Optogenetics combined with high pressure freezing
Traditionally electron microscopy only captures a "snap-shot" of a cell. But a snap-shot can be deceiving. Consider for a moment this photo. Is he falling? Floating? Or flying?
To understand dynamics of any process, one must know what came before and what came after. Looking at the micrograph below one might argue what is this particular cell doing.
Fig. 1: Micrograph showing fusing vesicles in the active zone. Arrow indicated fusing vesicle at a specific time after light stimulation –15 ms. PSD, postsynaptic density.
Is a vesicle fusing? Or is the cell removing membrane from the surface? What is the sequence of events that takes place in the particular cell? Such information cannot be extracted from random static images. However, by collecting images at a later time point (at 30 ms), we can demonstrate that the cell is in fact expelling the contents of vesicles.
Figure 2: Micrograph showing fusing vesicles in the active zone. Arrow indicated fusing vesicle at a specific time after light stimulation –30 ms.
Employing optogenetic tools a flash of light can trigger a dynamic cellular event. For example, by using the light activated ion channel (channelrhodopsin) a synaptic transmission can be stimulated in a neuron. By alignment of the triggered light stimulation with high-pressure freezing, regulated vesicle fusion and the subsequent vesicle recycling at synapses can be subsequently visualized in electron micrographs. By analyzing a sequence of images at specific time points, the progression of events can be assembled.
Flash-and-freeze approach can be expanded to other cellular events. A variety of light-sensitive tools are becoming available to stimulate particular cellular activities.
Optogenetics in combination with high pressure freezing adds another dimension to electron microscopy – time.
…. And if you are still curious, the ski acrobat was in fact flying.
References
- Watanabe S, Rost BR, Camacho-Pérez M, Davis MW, Söhl-Kielczynski B, Rosenmund C and Jørgensen EM: Ultrafast endocytosis at mouse hippocampal synapses. Nature 504: 242–47 (2013); doi: 10.1038/nature12809.
- Watanabe S, Davis MW and Jørgensen EM: Flash-and-Freeze Electron Microscopy: Coupling Optogenetics with High-Pressure Freezin. Neuromethods 84: 43–57 (2013); doi: 10.1007/978-1-4614-9179-8_3.
