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

2. Tomographic view of specimen

The depth discrimination feature of confocal microscopy enables user to look specimen along certain axis, usually Z-axis, slice by slice at a chosen distance interval to reveal the structural differences or fluorescence distribution differences inside the specimen. Somehow like a Röntgenist examines patients with CT: "computerized X-ray tomography", the tomography of a cell or thick tissue can be obtained in the similar way. That is why people sometimes refer this as "Microscopic CT", although not accurate as a full description for all the function of confocal microscopy, it is very suitable for this application. This function is very useful especially when you think the spatial distribution of the structures make sense in addition to its positive staining, even if you are not interested to get a Z-series for 3D reconstruction at all.

The four pictures below are taken from the same cell at different Z-position from top to bottom untill the cell attachment plane. It reveals differences in both internal structures or cell surface features among optical sections which can not be seen in normal wide field microscopy.

Not just the structure of a single cell, the relationship of adjacent cells at different plane can also be viewed clearly like below:


This technique can also be used to differentiate whether a diffused staining is membrane staining and pan-cytoplasm staining.
The left picture shows a staining pattern which is difficult to tell its nature. Cutting it deeper along Z-axis reveals a circular profile which enables you say with sure this is a membrane staining.


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This page was last updated 23.03.2004