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Molecular and technical advances for modern confocal imaging
Sponsored,vendor-submitted protocol   Sponsored by Olympus America Inc.    Published in BioTechniques Protocol Guide 2007 (p.65)


As well as the clear technological advances made in recent years, microscopy has also been significantly enhanced by the appearance of new fluorescent proteins. Applications such as FLIP, FRET and FRAP have all greatly improved our understanding of biological pathways. Further to this, new photoswitchable molecules have become available, which can be modulated using light of defined wavelengths, alternative to those used for imaging. This is therefore best achieved through the use of a secondary laser scanner such as the SIM scanner on the Olympus FluoView FV1000 confocal laser scanning microscope (cLSM), whilst the main scanner captures images to document the effects. The bleaching applications

Techniques like Fluorescence Loss In Photobleaching (FLIP) and Fluorescence Recovery After Photobleaching (FRAP), require bleaching of fluorescent molecules in target areas, to investigate intracellular protein movements. With FLIP, bleaching is applied throughout the experiment and the movement of molecules assessed by the resultant loss of fluorescence surrounding the target area, due to movement of molecules into the bleaching area. With FRAP, the target area is bleached for a fixed amount of time and the recovery of fluorescence is used to determine protein movements, mobility or dynamics. With the innovative SIM scanner module of the FV1000, photo-bleaching can be activated during ongoing image recording and therefore, even fast recovery reactions are not missed, as often occurs with standard single scanner cLSM set-ups. Magic Molecules

SIM scanner technology is also advantageous when using new fluorescent imaging molecules that give researchers selective control of the activation and deactivation of fluorescence by photo-manipulation. The fluorescence intensity of Dronpa1, (an advanced GFP-based molecule), for example, can be reduced by a strong 488nm laser and readily restored with a faint 405nm laser. As a result, repeated selective photoactivation experiments can be completed on the same cell providing excellent information about protein diffusion for example. Kaede,2, a recombinant fluorescent protein from Trachyphyllia geoffroyi, emits a green light under fluorescent illumination. Violet or UV illumination converts the fluorescence so that the molecules emit at a longer red wavelength. When violet/UV illumination is directed onto a Kaede-expressing cell, diffusion of the reddish Kaede can be monitored, providing an easy and accurate method to study dynamic processes in living cells (Image 1). Image 1.

Image 1. (Click to enlarge)

In a similar process, the 405nm laser can be used to activate (un-cage), caged derivatives of compounds such as ATP (Image 2), initiating physiological processes at defined cell locations.

Image 2.

Image 2. (Click to enlarge)

In summary

These amazing molecular and physiological processes can all be controlled very precisely using the SIM scanner on the Olympus FluoView FV1000. More importantly, this is all completed whilst the main scanner captures images to document the resultant changes. This combination of 2 scanners integrated within a cLSM provides researchers with an excellent tool for live cell biology.

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