Super-resolution imaging: early days w/ Video-enhanced DIC, TIRF, PALM, STORM, etc.

Transcription

1 15/05/2012 Super-resolution imaging: early days w/ Video-enhanced DIC, TIRF, PALM, STORM, etc. Prof. Dr. Rainer Duden 1

2 Using conventional light microscopy resolution is limited to ~220 nm in X-Y and ~500 nm in Z The PSF problem = Punkt-Bild-Funktion

3 Visualization of small structures One can visualize very small structures (i.e. with diameters much less than Abbe s diffraction limit of ~250 nm) without actually resolving them!! [but only under special, favorable conditions]

4 Digitally-enhanced DIC video microscopy - an exiting 1980 s innovation Good for specimen with low contrast, like living cells or biochemical preparations Image is captured by TV camera; background is digitally subtracted Image is then tweaked by adjusting contrast Darkest part of image is made black Lightest part of image is made white All parts in between made shades of gray

5 Classic test specimen (diatoms) as a test for VE-DIC

6 Example: View by eye VE-DIC of isolated microtubule preps Analog Contrast Enhancement Live Image De-focus sightly; acquire Background Image and store into Frame Buffer subtract Background from Live Image at video rates Increase Contrast digitally 6

7 Dr. Ron Vale (UCSF), 2009 Keith Porter Lecturer at the 49th Meeting of the American Society of Cell Biology (ASCB) in San Diego

8 Actin filaments Cytoskeletal Tracks Microfilaments are found around the periphery of the cell and as stress Microtubules fibers. Microtubules are found in arrays radiating from the MTOC. Intermediate filaments Intermediate filaments are like cables stretching across the cell

9 Dr. Ron Vale (UCSF), 2009 Keith Porter Lecturer at the 49th Meeting of the American Society of Cell Biology (ASCB) in San Diego

10 2. fractionate/purify motility factors using microscopy as an assay Dr. Ron Vale (UCSF), 2009 Keith Porter Lecturer at the 49th Meeting of the American Society of Cell Biology (ASCB) in San Diego

11 Organelles move along curvi-linear tracks in squid axoplasm - what molecules are involved?

12 Dr. Ron Vale (UCSF), 2009 Keith Porter Lecturer at the 49th Meeting of the American Society of Cell Biology (ASCB) in San Diego

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14 Biochemical fractionation of motor activity using VE-DIC as a functional assay Cell 42, (1985)

15 Long-distance cargo transport: the tracks consist of microtubules & the ATP-dependent motor classes involved are kinesins and dyneins dynein dynein receptor vesicle

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17 Total Internal Reflection Fluorescence Microscopy (TIRF)

18 TIRF imaging of EB3-GFP (MT + ends)

19 VSV-G-tsO45-G: arrival at the cell surface widefield = total fluorescence TIRF = vesicle / PM pool Toomre et al. (2000) J. Cell Biol. 149, 33-40

20 VSV-G-tsO45-GFP widefield = total fluorescence TIRF = vesicle / PM pool Toomre et al. (2000) J. Cell Biol. 149, 33-40

21 VSV-G-tsO45-GFP widefield = total fluorescence TIRF = vesicle / PM pool Toomre et al. (2000) J. Cell Biol. 149, 33-40

22 VSV-G-GFP Surface arrival imaged with TIRF Toomre et al. (2000) J. Cell Biol. 149, 33-40

23 TIRF-M of VSV-G-GFP Surface plot NIH image of TIRF movie Toomre et al. (2000) J. Cell Biol. 149, 33-40

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27 Photo-switchable fluorescent proteins for use in super-resolution imaging Source: Trends in Cell Biology, Volume 19, Issue 11, Pages

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29 Fig. 1. The principle behind PALM Betzig et al., (2006) Science 313,

30 Movie illustrating PALM data acquisition taken from: Betzig et al., Science 313, (2006)

31 Comparative summed-molecule TIRF (A) and PALM (B) images of the same region within a cryo-prepared thin section from a COS-7 cell expressing lysosomal protein CD63 tagged with PA-FP Kaede Betzig et al. (2006) Science 313,

32 Fig. 3. Comparative summed-molecule TIRF (A), PALM (B), TEM (C), and PALM/TEM overlay (D) images of mitochondria in a cryoprepared thin section from a COS-7 cell expressing deosfp-tagged cytochrome-c oxidase import sequence Betzig et al., Science 313, (2006)

33 Super-resolution PALM image of a cell expressing farnesylated tdeosfp at the PM, rendered with z axis color-coding Source: Trends in Cell Biology, Volume 19 (11),

34 Survey of PA-FPs & FPs useful for PALM: Trends Cell Biol. 19,

35 Photo-switchable FPs and dyes Nat. Rev. Mol. Cell Biol. 9,

36 Stochastic Optic Reconstruction Microscopy (STORM)

37 STORM principle from: Zhuang WEBsite

38 Imaging of Microtubules and clathrin coated pits using STORM vs. confocal

39 Imaging of Microtubules and clathrin coated pits using STORM

40 Imaging of Microtubules using STORM vs. confocal

41 Imaging of Microtubules using STORM vs. confocal taken from: Huang, Bates, Zhuang (2009) Annu. Rev. Bochem. 78,

42 STORM imaging of nuclear envelope, microtubules and chromatin in a prophasic cell

43 Dual-color STORM imaging of immunostaining for mitochondrial outer membrane protein tom20 (left panel) and mitochondrial matrix protein Hsp60 taken from: Huang, Bates, Zhuang (2009) Annu. Rev. Biochem. 78,

44 Structured Illumination Surpassing the lateral resolution limit by a factor of two using structured illumination. Gustafsson (2000) J. Microsc. 198, 82. Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution. Gustafsson (2005) PNAS 102, nm fluorescent beads Resolution extension through Moire effect Actin in Hela Cell Sine wave pattern generated by a laser illumination captured at 5 phases and 3 angles and multiple Z sections Non linear pattern -by saturation

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48 CAVEATS! : Steps in Labeling of Samples for STORM Chemical fixation Permeabilization with mild detergent Blocking Incubation with primary antibody Washing Incubation with secondary antibody/antibodies congugated with reporter (fluorescent probe) Washing, optional counterstaining Mount and view

49 CAVEATS! : Chemical Fixation Antigenic sites are easily denatured or masked during chemical fixation Glutaraldehyde gives good fixation but may mask antigens, plus it is fluorescent Paraformaldehyde often better choice, but results in poor morphology, especially for electron microscopy May use e.g., 4% paraformaldehyde with 0.5% glutaraldehyde as a good compromise Permeabilization (necessary for antibody access!) may alter cellular structures, but many antigenic sites may still remain inaccessible Signal-to-Noise Problem!!

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51 On-going mission: create the perfect cell-friendly microscope for high resolution fluorescnce imaging Nature Methods 8(5), Eric Betzig, Janelia Farm, HHMI, USA

52 The photon budget - a huge problem for live cell imaging at higher resolution and/or speeds from: Planchon et al. (2011). Nat. Methods 8(5),

53 XO = excitation objective S = specimen EO = epiobjective for view finding SC = medium-filled specimen chamber DO = detection objective Planchon et al. (2011) Nat. Methods 8(5),

54 Planchon et al. (2011) Nat. Methods 8(5),

55 Physiology Summer Course 19/06-4/07, 2011

56 The Bessel beam microscope at Woods Hole MBL Dr. Liang Gao

57 Dynamics of Filopodia at apical cell surfaces

58 Long-term imaging of Mitochondria

59 Mitotic chromosome separation imaged using Histone-2BmEmerald

60 Fibroblast actin GFP cell3 rotate view