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


Q1. When do I need confocal microscope for my fluorescence specimen?
    A1: see here.

Q2. Why my specimen looks different under confocal compared to under conventional microscope?
    A2: That is quite normal in confocal microscopy. Due to the Optical sectioning effect of confocal, you see only one layer of your specimen under confocal. The thickness of that layer under confocal, depending on the pinhole size, laser wavelength and objective NA you choose, is generally at range between 500-800 nm. But your specimen, even if it is a monolayer of cultured cell, has total thickness of 10-20 m. What you see is only 1/20 or less of your cell.
In conventional wide field microscope, you see an overall overlapped image from top to bottom.
Unless your cell or specimen is homogeneous in size, shape, or in internal structure along its whole z-direction, you should see some difference.
But if you try to roll the focus through the whole z-axial, you can encounter all the structures you see under conventional microscope, thought they are not necessarily in the same plane.

Q3. Why the picture looks even worse under confocal compared to picture under conventional FL-Mic?
    A3: Yes, that is a common phenomena or a problem of confocal. Confocal effects comes from pinhole and its massive rejecting out-of-focal-plane signal. The total signal reaches PMT detecter is less than 1/20 of the total  signal as mentioned in A2 above. The reduced signal influx, leads to not only weakened intensity, but also deteriorated SNR (Signal to Noise Ratio) according to the formula , see section SNR in confocal. The weakened signal can be amplified by increasing PMT gain, the deteriorated SNR, however, can not be compensated by PMT amplification at all. It has to be compensated by using approaches which increase signal influx such as: more average or accumulation, lower scan format for bigger pixel size, slower scan speed, bigger pinhole, etc.. (See related section in this tutorial). If all these fail, that means your specimen is not suitable for confocal. Your specimen does not have enough signal intensity, or more important, enough SNR to tolerate the massive signal rejecting from confocal.

Q4. Which laser line I can use if there is no exact match of laser line with the excitation peak value of my fluorophore?
    A4: Check Table 1, choose laser line exciting your fluorophore at the highest percentage. Usually, 50-80% is very good. If not available,  25%-50% is acceptable, but you might need higher AOTF, laser power or PMT setting. If there is similar percentage at different laser lines, lower wavelength usually gives out better excitation.

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This page was last updated 23.03.2004