Fluorescent microscopy of living cells
| Authors | |
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| Year of publication | 2007 |
| Type | Conference abstract |
| MU Faculty or unit | |
| Citation | |
| Description | Fluorescent protein technology has started by using aequorea victoria fluorescent protein derivatives. These have become the essential tools for the cell biology research. Enhanced green fluorescent protein (EGFP) has been mutated to cyan (ECFP) and yellow (EYFP) fluorescent variants, to allow the simultaneous observation of several protein fusions in live cells especially for co-localization studies and to use the biophysical properties of these fluorophores to detect protein-protein interactions and conformational changes by fluorescence resonance energy transfer (FRET) technique. The discovery and development of red fluorescent protein variants have led to fluorophores that can be distinctly imaged together with other fluorescent proteins to achieve two- or three-color imaging. The practical use of these fluorophores has many challenges: photobleaching of these proteins; signal bleeding due to overlapping emission/excitation spectra; and relatively low amount of fluorescent signal from some of the proteins. Time-lapse microscopy of living cells is especially sensitive to photobleaching due to frequent exposures of sample, cells viability on microscopic stage, and cell movement and changes. In this presentation, we will discuss various experimental methods used in living-cells fluorescent microscopy and we will also discuss numerous challenges and approaches to image analysis of obtained image data. This work is supported by the Grant Agency of the Czech Republic (grant number 204/03/D031) and by The Ministry of Education, Youth and Sports of the Czech Republic (project numbers MSM0021622419 and LC535). |
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