Correlative Light/Electron Microscopy

Transcription

1 Correlative Light/Electron Microscopy Dr. Guenter Resch Leica Beijing HPF Workshop, October 2014

2 Contents Overview Leica EM PACT2 CLEM Workflow Leica EM HPM100 CLEM Workflow Leica Cryo-LM Stage

3 Section 1 Overview

4 Overview Imaging Modalities Widefield light microscopy Total internal reflection microscopy Super-resolution light microscopy X-ray tomography Scanning EM Transmission EM AFM... Each modality has its specific advantages and drawbacks CLEM HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 4 / 27

5 Overview Correlative Microscopy Correlative microscopy is the inspection of an identical sample (cell) with two or more modalities. CLEM = Correlative Light and Electron Microscopy (TEM and SEM) Not a single technique, but a collection of many different approaches CLEM HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 5 / 27

6 Overview Advantages and Drawbacks of LM + Quick overview + Live samples, dynamics + Fluorescent dyes or endogenous tags (GFP) -- Resolution -- Context (fluorescence) CLEM HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 6 / 27

7 Overview Advantages and Drawbacks of EM -- Lack of overview -- Fixed (and dehydrated) specimens + Resolution + Cellular context IMP-IMBA Research Centre, Vienna CLEM HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 7 / 27

8 Overview Correlative Pre-Fixation LM/EM LM observation Fixation and EM preparation EM observation Wait for event Observe dynamics CLEM HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 8 / 27

9 Overview Correlative Pre-Fixation LM/EM: Challenges Time resolution observation fixation Yield Relocation of feature (Alignment of images) CLEM HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 9 / 27

10 Overview Correlative Post-Fixation LM/EM Fixation and EM preparation LM observation EM observation Localisation of sparse features ( needle in haystack ) Functional context of feature Overlay of fluorescent signal CLEM HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 10 / 27

11 Overview Correlative Post-Fixation LM/EM: Challenges Signal compatible with LM and EM Preservation of fluorescence through EM preparation Preservation of specimen Imaging quality and flexibility Relocation of feature Alignment of images CLEM HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 11 / 27

12 Section 2 Leica EM PACT2 CLEM Workflow

13 Leica EM PACT2 CLEM Workflow Workflow Samples grown on sapphire discs with index pattern EM PACT2 RTS designed to shorten transfer times Paul Verkade Thomas Müller-Reichert Special transfer holder on light microscope Cveta Tomova, Leica Microsystems CLEM HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 13 / 27

14 Leica EM PACT2 CLEM Workflow Workflow Samples grown on sapphire discs with index pattern EM PACT2 RTS designed to shorten transfer times Paul Verkade Thomas Müller-Reichert Special transfer holder on light microscope Leica Microsystems CLEM HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 13 / 27

15 Leica EM PACT2 CLEM Workflow Preload Rapid Transfer System Leica Microsystems CLEM HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 14 / 27

16 Leica EM PACT2 CLEM Workflow Live Cell Carrier Leica Microsystems CLEM HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 15 / 27

17 Leica EM PACT2 CLEM Workflow Live Cell Carrier Leica Microsystems CLEM HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 15 / 27

18 Leica EM PACT2 CLEM Workflow Light Microscopy Live cell carrier mounted in CLEM rapid loader Specimen in petri dish with glass bottom Leica Microsystems CLEM HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 16 / 27

19 Leica EM PACT2 CLEM Workflow TEM Preparation Quick transfer of sample to EM PACT2 RTS Freezing (5 s) Freeze substitution Target trimming TEM Leica Microsystems CLEM HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 17 / 27

20 Section 3 Leica EM HPM100 CLEM Workflow

21 Leica EM HPM100 CLEM Workflow Overview Live sample on sapphire discs 3 mm ( Monday) and 6 mm system available Sapphire discs with pattern for relocation Readyly assembled for freezing Leica Microsystems CLEM HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 19 / 27

22 Leica EM HPM100 CLEM Workflow Overview Live sample on sapphire discs 3 mm ( Monday) and 6 mm system available Sapphire discs with pattern for relocation Readyly assembled for freezing Cveta Tomova, Leica Microsystems CLEM HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 19 / 27

23 Leica EM HPM100 CLEM Workflow Components of CLEM System Cveta Tomova, Leica Microsystems CLEM HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 20 / 27

24 Leica EM HPM100 CLEM Workflow 6 mm CLEM System Cveta Tomova, Leica Microsystems CLEM HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 21 / 27

25 Leica EM HPM100 CLEM Workflow 3 mm CLEM System Cveta Tomova, Leica Microsystems CLEM HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 22 / 27

26 Leica EM HPM100 CLEM Workflow Light Microscopy Only dry objectives, minutes Andres Kaech, University of Zurich -- Cveta Tomova, Leica Microsystems CLEM HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 23 / 27

27 Leica EM HPM100 CLEM Workflow Light Microscopy Only dry objectives, minutes Hela GFP-Tubulin, H2B mcherry; Andres Kaech, University of Zurich CLEM HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 23 / 27

28 Leica EM HPM100 CLEM Workflow TEM Preparation Andres Kaech, University of Zurich -- Cveta Tomova, Leica Microsystems Locate certain feature, wait for certain stage Quick transfer of sample to EM HPM100 <Video 1:55> Freezing (min. 7 s) Freeze substitution Target trimming TEM CLEM HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 24 / 27

29 Section 4 Leica Cryo-LM Stage

30 Leica Cryo-LM Stage Separate Presentation CLEM HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 26 / 27

31 Thank you for your attention. Nexperion e.u. Solutions for Electron Microscopy nexperion