Light Stimulation and High Pressure Freezing
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1 Light Stimulation and High Pressure Freezing Dr. Guenter Resch Leica Beijing HPF Workshop, October 2014
2 Contents Optogenetics Light Stimulation on Leica EM PACT2 Light Stimulation on Leica EM HPM100 Application Example Summary
3 Section 1 Optogenetics
4 Optogenetics Rationale Many biological processes are highly dynamic (millisecond range) Consequences for visualisation with electron microscopy: Chemical fixation Bad temporal resolution due to slow action of fixatives High pressure freezing Bad temporal resolution due to transfer to instrument Light Stimulation HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 4 / 23
5 Optogenetics Rationale Many biological processes are highly dynamic (millisecond range) Consequences for visualisation with electron microscopy: Chemical fixation Bad temporal resolution due to slow action of fixatives High pressure freezing Bad temporal resolution due to transfer to instrument Trigger for precise control of events combined with instantaneous fixation Light Stimulation HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 4 / 23
6 Optogenetics Rationale Optogenetics: light pluses can selectively activate/inactivate cellular events via light activated ion channels (channelrhodopsin) caged neurotransmitters caged second messengers photo-switchable ligands photoactivatable proteins Light Stimulation HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 5 / 23
7 Optogenetics Combining Optogenetics with HPF HPF method of choice for fast fixation of tissues Combination of timed light pulses and freezing in high pressure freezers Neurobiology (ion channels, neurotransmitters,... ) Light sensitive cells (cones, rods) Cytoskeletal reorganization (second messengers) Membrane trafficking Light Stimulation HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 6 / 23
8 Section 2 Light Stimulation on Leica EM PACT2
9 Light Stimulation on Leica EM PACT2 Shigeki Watanabe/Jorgensen Lab Watanabe et al. (2014) Neuromethods Modification of the Leica EM PACT2 specimen pods/bayonet Commercially available from Marine Reef International Leica Microsystems Light Stimulation HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 8 / 23
10 Light Stimulation on Leica EM PACT2 Shigeki Watanabe/Jorgensen Lab Watanabe et al. (2014) Light Stimulation HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 9 / 23
11 Light Stimulation on Leica EM PACT2 Shigeki Watanabe/Jorgensen Lab Watanabe et al. (2014) Light Stimulation HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 9 / 23
12 Section 3 Light Stimulation on Leica EM HPM100
13 Light Stimulation on Leica EM HPM100 Features Optional accessory from Leica Temporal resultion of 5 ms Compatible with many different light sources Leica Microsystems Light Stimulation HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 11 / 23
14 Light Stimulation on Leica EM HPM100 Integration into HPM100 I Optical fiber connected directly with high pressure chamber I Transparent upper/lower halves I Sample between sapphire discs I Light source and control box Leica Microsystems Light Stimulation HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 12 / 23
15 Light Stimulation on Leica EM HPM100 Integration into HPM100 Optical fiber connected directly with high pressure chamber Transparent upper/lower halves Sample between sapphire discs Light source and control box Leica Microsystems Light Stimulation HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 12 / 23
16 Light Stimulation on Leica EM HPM100 Integration into HPM100 Optical fiber connected directly with high pressure chamber Transparent upper/lower halves Sample between sapphire discs Light source and control box Leica Microsystems Light Stimulation HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 12 / 23
17 Light Stimulation on Leica EM HPM100 Integration into HPM100 Optical fiber connected directly with high pressure chamber Transparent upper/lower halves Sample between sapphire discs Light source and control box Leica Microsystems Light Stimulation HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 12 / 23
18 Light Stimulation on Leica EM HPM100 Integration into HPM100 Optical fiber connected directly with high pressure chamber Transparent upper/lower halves Sample between sapphire discs Light source and control box Leica Microsystems Light Stimulation HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 12 / 23
19 Light Stimulation on Leica EM HPM100 HPM100 Light Stimulation Setup Leica Microsystems Light Stimulation HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 13 / 23
20 Light Stimulation on Leica EM HPM100 Triggering Light Pulses Automatically via freeze button Manually via control box (buttons or external, programmatic control for stimulation and freezing) Light Stimulation HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 14 / 23
21 Section 4 Application Example
22 Application Example Watanabe et al. (2013) Light Stimulation HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 16 / 23
23 Application Example Scientific Question How are synaptic vesicles (membrane and proteins) recycled to sustain neurotransmission? Clathrin-mediated ( 20 s) Kiss-and-run ( 1 s)... Light Stimulation HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 17 / 23
24 Application Example Method Cultured mouse hippocampal neurons expressing channelrhodopsin were stimulated optically after being kept in dark (blue light, 10 ms) Frozen in EM HPM100 with light stimulation at different time intervals (15 ms - 10 s) Freeze substitution, ultrathin sectioning, TEM/tomography Statistical evaluation of vesicles at synaptic membrane Light Stimulation HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 18 / 23
25 Application Example Results Watanabe et al. (2013) Light Stimulation HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 19 / 23
26 Application Example Results Watanabe et al. (2013) Light Stimulation HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 19 / 23
27 Application Example Results Watanabe et al. (2013) Light Stimulation HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 19 / 23
28 Application Example Results Main peak of endocytosis after 100 ms in lateral regions Timing incompatible with clathrin mediated endocytosis Timing and location incompatible with kiss-and-run model Ultrafast endocytosis as primary mechanism of recycling Light Stimulation HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 20 / 23
29 Section 5 Summary
30 Summary Recommended Reading Watanabe S, Davis MW and Jorgensen EM: Flash-and-Freeze Electron Microscopy (2014): Coupling Optogenetics with High-Pressure Freezing. Neuromethods 84. Watanabe S, Rost BR, Camacho-Perez M, Davis MW, Söhl-Kielczynski B, Rosenmund C and Jorgensen EM (2013): Ultrafast endocytosis at mouse hippocampal synapses. Nature 504. Light Stimulation HPF Workshop, Oct 2014 (C) Nexperion e.u., 2014 Slide 22 / 23
31 Thank you for your attention. Nexperion e.u. Solutions for Electron Microscopy nexperion
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