SUPER-RESOLUTION MICROSCOPY. Dr. Nathalie Garin

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1 SUPER-RESOLUTION MICROSCOPY Dr. Nathalie Garin

2 Content Motivation for superresolution Superresolution, nanoscopy, : definition Structured Illumination Microscopy (SIM) Localization microscopy STimulated Emission Depletion (STED) Combined technics

$The diffraction limit Rolf Borlinghaus / Leica PSFs melt$

3 The diffraction limit Rolf Borlinghaus / Leica PSFs melt together not a good representation of the structure

4 Why Superresolution Microscopy? x 2nsin Diffraction Limit Cell Bacterium Mitochondrium Influenza Virus Titin GFP

5 Content Motivation for superresolution Superresolution, nanoscopy, : definition Structured Illumination Microscopy (SIM) Localization microscopy STimulated Emission Depletion (STED) Combined technics

6 Abbe limit: Pushing or breaking? Nanoscopy Confocal superresolution Confocal

7 Content Motivation for superresolution Superresolution, nanoscopy, : definition Structured Illumination Microscopy (SIM) Localization microscopy STimulated Emission Depletion (STED) Combined technics

8 Slide courtesy: G. Wright, Singapor H. Horn, Journal of Cell Biology (2013) KASH5 SCP3 Horn et al., (2013) JCB 202:

9 f (x) a 0 Fourier series (1D) n 1 a n cos(n x L ) n Vincent Studer IINS - Bordeaux

10 Vincent Studer IINS - Bordeaux f ˆ ( k r ) f ( r ) Fourier transform Real Space f ( r ) r (x,y,z) (,z) r f ( )e r 2i r k. r r d 2 f ˆ ( k r )e 2i r k. r d r 2 k FT Inverse FT Fourier space f ˆ ( r r (x,y,z) (,z) r r k (k x,k y,k z ) ( k r,k z ) k ) r k (k x,k y,k z ) ( k r,k z ) k y k x

Creating a Moiré Fringe Two superimposed patterns (in this case

interfere with each other and produce a third, characteristic

The Moiré fringes have a lower spatial frequency than the

11 Creating a Moiré Fringe Two superimposed patterns (in this case the illumination pattern and the structures in the sample) interfere with each other and produce a third, characteristic pattern: the Moiré fringes. The Moiré fringes have a lower spatial frequency than the original structures within the sample. Therefore, the fringes can be transmitted by a normal objective lens.

12 Vincent Studer IINS - Bordeaux TF x OTF= TF x OTF=

13 Optical Sectioning Vincent Studer IINS - Bordeaux

14 Optical Sectioning Vincent Studer IINS - Bordeaux

15 Optical-Sectioning Vincent Studer IINS - Bordeaux

16 3D-SIM Applied Precision Gustafsson et al BioPhy J keratin-14

17 SIM: take home message Wide field technique Works with any fluorophore Sub-diffraction resolution (130 nm x 350 nm, Zeiss website) Un modulated areas of the sample due to out of focus fluorescence do not contribute to the signal but they add shot noise! Problem with thick samples. > TIRF SIM is therefore of great interest for low level fluorescence where shot noise is important

18 Content Motivation for superresolution Superresolution, nanoscopy, : definition Structured Illumination Microscopy (SIM) Localization microscopy STimulated Emission Depletion (STED) Combined technics

19 Galectin-3 accumulates in Flotillin-positive endosomes Slide courtesy: Ralf Jacob, Marbrug

$The solution with diffraction limited resolution$ $Diffraction limited resolution FWHM of the PSF$

20 The solution with diffraction limited resolution Localization resolution x 2NA N (N= number of photons) Diffraction limited resolution FWHM of the PSF Thompson et al., Biophys. J WF single molecule superresolution

22 Movie courtesy: Claudio Dellagiacoma, EPFL, Lausanne

23 Localization microscopy acquisition S1 T1 Fölling, J., Bossi, M., Bock, H., Medda, R., Wurm, C. A., Hein, B., Jakobs, S., Eggeling, C. and Hell, S. W., Nat Methods. 5(2008), S0 t ~ 3ns t ~ 100ms 10k to 100k frames calculate centre of mass Sum

24 Acronymes PALM PhotoActivation Light Microscopy N-STORM Stochastic Optical Reconstruction Microscopy Sam Hess upaint Universal Point Accumulation Imaging in the Nanoscale Topography

25 GSDiM / (D)STORM The dark state of fluorescence not bleaching Ground state depleted

26 3D: different possibilities Astigmatism Double helix biplane microscopy US B2

27 Localization techniques: take home message Widefield technique Calculated SR Works with any fluorophores Sub-diffraction resolution: o 20 nm in xy o 50nm in z Often in TIRF

28 Content Motivation for superresolution Superresolution, nanoscopy, : definition Structured Illumination Microscopy (SIM) Localization microscopy STimulated Emission Depletion (STED) Combined technics

(red) in confocal (left) and deconvolved STED image (right).

29 Does it colocalize? STED Confocal 500 nm Centrioles in U2OS cells visualized by indirect immunofluorescence. Co-localization of Centrin3 Alexa Fluor 594 (green) and Cep152 Alexa Fluor 647 (red) in confocal (left) and deconvolved STED image (right). Sample courtesy of Ella Fung, CRUK/MRC Oxford Institute for Radiation Oncology, UK. The anti-cep152 antibody was kindly provided by E. Nigg, Biozentrum, University of Basel, Switzerland (Sonne KF et. al. J Cell Science 2013). 29

$The diffraction limit Lens Scan Point$ prevents to separate objects closer than

30 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

$Breaking the diffraction limit Lens$ Hell, Inventor of STED-microscopy

31 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

32 STED Microscopy PSF shaping On state Off state

33 STED: a switch off process Pyridine 2

34 The driving forces: laser and dye development ~ 200 nm ~ 150 nm ~ 75 nm

35 Gated STED

Gated STED: The Principle Confocal STED Lifetime

resolution has a life time dependence Short lived states

of STED CW images Gated STED only records fluorescence

36 Gated STED: The Principle Confocal STED Lifetime distribution within STED CW focal spot For CW STED resolution has a life time dependence Short lived states cause somehow blurry appearance and reduce the contrast of STED CW images Gated STED only records fluorescence from long living states Improved resolution Clearer images/better contrast

37 Standard STED CW with pulsed Excitation Confocal Excitation Fluorescence Detection Time (ns) DNA Origami 70nm distance

38 Standard STED CW with pulsed Excitation STED CW Excitation Fluorescence Detection STED Time (ns) DNA Origami 70nm distance STED laser not used at full power

39 Gated STED Excitation Detection STED Time (ns) DNA Origami 70nm distance STED laser not used at full power

40 Gated STED Excitation Detection STED Time (ns) DNA Origami 70nm distance STED laser not used at full power

41 Deuterosomes: platform for centriole amplification 1mm Work done with C. Boutin, Institut de Biologie du Développement de Marseille Centriole marker: yellow/alexa488 Deuterosome marker: Cyan/Alexa568, magenta/alexa514

Cleared kidney sample: 45-55 µm inside Confocal 3D STED Sample courtesy

42 Cleared kidney sample: µm inside Confocal 3D STED Sample courtesy of David Unnersjö Jess, KTH, Stockholm More on clearing on Leica Science Lab

$or 775nm Sub-diffraction resolution: o <50 nm in xy o < 130 nm in z Ideal for thick and living samples$

43 STED: take home message Confocal technique Optical SR Works with any fluorophores depleted by 592, 660 or 775nm Sub-diffraction resolution: o <50 nm in xy o < 130 nm in z Ideal for thick and living samples

44 Content Motivation for superresolution Superresolution, nanoscopy, : definition Structured Illumination Microscopy (SIM) Localization microscopy STimulated Emission Depletion (STED) Combined technics

45 Lattice light sheet Technic combining: o Light sheet fluorescence microscopy o Bessel beam microscopy o Structured Illumination Microscopy (SIM)

46 Lattice light sheet

47 4Pi microscopy (Stefan Hell) Confocal focus cross section 4Pi focus cross section z z 500 nm 110 nm x Combining the Numerical Aperture of 2 opposing objectives Volume reduction by factor 3 5 compaired to confocal Interference side lobes are removed mathematically

48 4Pi measurements in living cells 4Pi microscopy Conventional fluorescence microscopy

49 Summary Gated STED 3D STED /GSDIM 70 < <130 < Modified from JBC Review 2010 Lothar Schermelleh, Rainer Heintzmann and Heinrich Leonhardt

50 From microscopy to nanoscopy Sample courtesy: Anne Aubusson-Fleury, CNRS, Gif sur Yvette, France

51 Some references Stefan W. Hell & Jan Wichmann (1994). "Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy". Optics Letters M. L. Bossi, J. Fölling, M. Dyba, V. Westphal, S. W. Hell (2006). "Breaking the diffraction resolution barrier in far-field microscopy by molecular optical bistability" New J. Phys. Lothar Schermelleh et al. (2010). A guide to super-resolution fluorescence microscopy J Cell Biol. Review Gustafsson, M. G. L. (2000). Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. Journal of Microscopy. Bi-Chang Chen et al. (2014). Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution. Science