Part 1 Principles
Confocal Microscopy tutorial
Part 2 application of confocal microscopy
5. Physiological study
Taking advantage of the high scan speed of line scan in confocal scanner and fast sampling speed on the PMTs (in contrast, digital camera has to take image in a frame of n x n matrix, the frame readout speed is limited), confocal microscope can be used to monitor the highly dynamic intra cellular events such as calcium release, concentration change of calcium and other ions like K, Na, Mg, Ze, PH change in the cytoplasm of a cell.
In terms of time resolution, the
Galvan-meter driven scanner scan up to 2000 lines per second, a resonance scanner
scan up to 8000 lines per second at bi-direction scan mode, which is fast enough
to monitor sub micro-second events. Compared to the fastest digital camera at 500 frames
per second, time resolution is 2 ms.
Since confocal microscope can use multiple PMTs as detector , simultaneously detecting signal from different emission channel is achieved without any time delay. Up to six detecting channels can be fitted to the single system. This is another advantage of confocal microscopy vs. digital camera based system. With UV laser installed, the ratio image of calcium can be easily acquired without need of extra device. In digital camera system, special configuration is needed for ratio image.
Taking advantage of the high intensity of laser, fast laser AOTF control and accurate scan register of confocal system, multiple ROI (region of interesting) scan can be configured on the same field with different excitation light on and off for overall background image and different ROIs. This makes point bleaching experiment, FRAP (fluorescence recovery after photo bleaching) and FRET (fluorescent resonance energy transfer) very easy to be implemented.
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