - 510
+
Oregon Green 488, DyLight 488 / any 514
dyes
488
514
505 - 580
520 - 580
NBD-X
458
535 – 580
Or
500 - 580
+
488
500 – 530
Or
500 -580
Atto 425
458
465 - 510
+
514
520 - 580
DyLight 488, Alexa 488 / Any 488
(excluding Oregon Green 488)
Oregon Green / Any 488 / Any 514 dyes
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Selected spectra
ATTO 425
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ATTO 425 (Ex / Em)+ DyLight 488 (Ex / Em)
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STED CW: Sample Preparation
Living up to Life
Question?
Need any help before / during sample preparation?
Please contact me directly at: 866-830-0735, Option 3
or on my cell phone (given directly during the talk).
Myriam Gastard, PhD, Application and Tech Support, Exton, PA, 19341.
03/14/2011
www.leica-microsystems.com
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