Introduction to N-STORM

Transcription

1 Introduction to N-STORM Dan Metcalf Advanced Imaging Manager

2 Outline Introduction Principles of STORM Applications N-STORM overview

3 Biological Scale Mitochondrion Microtubule Amino Acid 1Å Kinesin 1nm 10nm 100nm 1µm 10µm 0.1mm 1Å 1nm 10nm 100nm 1 m 10 m 0.1mm Atomic Molecular Sub-cellular Super-Resolution Cellular Light Microscopy wikipedia.org/wiki/kinesin; cvcweb.ices.utexas.edu; Fotin et al., Nature 2004; hrsbstaff.ednet.ns.ca;

4 Biological Model Systems Understand molecular and biochemical reactions, behaviour, and interactions in as physiologically relevant system as possible Kines in Microt ubule Mitocho ndrion Cell models eg. HeLa or primary cells Small model organisms eg. Drosophila Large model organisms eg. mice In vitro cell free systems More physiologically relevant = more complex and technically challenging Correlate data across techniques and model systems to build up picture

5 Microscopy techniques Depth MP Confocal Speed Widefield SIM STORM Cost User-friendliness (robustness) Applications window (samples) Multi-colour & 3D capability Resolution

6 Outline Introduction Principles of STORM Applications N-STORM overview

7 Principles of STORM

8 Principles of STORM Conventional fluorescence Raw images STORM Image Localisation Stochastic Optical Reconstruction Microscopy = STORM Rust, Bates & Zhuang, Nat. Methods, 2006 Bates, Huang, Dempsey & Zhuang, Science, 2007

9 History of acronyms PALM GSDIM ipalm STORM dstorm DNA-PAINT qpaint sptpalm SAME Blinking fluorescence Multiple raw frames Localise & reconstruct DIFFERENT Labelling strategies Laser sequences

10 N-STORM Super-Resolution System

11 N-STORM Acquisition Sequence

12 Identify Molecules

13 40,000 frames, 1,502,569 localization points 500 nm 5 μm Microtubules

14 Widefield image Low laser power Overlays - Widefield

15 STORM acquisition 100 fold increase in laser power Overlays - Blinks

16 Overlays - STORM STORM image

17 Microtubules Widefield image STORM image

18 Clathrin

19 Clathrin

20 Clathrin

21 Outline Introduction Principles of STORM Applications N-STORM overview

22 Selected publication 1

23 Comparison of SIM and epi

24 Organisation of integrin - SIM

25 Organisation of integrin - STORM

26 Organisation of integrin - STORM

27 Organisation of talin & vinculin

28 Phosphoyrlation

29 Selected publication 2

30 qpaint (a) In DNA-PAINT, fluorescently labeled 'imager' strands (P*) transiently bind from solution to complementary 'docking' strands (P) attached to a target. Intensity vs. time traces show characteristic fluorescence on- and off-times

31 qpaint image With super resolution microscopy and qpaint analysis, researchers will be able to quantify individual molecules at specific locations in the cell. These images show varying copy numbers (shown are three, four, five, and six in the bottom images) of a protein residing in small so-called nuclear pore complexes that permit shuttling of various molecules in and out of the cell's nucleus. The arrows indicate pores with only a single protein that serve to calibrate the counting method. Credit: Wyss Institute at Harvard University.

32 Selected publication 3

33 Nuclear pore complexes Fig. 1.dSTORM of the NPC integral membrane protein gp210. (A) Comparison of widefield fluorescence (upper left corner) anddstorm image (lower right corner). gp210 proteins in nuclear envelopes isolated from Xenopus laevis oocytes were labeled by indirect immunofluorescence using the primary antibody X222 directed against an epitope located in the lumen of the nuclear envelope bordering the pore wall (Gajewski et al., 1996) and Alexa647 secondary antibodies. (B D) Higher magnifications of fluorescent circular structures to highlight the eightfold symmetrical arrangement of gp210 proteins in NPCs. (E) NPCs are generally seen as eightfold symmetrical ring structures in electron microscopy using negative staining. Scale bars: 1 μm (A), 250 nm (B), 150 nm (C E).

34 NPC diameters Distribution of gp210 and WGA in NPCs as revealed by dstorm. In each case two representative examples are shown to emphasize differences in labeling efficiency. (A) The integral membrane protein gp210 surrounding the NPC labeled by immunofluorescence with Alexa647. The cross-section profile (below) of the left ring structure yields a ring diameter of 146 nm. (B) Nucleoporins of the central channel labeled with WGA Alexa647. The cross-section profile of the left image yields a channel diameter of 40 nm. (C) Diameters were confirmed by double staining of gp210 and WGA-binding nucleoporins located in the central channel of the NPC. Here diameters of 152 and 35 nm were calculated from a cross-section profile of the left image for the outer and the inner ring, respectively. (D) Average values of outer ring and central channel diameters as calculated from 50 different NPC ring structures. The diameter of the gp210 ring structure was determined as 161±17 nm, and the diameter of the central channel to be 38±5 nm. In addition, the distribution of FWHM values extracted from the cross-sectional profiles for gp210 and WGA showed the FWHM to be 29±7 nm for gp210 and 15±4 nm for WGA. Scale bars: 150 nm (A,C) 50 nm (B).

35 NPC diameters Fig. 3.Image analysis of accumulated NPC dstorm data. (A) For gp210, 426 individual rings containing ~160,000 localizations were combined to yield an average diameter of 164±7 nm for the gp210 ring surrounding the NPC. (B) For WGA, 621 rings containing ~40,000 localizations were combined and a diameter of 41±7 nm was determined for the central channel of the NPC. (C) Superimposed image of both structures. Scale bars: 100 nm.

36 Multicolour NPC imaging Fig. 4.Two-color dstorm images of NPCs using WGA-ATTO520 and Alexa647-labeled secondary antibodies directed against an epitope of gp210 on the luminal side. (A) Comparison of conventional widefield fluorescence image (lower left corner) anddstorm image (upper right corner). (B D) Higher magnification reveals the typical eightfold symmetrical ring structure of gp210 proteins (violet) surrounding the NPC and N-acetyl glucosamine-containing nucleoporins in the central channel labeled with WGA-ATTO520 (green). Scale bars: 2.5 μm (A), 100 nm (B D).

37 Outline Introduction Principles of STORM Applications N-STORM overview

38 N-STORM Super-Resolution System Hardware 3 colour STORM 2D & 3D capability TIRF/HILO/Epi illumination EMCCD camera Software Low and high density algorithms Gaussian & cross visualisation Drift correction 3D and chromatic calibrations Molecule statistics

39 N-STORM overview Resolution dyes & labelling Depth & 3D strategies Astigmatism & HILO Multi-colour strategies N-STORM dye pairs Analysis Interpreting data

40 N-STORM overview Resolution dyes & labelling Depth & 3D strategies Astigmatism & HILO Multi-colour strategies N-STORM dye pairs Analysis Interpreting data

41 Sample Preparation - Dyes Dempsey et al., Nature Methods, 2011

42 Sample Preparation - Glass

43 Accuracy of Localisation

44 Microtubules Widefield image STORM image

45 Localisation Precisions

46 Data Quality Good localisation precision + Large number of molecules identified

47 Microtubules Widefield image STORM image

48 Sample Preparation - labelling Antibodies Fab fragments Nanobodies Direct conjugation Optimisation tips Use smaller labels Use dyes at 1:1 label

49 Drift Correction

50 N-STORM overview Resolution dyes & labelling Depth & 3D strategies Astigmatism & HILO Multi-colour strategies N-STORM dye pairs Analysis Interpreting data

51 Above Focus z (nm) 3D STORM (x, y, z) Above Focus In Focus 0 In Focus Below Focus Below Focus 2γ Tube Lens Cylindrical Lens DU-897 EMCCD Huang, Zhuang et al, Science (2008) Molecules localized in Z Molecules above focus maintain symmetry in Y Molecules below focus maintain symmetry in X Fitted to Gaussians similar to XY

52 3D STORM 600 z (nm) nm 5 μm Huang, Wang, Bates and Zhuang, Science, 2008

53 Image Acquisition

54 N-STORM overview Resolution dyes & labelling Depth & 3D strategies Astigmatism & HILO Multi-colour strategies N-STORM dye pairs Analysis Interpreting data

55 Option 1 find 2 or more dyes Dempsey et al., Nature Methods, 2011

56 Option 1 recommendations Channel nm laser Alexa 647 Cy5 Channel nm laser Alexa 568 Alexa 555 CF Biotium 555 meos2 Channel nm laser Not recommended GLOX buffer + MEA-HCL

57 Option 1 - Chromatic Warp Uncorrected Corrected

58 Option 2 - N-STORM Dye Pairs Composition Activator Reporter Antibody

59 Photo-Switchable Dyes Pairs Alexa405 Alexa647 CY2 Alexa647 CY3 Alexa647 3 kinds of Activator and 1 kind of Reporter are available

60 N-STORM Sequence Step 1: Excite Activator molecule to energize the Reporter to a ready state 561nm CY3 Alexa647 Target sample (molecule)

61 N-STORM Sequence Step 2: Excite Reporter and emitted light is captured by the camera. 647nm 670nm CY3 Alexa647 Target sample (molecule)

62 2 Colour STORM Clathrin Microtubules 1 μm Bates, Huang, Dempsey and Zhuang, Science, 2007

63 2 Colour STORM 200 nm Image by Harvard Univ.

64 200 nm 2 Color 3D STORM Clathrin Formin Wu et al., Nature Cell Biology, 2010

65 Multi-colour summary Option 1 different dyes Option 2 dye pairs Easy sample prep 2 colour imaging Channel 2 (561 nm) poorer quality Chromatic correction required Harder sample prep High quality 3 colour imaging No chromatic correction required Longer image acquisition Better resolution

66 N-STORM overview Resolution dyes & labelling Depth & 3D strategies Astigmatism & HILO Multi-colour strategies N-STORM dye pairs Analysis Interpreting data

67 ROI Statistics Molecule lists

68 Overlapping Peak Fitting Original data Detected molecules with conventional fitting Detected molecules with Fit Overlapping Peaks Denser samples &/or wider range of fluorophores

69 Overlapping Peak Fitting 351,560 mol. 1,065,088 mol.

70 Summary Introduction Principles of STORM Applications N-STORM overview