Sample preparation for 1- and 2-color STED CW
Sample preparation for 1- and 2-color STED CW
Sample preparation for 1- and 2-color STED CW
Create successful ePaper yourself
Turn your PDF publications into a flip-book with our unique Google optimized e-Paper software.
Living up to Life<br />
<strong>Sample</strong> <strong>preparation</strong> <strong>for</strong> 1- <strong>and</strong> 2-<strong>color</strong> <strong>STED</strong> <strong>CW</strong><br />
a-tubulin,(Rockl<strong>and</strong> Immuno). M. Gastard, Leica Microsystems, Inc. (personal communication)<br />
Myriam C. Gastard, PhD<br />
www.leica-microsystems.com 1
Super resolution Imaging techniques<br />
Living up to Life<br />
STimulated Emission Depletion (<strong>STED</strong>)<br />
Excitation<br />
spot<br />
Depletion power:<br />
Depletion<br />
low<br />
ring<br />
Overlaid<br />
medium<br />
spots<br />
Effective high<br />
spot<br />
FWHM<br />
ca. 200nm<br />
FWHM<br />
ca. 90nm<br />
Size of effective fluoresceing spot<br />
<strong>STED</strong><br />
Detection<br />
Excitation<br />
SP5 Scanning Head<br />
<strong>STED</strong> Module<br />
www.leica-microsystems.com 2
FWHM ~260nm<br />
FWHM < 65nm<br />
Living up to Life<br />
Resolution Enhancement by <strong>STED</strong><br />
FWHM ~260nm<br />
FWHM 65 nm<br />
<strong>STED</strong><br />
<strong>STED</strong><br />
<strong>STED</strong><br />
<strong>STED</strong><br />
confocal<br />
<strong>STED</strong><br />
<strong>STED</strong><br />
<strong>STED</strong><br />
<strong>STED</strong><br />
<strong>STED</strong><br />
<strong>STED</strong><br />
<strong>STED</strong><br />
<strong>STED</strong><br />
<strong>STED</strong> <strong>STED</strong> <strong>STED</strong> <strong>STED</strong><br />
A threefold improved resolution can make 9 spots out of 1 !<br />
7/20/2010<br />
www.leica-microsystems.com Page 3<br />
3
Measured Resolution<br />
Living up to Life<br />
confocal<br />
<strong>STED</strong><br />
average lateral resolution<br />
FWHM measured on<br />
fluorescent beads<br />
(n =30; confocal/ 60;<strong>STED</strong>)<br />
Confocal:<br />
<strong>STED</strong>:<br />
260 nm<br />
64 nm<br />
www.leica-microsystems.com 4
Living up to Life<br />
<strong>STED</strong>: Spectral Conditions<br />
Excitation<br />
<strong>STED</strong><br />
Detection B<strong>and</strong><br />
Requirements <strong>for</strong> <strong>STED</strong> <strong>CW</strong>:<br />
• Dye excitable with Argon laser<br />
• Still significant emission at l <strong>STED</strong>c<br />
• No Excitation at l <strong>STED</strong>c<br />
350 400 450 500 550 600<br />
650<br />
www.leica-microsystems.com 5
Resolution: Reality check by <strong>STED</strong><br />
Living up to Life<br />
<strong>STED</strong> <strong>CW</strong> resolution (in red) vs confocal resolution (in green): AKTpS473 (Rockl<strong>and</strong> Immuno, Inc, PA)<br />
REMEMBER<br />
Do NOT expect to have a resolution below 100 nm if the protein(s) or structure of interest<br />
are bigger than 100 nm !<br />
www.leica-microsystems.com 6
Living up to Life<br />
STimulated Emission Depletion (<strong>STED</strong>)<br />
ACTIN<br />
MICROTUBULES<br />
Confocal<br />
Confocal<br />
<strong>STED</strong><br />
<strong>STED</strong><br />
www.leica-microsystems.com 7
Living up to Life<br />
1- <strong>and</strong> 2- <strong>color</strong> <strong>STED</strong> <strong>CW</strong> dye choices<br />
Confocal<br />
<strong>STED</strong> <strong>CW</strong> + deconvolution<br />
M. Gastard, Leica Microsystems, Inc. (personal communication)<br />
www.leica-microsystems.com 8
2-<strong>color</strong> <strong>STED</strong> <strong>CW</strong><br />
Living up to Life<br />
What is the challenge<br />
458<br />
488<br />
592 depletion laser<br />
Shift stock<br />
www.leica-microsystems.com 9
1 <strong>and</strong> 2-<strong>color</strong> <strong>STED</strong> <strong>CW</strong><br />
Living up to Life<br />
Secondary Antibodies<br />
Best Good Fair<br />
1-<strong>color</strong> <strong>STED</strong> <strong>CW</strong><br />
DyLight 488 Alexa 488 FITC<br />
Chromeo 488 Atto 488 Oregon<br />
+<br />
=<br />
Alexa 430 (only in<br />
combination with<br />
DyLight, Chromeo, Alexa<br />
or Atto 488 <strong>and</strong> only with<br />
2-<strong>color</strong> <strong>STED</strong> <strong>CW</strong>)<br />
Other dyes are in<br />
testing <strong>and</strong> will be<br />
released soon.<br />
2 -<strong>color</strong> <strong>STED</strong> <strong>CW</strong><br />
www.leica-microsystems.com 10
2-<strong>color</strong> <strong>STED</strong> <strong>CW</strong><br />
Living up to Life<br />
Fluorescence Proteins<br />
Best<br />
Good<br />
Depending on the amount of<br />
the protein of interest, the<br />
brighter being the better.<br />
DO NOT USE<br />
mCitrine, mVenus<br />
(514 nm excitation)<br />
eYFP<br />
(514 nm excitation)<br />
DAPI<br />
(must be avoided<br />
completely)<br />
eGFP<br />
(488 nm excitation)<br />
mCerulean (458 nm<br />
excitation)<br />
eCFP (458 nm<br />
excitation)<br />
www.leica-microsystems.com 11
Living up to Life<br />
<strong>STED</strong> <strong>CW</strong>: “Must know”<br />
a-tubulin (Rockl<strong>and</strong> Immuno, Inc.) , Alexa 430 (Invitrogen)<br />
M. Gastard, Leica Microsystems, Inc. (personal communication)<br />
www.leica-microsystems.com 12
Living up to Life<br />
<strong>STED</strong> <strong>CW</strong>: “Must Remember”<br />
Cells or tissue<br />
Coverslip #1.5 or glass bottom<br />
dishes<br />
Fluorescence (1or 2-<strong>color</strong>)<br />
Coverslip # 1.5<br />
Chosen protocol<br />
<strong>Sample</strong> thickness is < 30mm<br />
• Prolong + Antifade<br />
• Moviol + Antifade<br />
Mounting media<br />
<strong>Sample</strong> thickness is > 30mm<br />
• TDE + Antifade<br />
• Moviol + Antifade<br />
www.leica-microsystems.com 13
Living up to Life<br />
<strong>STED</strong> <strong>CW</strong>: Thiodiethanol <strong>preparation</strong> <strong>for</strong> tissue mounting<br />
(TDE, Sigma, #88559)<br />
6 Well-plate<br />
TDE 25%<br />
TDE 50% TDE 75%<br />
TDE 97% + Antifade<br />
• 15-30 minutes incubation at each step (depending on the sections thickness).<br />
• Mix VERY WELL the antifade (pipette in <strong>and</strong> out) with the TDE 97% prior to mount the tissue<br />
sections (eliminate the bubbles by centrifugation).<br />
• Use nail polish (high quality un<strong>color</strong>ed nail polish) on the #1.5 coverslip corner to keep it in<br />
place <strong>for</strong> the night (must be kept flat).<br />
• Seal the coverslip edges the following morning with nail polish.<br />
www.leica-microsystems.com 14
Living up to Life<br />
<strong>STED</strong> <strong>CW</strong>: Antifading reagents<br />
• P-phenylenediamine (PPD): Probably the most effective antifade (0.1-0.01%).<br />
• N-propyl gallate (NPG, 2%): Not very soluble; non-toxic <strong>and</strong> can be used on live cells.<br />
• DABCO (2.5%): Not as effective as the PPD, but is less toxic.<br />
www.leica-microsystems.com 15
Living up to Life<br />
<strong>STED</strong> <strong>CW</strong> Protocol: To Follow <strong>and</strong> to Apply each <strong>and</strong> every time<br />
www.leica-microsystems.com 16
<strong>STED</strong> <strong>CW</strong>: 2-<strong>color</strong> protocol<br />
Living up to Life<br />
This is just an example <strong>and</strong> we are encouraging you to stay as close as possible to your usual protocol. Only<br />
adjust the “Must Know” to your protocol.<br />
• Cells grow on # 1.5 coverslip<br />
• Cells fixed with PAF 4% 10 min., RT<br />
• Rinse 3x in PBS<br />
• Block with PBS/Triton X 0.2%/Normal goat serum 10%, 30 min in rotation at RT<br />
• Incubate in primary Ab in PBS/Tx/NGS 1 hr, RT. The first primary (using the Alexa 430 as secondary) must have a stronger<br />
fluorescence compared to the second primary Ab.<br />
• Rinse 3x in PBS<br />
• Block <strong>for</strong> 30 min.<br />
• Incubate in secondary, Alexa 430, overnight, on rotation at 4 degree C.<br />
• Rinse, rinse, rinse!!!! At least 3x in PBS, in rotation at RT<br />
• Block in PBS/Tx/ normal serum (in accordance with the “second” secondary Ab species<br />
• Incubate in 2 nd primary Ab <strong>for</strong> 1hr at RT<br />
• Rinse in PBS<br />
• Block<br />
• Incubate in DyLight 488, 1 hr, RT<br />
• Rinse 3x in PBS<br />
• Rince once in tap water<br />
• Mount in Prolong Gold (with antifade). Let cure at least overnight (at best 48 hrs to reach the maximum RI).<br />
• Keep the slide at 4 degree C <strong>and</strong> protected from the light.<br />
• Enjoy the <strong>STED</strong> <strong>CW</strong> imaging!<br />
Myriam Gastard, PhD, personal communication, Leica Microsystems, Inc. USA<br />
www.leica-microsystems.com 17
<strong>STED</strong> <strong>CW</strong>: “Must See”<br />
Living up to Life<br />
1-<strong>color</strong> <strong>STED</strong> <strong>CW</strong><br />
Confocal<br />
<strong>STED</strong> <strong>CW</strong> +<br />
deconvolution<br />
Myriam Gastard, Leica Microsystems, Inc. (personal communication)<br />
www.leica-microsystems.com 18
Living up to Life<br />
Confocal<br />
<strong>STED</strong> <strong>CW</strong> + deconvolution<br />
M. Gastard, Leica Microsystems, Inc. (personal communication)<br />
www.leica-microsystems.com 19
2-<strong>color</strong> <strong>STED</strong> <strong>CW</strong><br />
Living up to Life<br />
a-tubulin (Alexa 430, green) + DARPP (DyLight 488, red)<br />
Myriam Gastard, PhD, personal communication. Leica Microsystems, Inc. USA.<br />
www.leica-microsystems.com 20
Change language