DESIGN & INSTALLATION OF AN ATTACHMENT FOR GFP PHOTOBLEACHING

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(Supported by the NIH/NCRR/P41 RR01219 grant and by NIH GMS 40198 to C. Rieder). The routine ability to expresses GFP-tagged fusion proteins in cells promises to become an important tool for understanding the function of many new proteins identified by the various genome projects. This year we improved our low light level fluorescence imaging system so that it can be used to conduct GFP (and other fluoropore) photobleaching studies. To do this we purchased an inexpensive Uniphase multi-line Argon laser and modified the microscope so that the beam of this laser could be directed through the epi-port and then focused onto the specimen through the objective. As a result we can now create a 1-2 (m diameter circular photobleached pattern in any cell containing GFP or other suitable fluorophores. This enhancement provides the capacity to measure the motility of GFP-molecules, and their turnover, using standard fluorescence recovery after photobleaching (FRAP) methods. This im provement was driven by the in-house need to selectively photobleach centrosome-associated GFP-labeled gamma tubulin.