Confocal imaging optically reduces out of focus light, thereby improving image contrast. FRAP (fluorescence recovery after photobleaching) is a technique to measure the binding and diffusion rates of proteins. FRET (Fluorescence Resonance Energy Transfer). FRET is a technique to detect protein interaction and formation of complexes between different proteins tagged with fluorescence markers. Confocal microscope is the best suitable microscope for these techniques.
The facility currently uses two confocal microscopes.
The Leica TCS spectrophotometer confocal inverted microscope is mainly used for optical sectioning in multiple channels, and time lapse experiments. Four lasers are available for imaging dyes in either the uv, green, red or infrared (laser lines: 351, 363, 488, 514, 568, 633 nm). The spectrophotometer feature of this instrument permits fine-tuning of the spectral band over which emitted fluorescence is collected, thereby permitting the microscope to be tuned to image a variety of dyes. Data collection is controlled by an NT-based PC. Images are collected by a PMT with 8 bit resolution level (256 grayscale levels). The Leica Confocal software may be used for creating overlays, and for simplified 3D volume rendering. Images may be exported to Adobe Photoshop, Scion Image and other programs accepting TIFF files.
The Zeiss 510 confocal inverted microscope is used mainly for FRAP, FRET and imaging of YFP and CFP. Three lasers are available for imaging green, red and infrared (laser lines: 454, 488, 514, 543, 633 nm). UV dyes (DAPI, Hoechst) may be imaged in suboptimal conditions excited with a laser line at 454 nm. The main advantages of this confocal microscope are 12 bit resolution, the fast scanning and the ability to select and bleach regions of interest of any shape. Data collection is controlled by an NT-based PC. Images are collected by a PMT with 12 bit resolution level (4095 grayscale levels). The LSM510 software may be used for creating overlays, projections and for quantification of intensities. Images may be exported to Adobe Photoshop, Scion Image and other programs accepting TIFF files.