Leica TCS STED CW. The Fast Track to Superresolution. Leica TCS STED CW

Transcription

1 The Fast Track to Superresolution

2 content Motivation Concept Realisation Applications - why do researchers need the TCS STED CW? - what is the TCS STED CW based on? - how does the TCS STED CW work? - what s possible with the TCS STED CW?

3 the situation / the desire

4 the desire fast reliable results- not only for experienced Biophysicists a system suitable for daily research maximum flexibility >> turning superresolution microscopy into a piece of the standard workflow

5 what s needed in practical terms? superresolution must work. accessible and reliable for biologists based on standard protocols live cell imaging with FPs inside the 3D sample

6 what s required to achieve that? a rock solid platform: From 22 frames/s to 512x512 image The true confocal point scanning TCS SP5 II Best resolution, contrast, depth imaging with single point scanning system Highest flexibility for multi-labelling due to AOBS, 5 SP detectors, APD etc.

7 what s required to achieve that? an excellent optical superresolution concept >> STED microscopy

8 motivation for Superresolution in general

9 What is resolution? Taken from: How close can 2 particles be to seen as two, not as one?

10 At the Resolution Limit Rolf Borlinghaus / Leica PSFs melt together not a good representation of the structure

11 The diffraction limit Lens Scan Point Detector x 2nsin Consequence: Diffraction prevents to separate objects closer than 200nm (= ½ wavelength of visible light) Ernst Abbe, 1873

12 How can we see more???? 0.4 x NA Confocal microscope 1. Use a shorter wavelength >> electron microscopy >> too many drawbacks 2. Maximize the numerical aperture of the objective >>technically limited to ~1.5 Something else is needed!!!!

13 1000 m How far can we go with light microscopy? Green: Nucleus (Alexa 488) Red: Axon (Cy 3) Blue: Axon Ending at MJ94 pos. Neurons (Cy 5) Scanner: TCS SP2 AOBS Objective: 10x / 0,4 (Oil) Magnification: 200x (Screen Size) Dr. Christoph Melcher Forschungszentrum Karlsruhe Institut für Toxikologie und Genetik

14 500 m Green: Nucleus (Alexa 488) Red: Axon (Cy 3) Blue: Axon Ending at MJ94 pos. Neurons (Cy 5) Scanner: TCS SP2 AOBS Objective: 10x / 0,4 (Oil) Magnification: 400x (Screen Size) Dr. Christoph Melcher Forschungszentrum Karlsruhe Institut für Toxikologie und Genetik

15 150 m Green: Nucleus (Alexa 488) Red: Axon (Cy 3) Blue: Axon Ending at MJ94 pos. Neurons (Cy 5) Scanner: TCS SP2 AOBS Objective: 63x / 1,3 (Glycerin) Magnification: 1200x (Screen Size) Dr. Christoph Melcher Forschungszentrum Karlsruhe Institut für Toxikologie und Genetik

16 50 m Green: Red: Blue: Cytoskeleton (Actin) (Bodipy FL) Mitochondria (Mitotracker Red) Nucleus (Dapi) Objektive: 63x / 1,4 (Oil) Scanner: confocal Magnification: 3600x 5 m Ulf Schwarz Leica Microsystems

17 20 m Green: Red: Blue: Cytoskeleton (Actin) (Bodipy FL) Mitochondria (Mitotracker Red) Nucleus (Dapi) Objektive: 63x / 1,4 (Oil) Scanner: TCS SP2 AOBS Magnification: 9000x 2 m Ulf Schwarz Leica Microsystems

18 Further magnification does not give more information in optical microscopy!

19 the concept behind

20 The diffraction limit Lens Scan Point Detector x 2nsin Consequence: Diffraction prevents to separate objects closer than 200nm (= ½ wavelength of visible light) Ernst Abbe, 1873

21 Breaking the diffraction limit Lens Scan Point Detector Stefan W. Hell, Inventor of STED-microscopy Reducing the area of effective excitation, in combination with a scanning microscope breaking the diffraction limit becomes possible Page 26 5/17/2010

22 The principle Sample Excitation PSF Exc. Det. Point Detector STED Lens Phase modulation Y Phase + /2 X Phase +0 Entrance Pupil X Z + STED PSF X Y = Effective PSF

23 STED: Spectral Conditions Excitation STED Detection Band requirements: Acceptable photobleaching No Excitation at STED Selected, optimized dyes and wavelength needed, Recommendation: ATTO647N & ATTO655

24 Stimulated Emission Depletion Excitation: - internal Ar gas laser (488, 514 nm) Depletion: - external visible fiber laser (592 nm) The STED-doughnut detected by Photomultiplier(s) or Avalanche Photodetector spectrally filtered out (emitted photons are identical to 592nm photons)

25 Measured resolution confocal STED average lateral resolution FWHM measured on fluorescent beads (n =30; confocal/ 60;STED) Confocal: STED: 260 nm 64 nm

26 the solution

27 One solution: STED pulsed (Ti-Sa) 2007

28 One solution: 2009 The

29 the STED CW new! reduced alignment required - higher stability - faster auto alignment

30 Pulsed and CW the difference? PULSED STED STED with continuous wave lasers perfectly synchronized excitation (100 ps) and depletion pulses (300 ps) dedicated excitation (640nm) and depletion laser (IR-laser, nm tuning range for STED) 80 Mhz repetition rate each excitation pulse followed by a depletion pulse continuous excitation and depletion >> persistent competition in the doughnut area flexible excitation (Ar laser, 488 & 514 nm) 592 nm

31 why more signal per time with cw lasers? (avg. fluorescence lifetime 1-5 ns) excitation pulse detectable signal is emitted dark interpulse period dark interpulse period t = 12,5ns (80 Mhz rep. rate of the lasers) no dark interpulse periods but continuous generation and readout of fluorescence signal in STED CW Page 36

32 the solution The Fast Track To Superresolution Fast means fast recordings (excellent S/N ratio with 1fps) fast (=immediate) results a fast workflow

33 Freedom to choose - well-known fluorescent dyes Alexa 488 FITC Oregon Green and more, e.g. Chromeo 488 (e)yfp, Citrin, Venus no need to waste time to change well established protocols to do SR imaging! Page 38

34 the solution Observe what s inside - with confocal superresolution optical sectioning! PALM ends here (100nm) z based on confocal basis superior start superior results!

35 the solution Subdiffraction Imaging - devoid of algorithms purely optical superresolution the only method that does need to postprocess recorded data! time saving: imaging publishing reliability less artifacts image storage this belongs to structured Illumination!

36 the solution Watch live cell dynamics in the sub 100nm scale! the only commercial solution that enables to do dynamic recordings in subdiffraction resolution fast scans possible live celll inside the cell! with FPs and membrane permeant fluorophores (Oregon Green)

37 what are the top strengths? you simply try the sample you are working with: the times of today I have to prepare my sample for a superresolution experiment which I will start after lunch and finish around dinner are over! Now it s: mhhh, I have been using this specimen for quite a while doing confocal and Widefield, but something remains blurry- let s shine some STEDlight on it! and you simply turn on the STED-laser that s all! >>change in paradigm!!! you can use genetically encoded markers standard ones live cell imaging is possible BUT WITH limitations

38 the applications

39 top applications? everything that is small (<120nm) Vesicles, protein-cluster, receptor complexes, cytoskeleton etc. (see chart at the end) everything that is small and not attached to the coverslip everything that is small and not attached to the coverslip and moving

40 Live Cell Imaging is possible! confocal confocal STED t=0 sec 1 µm t=0 sec STED 3µm t=18 sec t=36 sec t=54 sec t= 72 sec 3µm

41 confocal superresolution Maximum projection STED Maximum projection confocal 4 µm z-pos: 0.9 µm z-pos: 2 µm z-pos: 4 µm z-pos: 6 µm Superresolution pixel by pixel in each single optical slice!

42 Actin Fibers (Alexa 488, Elise Stanley, Toronto) STED confocal

43 ß-Tubulin Oregon Green confocal confocal STED STED

44 Keratinocytes-YFP (Reiner Windoffer RWTH Aachen Univ.) confocal STED

45 nuclear proteins Alexa488; D. Gruenwald; Einstein Univ., New York confocal STED

46 resolution measurements with bioparticles confocal STED

47 Neuroscience Bruchpilot 2 µm confocal STED Immunfluorescent staining of neuromuscular junctions of a drosophila larvae. Labels: Bruchpilot (Chromeo488, red), Res(green, Cy3). Courtesy Stephan Sigrist and Wernher Fouquet, Freie Universitaet Berlin 500 nm

48 superior start >> superior results widefield image confocal confocal zoomed in deconvolved STED zoomed in

49 the system

50 the system

51 the inside

52 The «standard» software LAS AF

53 the software

54 Green light for Superresolution Software workflow: intuitive Easy to operate Fully flexible Feedback on correct settings balancing act: maximum flexibility vs. optimal user guidance Traffic light concept gives feedback about appropriate STED conditions

55 The - facts lateral resolution <80 nm FWHM purely optical further improvement possible- with integrated deconvolution confocal platform enables imaging deep inside the sample and to do 3D stacks conventional fluorophores (Alexa 488, Oregon Green etc.) & fluorescent proteins can be used (eyfp, Venus ) very fast recordings possible (even with resonant)

56 TCS STED - TiSa All-in-One solution SP5-MP-STED Two-color-option Exc 531nm AND 640nm Depletion nm TCS STED - CW Live cell imaging with fluorescent proteins possible Standard dyes can be used Exc 488/514nm Depletion 592nm

57 STED-take home There are different ways to realize a STED system, mainly differing in the used depletion laser: - Pulsed STED (Ti-Sa) - STED CW - Not shown (no commercial): continuum laser, T-Rex General concept: saturated depletion by a powerful laser depopulates the excited state, leaving the inner part of the doughnut unaffected Resolution diffraction unlimited (but practically limited, of course)