Part 1 Principles
1. Fluorescence microscope
2. Filterset in FL-Mic
3. How concocal differs?
. What is confocal?
5. Resolution in confocal
6. Optical sectioning
7. Confocal image formation
    and time resolution
8. SNR in confocal
9. Variations of confocal

10. Special features from
     Leica sp2 confocal

Part 2 Application
1. Introduction
2. Tomographic view
    (Microscopical CT)

3. Three-D reconstruction
4. Thick specimen
5. Physiological study
Fluorescence detecting
       General consideration
Multi-channel detecting
       Background  correction
       Cross-talk correction
            Cross excitation
            Cross emission
            Unwanted FRET

Part 3 Operation and

 1. Getting started
 2. Settings in detail
     Laser line selection
      Laser intensity and 
         AOTF control

      Beam splitter
      PMT gain and offset 
      Scan speed
      Scan format, Zoom
        and Resolution

     Frame average, and
         Frame accumulation
     Pinhole and Z-resolution
     Emission collecting rang
        and Sequential scan

When Do you need confocal?
Are you abusing confocal?

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.
But it should be pointed out, in frame mode, digital camera has much higher speed than confocal system (refers to confocal image generation). It is a matter of value speed more or image quality more. Digital camera has a better balance in this regard.

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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This page was last updated 23.03.2004