Lecture 16. OMX - Structured Illumination Microscopy Ian Dobbie x Microscopy Course Lecture 16 1

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1 Lecture 16 OMX - Structured Illumination Microscopy Ian Dobbie x13323 Microscopy Course Lecture 16 1

2 Super-resolution fluorescence microscopy Specificity Sensitivity Non-invasive (in situ & in vivo) Multi-dimension (x, y, z, λ, t,...) Relative localisation & dynamics Single cell to high throughput Spatial resolution is diffraction limited Typical widefield image... Magnification alone does not give more details Microscopy Course Lecture 16 2

3 ...warmup: What determines the resolution of an optical microscope? x/ x/ x/ what objective would you take... Microscopy Course Lecture 16 3

4 ... a bit more difficult...? x/ x/1.0 40x/1.1 12,800 3,004 8,816 What's the difference in brightness?... what objective would you take... Microscopy Course Lecture 16 4

5 Numerical aperture determines... Brightness Fepi = 10 4 NA 4 / Mag 2 (epifluorescence) Lateral Resolution dx,y = 0.61 λ / NA ( nm) Axial Resolution dz = 2 λ / NA 2 ( nm) Only applies under optimal conditions BUT... spherical aberrations (refractive index mismatch, sample) chromatic aberrations stray light out-of-focus blur detector noise... Effective resolution is worse (max. 250 nm lateral and 1 μm axial)...improved to some extent by confocal imaging or deconvolution Microscopy Course Lecture 16 5

6 Super-resolution fluorescence microscopy How Structured Illumination (SI) improves not only resolution... & how it is realized in OMX system Comparison of super-resolution methods (Pros & Cons) 3D structured illumination microscopy Microscopy Course Lecture 16 6

7 Superresolution microscopy - three major concepts Super&resolu*on,light,microscopy:, Imaging,beyond,Abbe s,diffrac*on,limit, Structured,illumina*on, SIM-Methods: S*mulated,emission,deple*on, (STED), Apotome (conventional SIM) 2D-SIM 3D-SIM (linear SIM) Localization microscopy Photoac*va*on, Stochas*c,op*cal, Photoac*va*on,localiza*on, TIRF-SIM microscopy,(palm), reconstruc*on,microscopy, (STORM), SSIM (non-linear SIM) NL-SIM Microscopy Course Lecture 16 7

8 Resolving power of commercial super-resolution systems ^ 3D-SIM resolves ~8-fold smaller volumes than conventional (confocal) microscopes Schermelleh, Heintzmann, Leonhardt, JCB, 2010 Microscopy Course Lecture 16 8

9 Not only resolution matters, but... also context (II) What could this be? 3D information (z-res., optical sectioning, z-depth) Microscopy Course Lecture 16 9

10 Not only resolution matters, but also context (II) Prague National Museum Microscopy Course Lecture 16 10

11 3D-SIM: 3-color 3D optical sectioning 8x enhanced volumetric resolution µm depth 11 Microscopy Course Lecture 16

12 How does it work? Microscopy Course Lecture 16 12

13 The basic principle: Abbe s view Sample = Structure Periodicity a Microscopy Course Lecture 16 13

14 The basic principle: Abbe s view Image Objective lens α Sample a Lightwaves Microscopy Course Lecture 16 14

15 The basic principle: Abbe s view Image Objective lens a -1 α 0 Lightwaves +1 Sample highest frequencies (biggest α) smallest structures Microscopy Course Lecture 16 15

16 Image = superposed periodicities Real space (xy) Real space FFT (Fourier Transformation) Frequency space (kx, ky) Frequency space k y k x y k y x k x Microscopy Course Lecture 16 16

17 Image = superposed periodicities Real space (xy) Real space FFT (Fourier Transformation) Frequency space (kx, ky) Frequency space k y k x y k y x k x Microscopy Course Lecture 16 17

18 Image = superposed periodicities Real space (xy) Real space Real space FFT (Fourier Transformation) (Fourier Transformation) Frequency space (kx, ky) Frequency space Frequency space k y k x y k y x low N.A. k x Microscopy Course Lecture 16 18

19 Image = superposed periodicities Real space (xy) Real space FFT (Fourier Transformation) Frequency space (kx, ky) Frequency space Cut-off frequency k y 2 N.A. / λ k x y k y x high N.A. k x Microscopy Course Lecture 16 19

20 SIM principle - super-resolution by Moiré interference Moiré fringes Schermelleh, Carlton et al. (2008), Science 320 Fourier transform of the measured image unknown structure known illumination function Microscopy Course Lecture 16 20

21 Generating 2D-structured illumination multimode fiber despeckled laser light 405 nm, 488 nm, 594 nm Δx,ΔΦ polarizer 0 optical grating dichroidic mirror CCD back focal plane Y sample Δz focal plane X adapted from Gustafsson et al. (2008) BiophysJ, 94 Microscopy Course Lecture 16 21

22 Generating 3D-structured illumination multimode fiber despeckled laser light 405 nm, 488 nm, 594 nm Δx,ΔΦ polarizer optical grating axial modulation dichroidic mirror image plane CCD back focal plane Y sample Δz focal plane X adapted from Gustafsson et al. (2008) BiophysJ, 94 Microscopy Course Lecture 16 22

23 OMX 3D-SIM microscope system Cabinet Laser optics table Optical grating for SI Sample holder OMX V3 Electronics rack 100x/1.4 NA Max. mech. stability Highest sensitivity High-sensitive EMCCD camera (3x) Fixed filter drawer Mess OMX V2 Microscopy Course Lecture 16 23

24 3D-SIM in practice Raw SI data Actin (Phaloidin-Alexa488) in Drosophila macrophage (Eva Wegel) Microscopy Course Lecture 16 24

25 Conventional SIM: Apotome uses coarse SI to remove out-of-focus blur Poor man s confocal No super-resolution Microscopy Course Lecture 16 25

26 3D-SIM in practice Raw SI data SI reconstructed data (xy) I (Black 0 box) = sin I 1 k 1 + cos sin I 1 k 1 cos I em (r) =I ex (r) S(r) I em ( k)=i 0 { k ( k k0 ) e +j ( k + k 0 ) e j I I(r) 2 2k sin E l e jkl r 0 2= l I 2 2k sin 0 sin I 3 k 1 cos sin m I 3 k 1 + cos l D(r) = El e jkl r E l e Maths jkl r l S(k),S(k + k 0 ),S(k k 0 ) [(H I m ) (S J m )](r) m= El E q e j(kq l,q kl )r H(r r ) I m (z z ) S(r) J m (r xy )dr 15 images / plane (5 phases + 3 angles) 135 images / 1 µm z-stack / λ Exposure time ms (xz) Tubulin in Drosophila macrophage Eva Wegel Microscopy Course Lecture 16 26

27 Doubling frequency support by SI 3 phase shifts (2π/3) 3 phase shifts (2π/3) 5 phase shifts (2π/5) 5 phase shifts (2π/5) +2 Moiré information (shifted) +1 0 order -1-2 Microscopy Course Lecture 16 27

28 1st angle Doubling frequency support by SI SI raw SI reconstructed Wide-field 2nd angle2 3rd angle projected widefield 5 µm 5 µm Microscopy Course Lecture 16 28

29 Doubled frequency support = 2-fold resolution in xy and z SI raw SI reconstructed 1st angle 2nd angle2 3rd angle projected widefield 5 µm Microscopy Course Lecture 16 29

30 Doubled frequency support = 2-fold resolution in xy and z Widefield SI reconstructed 5 phases, 3 angles optical sectioning with 2x2x2-fold frequency support k z k y k x 5 µm Microscopy Course Lecture 16 30

31 3D optical sectioning capacity Example: 170 nm Fluospheres z=0 z=125 μm z=250 μm z=375 μm Microscopy Course Lecture 16 31

32 3D SIM example: Prophase Lamin B DAPI 3D volume rendering Schermelleh, Carlton et al. (2008), Science 320 Microscopy Course Lecture 16 32

33 3D SIM example: chromatin 3D-3D-SIMSIM 3D-SIM Widefield mouse C2C12 cell (DAPI staining) mouse C2C12 cell (DAPI staining) Microscopy Course Lecture 16 33

34 3D SIM example: chromatin 3D-3D-SIMSIM Widefield DAPI anti-npc 1 µm mouse C2C12 cell (DAPI staining) Microscopy Course Lecture 16 34

35 3D-SIM resolves chromatin domains and interchromatin channels, leading towards nuclear pores CLSM 3D-SIM 5 µm 1 µm Mouse C2C12 cell Schermelleh et al. (2008), Science 320 Lothar Schermelleh Barcelona Microscopy Course Lecture 16 35

36 Can we go live? Microscopy Course Lecture 16 36

37 Live cell 3D-SIM with OMX Blaze Inferometric pattern generation + scmos cameras 10 x faster imaging H2B-GFP (unfixed) RecA-GFP (E.coli) 2 µm 7 µm z-stack (56 sections, 5 ms exposure) 2 s / 3D-frame (1 µm z-stack = 120 images ; 100 ime points) Christian Lesterlin (D. Sherratt Lab) Microscopy Course Lecture 16 37

38 3D-SIM, just another tool in the repertoire? It s not that simple The untold story Microscopy Course Lecture 16 38

39 SI reconstruction artifacts Good SI reconstruction RPE-1 cell, DAPI staining Microscopy Course Lecture 16 39

40 SI reconstruction artifacts Stripes High frequency noise Halo / Doubling C127 cell nuclei, chromatin staining Bleaching, Drift or vibrations Moving particles (locally constrained) Low contrast-to-noise, Low modulation contrast Spherical aberration, Refractive index mismatch Microscopy Course Lecture 16 40

41 Quality control: Reconstruction artifacts H3K4me3 RNA Pol II DAPI y x Bad SI reconstruction Good SI reconstruction z x Microscopy Course Lecture 16 41

42 Quality control by Fourier analysis (FFT green) Bad SI reconstruction Good SI reconstruction z x Microscopy Course Lecture 16 42

43 Balance between contrast and bleaching Signal System sensitivity Excitation intensity Background Labeling density Camera noise Brightness Autofluorescence Unspecific / unbound label Stray light / Out-of-focus blur Bleaching (less than ±30-50%) Photostability Anti-fade Z-hight Discrete, isolated structures Restricted z-hight, low background Deconvolution Centrosomes 3D-SIM Tolerant to low intensities (>1.000 gray levels) EM 5MHz (gain 3000) Complex structures Extended z-hight, out-of-focus blur contribution Chromatin (DAPI) SI-raw 3D-SIM High intensities required (> gray levels) Conventional 5MHz Microscopy Course Lecture 16 43

44 How to get the best image? Quality is paramount Labeling Dyes (spectra, photostability) Labeling method (FPs, IF, FISH,...) Labeling specificity (antibodies) Signal-to-noise / background Sample Optical quality (coverslip, cleanness) Refractive index mismatch Embedding medium, RI immersion Imaging depth SI Postprocessing PSF/OTF (λ-, depth-, RI-dependent) Channel alignment (...) OMX Hardware Mechanical stability Photon efficiency SI modulation contrast Camera (CCD, EMCCD, scmos) Dataset x, y, z, λ,(t) Statistical analysis Quality control Colocalization (?) Segmentation Distance measurements (...) Microscopy Course Lecture 16 44

45 3D-SIM - the pros & cons + Multi-color, standard dyes (e.g., DAPI, Alexa, GFP...) + 3D with 2x resolution in XY and Z (8x volumetric) + Massive contrast improvement / high dynamic range + Z-sectioning over larger volumes (10 µm in z) + Sensitive (EMCCD/sCMOS) and fast (OMX Blaze) live cell imaging + Fast imaging of a large field of view (40 x 40 µm) o Only moderate lateral resolution improvement - Mathematical reconstruction artifacts Context Versatility - High requirements on sample quality and system calibration 45 Microscopy Course Lecture 16

46 No free lunch - trade-offs in super-resolution microscopy (Wide-field) Photon budget Spatial resolution (Contrast) (Confocal) STED ± 60 nm (xy) 3D-SIM ± 120 nm (xy); ± 300 nm (z) Multi-D: z-depth, λ, t Christian Eggeling Low Photodamage (Bleaching) Temporal Resolution (Speed) Localisation Microscopy The best SR-technique is determined by demands of the biological application Spatial resolution is only part of the equation ± 20 nm (xy accuracy); 50 nm (xy, structural res.) Rainer Kaufmann 46 Microscopy Course Lecture 16

47 SIM rocks 47 to Jürgen Neumann, Pete Carlton, Lothar Schermelleh for sharing slides Microscopy Course Lecture 16