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 1 Principles of Confocal microscopy

1. Basics of conventional fluorescence microscope

In Fluorescence Microscopy, reflected light gives much better results, so almost all fluorescence microscope take this configuration.

The configuration of a reflected light fluorescence (also known as epi-fluorescence) microscope is schematically shown on the left:
The light from source (1) pass through neutral density filter, aperture, field stop (2,3,4) for Köhler illumination adjustment, reaches excitation filter(5). After filtering away unwanted wavelength,  excitation light goes further and reaches a special and important filter called beam splitter (6), BSP for short. The excitation light selected by BSP is reflected to the specimen via objective (7) and excites fluorophores within specimen (8). The fluorescence emission from the specimen comes back through the same path to BSP again. This time, the light should not be reflected away but passes through it and reaches emission filter(9). Being further filtered by emission filter, the emission light is either focused on the front focal plane of binocular or projected to infinite by tube lens in case of infinite corrected objectives(10). The final image is further magnified by binocular(11), detected by your retinal, or sent to a camera port, detected by devices coupled above the port: CCD camera or a photographic film camera.
Filters 5, 6, 9 are usually installed together into a cube, called filter-set. It is the pivotal part of fluorescence microscope and will be discussed more in next section: filter-set.


In a conventional reflected fluorescence light microscope, the light is usually from mercury arc lamp which provides a mixture of wave length from UV to red, like the figure on the left depicted. A emission filter is used to separate desired wavelength. Contrary  to common assumption, the peaks distributed mostly in UV and violet-blue area. For the frequently used fluorophore FITC, which need excitation at 490 nm, there is no beak but only continuous wave at less than 10% intensity. But this seems to be enough for FITC due to its high molar extinct coefficient and high quantum yielding.

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This page was last updated 23.03.2004