Living up to Life Sample preparation for 1- and 2-color STED CW a-tubulin,(Rockland Immuno). M. Gastard, Leica Microsystems, Inc. (personal communication) Myriam C. Gastard, PhD www.leica-microsystems.com 1 Super resolution Imaging techniques Living up to Life STimulated Emission Depletion (STED) Depletion power: Excitation Depletion low spot ring FWHM ca. 200nm Overlaid medium spots Effectivehigh spot FWHM ca. 90nm Size of effective fluoresceing spot STED Detection Excitation SP5 Scanning Head STED Module www.leica-microsystems.com 2 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 ! www.leica-microsystems.com Page 3 3 7/20/2010 Measured Resolution confocal Living up to Life STED average lateral resolution FWHM measured on fluorescent beads (n =30; confocal/ 60;STED) Confocal: STED: www.leica-microsystems.com 260 nm 64 nm 4 Living up to Life STED: Spectral Conditions Excitation STED Detection Band Requirements for STED CW: • Dye excitable with Argon laser • Still significant emission at lSTEDc • No Excitation at lSTEDc 350 400 450 500 550 600 650 www.leica-microsystems.com 5 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 ! www.leica-microsystems.com 6 Living up to Life STimulated Emission Depletion (STED) ACTIN MICROTUBULES Confocal Confocal STED STED www.leica-microsystems.com 7 Living up to Life 1- and 2- color STED CW dye choices Confocal STED CW + deconvolution M. Gastard, Leica Microsystems, Inc. (personal communication) www.leica-microsystems.com 8 2-color STED CW Living up to Life What is the challenge? 458 488 592 depletion laser Shift stock www.leica-microsystems.com 9 Living up to Life 1 and 2-color STED CW Secondary Antibodies 1-color STED CW + Best Good Fair DyLight 488 Alexa 488 FITC Chromeo 488 Atto 488 Oregon Alexa 430 (only in combination with DyLight, Chromeo, Alexa or Atto 488 and only with 2-color STED CW) Other dyes are in testing and will be released soon. = 2 -color STED CW www.leica-microsystems.com 10 Living up to Life 2-color STED CW Fluorescence Proteins Good Best Depending on the amount of the protein of interest, the brighter being the better. mCitrine, mVenus (514 nm excitation) eYFP (514 nm excitation) DO NOT USE DAPI (must be avoided completely) eGFP (488 nm excitation) mCerulean (458 nm excitation) eCFP (458 nm excitation) www.leica-microsystems.com 11 Living up to Life STED CW: “Must know” a-tubulin (Rockland Immuno, Inc.) , Alexa 430 (Invitrogen) M. Gastard, Leica Microsystems, Inc. (personal communication) www.leica-microsystems.com 12 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 < 30mm Sample thickness is > 30mm • Prolong + Antifade • TDE + Antifade • Moviol + Antifade • Moviol + Antifade www.leica-microsystems.com 13 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. www.leica-microsystems.com 14 Living up to Life STED CW: Antifading reagents • P-phenylenediamine (PPD): Probably the most effective antifade (0.1-0.01%). • N-propyl gallate (NPG, 2%): Not very soluble; non-toxic and can be used on live cells. • DABCO (2.5%): Not as effective as the PPD, but is less toxic. www.leica-microsystems.com 15 Living up to Life STED CW Protocol: To Follow and to Apply each and every time www.leica-microsystems.com 16 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 • 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. The first primary (using the Alexa 430 as secondary) must have a stronger fluorescence compared to the second primary Ab. • Rinse 3x in PBS • Block for 30 min. • Incubate in secondary, Alexa 430, overnight, on rotation at 4 degree C. • 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 Gold (with 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 17 STED CW: “Must See” Living up to Life 1-color STED CW STED CW + deconvolution Confocal Myriam Gastard, Leica Microsystems, Inc. (personal communication) www.leica-microsystems.com 18 Living up to Life Confocal STED CW + deconvolution M. Gastard, Leica Microsystems, Inc. (personal communication) www.leica-microsystems.com 19 2-color STED CW Living up to Life a-tubulin (Alexa 430, green) + DARPP (DyLight 488, red) Myriam Gastard, PhD, personal communication. Leica Microsystems, Inc. USA. www.leica-microsystems.com 20