Olympus FV1000 manual Laser Light Stimulation, FLIP-Fluorescence Loss in Photobleaching

Laser Light Stimulation

The SIM scanner system combines the main scanner with a laser light stimulation scanner.

Control of the two independent beams enables simultaneous stimulation and imaging, to capture reactions during stimulation.

Multi-stimulation software is used to continuously stimulate multiple points with laser light for simultaneous imaging of the effects of stimulation on the cell.

FLIP—Fluorescence Loss in Photobleaching

Fluorescence loss in photobleaching (FLIP) combines imaging with continuous bleaching of a specific region to observe the diffusion of a target protein within a cell. The changes in the image over time make it possible to observe the location of structural bodies that inhibit the diffusion of the molecule.

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Time (ms)

Specimen: HeLa cell, GFP (free), 488 nm excitation (multi-argon laser)

Image acquisition time: 100 ms/ bleach time: 100 s continuously, 405 nm bleaching

FRAP—Fluorescence Recovery after Photobleaching

Exposure of fluorescent-labeled target proteins to strong laser light causes their fluorescence to fade locally. Fluorescence recovery after photobleaching (FRAP) is used to observe the gradual recovery of fluorescence intensity caused by protein diffusion from the area surrounding the bleached region. By examining the resulting images, it is possible to characterize the diffusion speed of the molecule, and the speed of binding and release between the molecule and cell structures.

intensity		Example: Fluorescence recovery without interactions
		its fluorescence at a high speed due to Brownian motion.
		If the protein can freely diffuse, the bleached region recovers
Fluorescent	Time

Specimen: Hippocampal neurons, Shank-GFP stain, 488 nm excitation (multi-argon laser)

Image acquisition time: 100 ms Bleach time: 80 ms, 488 nm excitation (Sapphire 488 laser)

Data courtesy of: Dr. Shigeo Okabe

Department of Anatomy and Cell Biology, Tokyo Medical and Dental University

Fluorescent intensity

Time

Example: Fluorescence recovery with interactions

If the protein is strongly bound to a structure or forms part of a large protein complex, the bleached region recovers its fluorescence at a slower rate relative to the unbound state.

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					Time (ms)

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