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

3. the differences between conventional and confocal microscope

Extended light versus point light source illumination

In conventional wide field microscope, ordinary extended light is used as light source, the specimen is lit laterally and vertically at the same time as shown in the illustration. The resulting image is affected by all the lit spots from the whole illuminated field, although it is centered at a given focal plane and local spot. These illuminated dots interfere with each other laterally and the stray light compromise image contrast. Image contrast, defined as the difference between the minimum and maximum intensity of two points in the image, is an important factor for an optical device to achieve its resolution, without proper contrast, the signal has little difference with background and the resolution of the an optical lens can not be realized. Improved contrast helps an optical device to reach its maximum resolution.

Whole length image versus optical section

If a point light source illumination is used, only one point in the specimen is lit at a time and the resulting image therefore is void of those lateral interference of dots in extended light illumination. But here the lit spots from above and under-focal plane still exists since light pass through it. The images from out-focal-planes overlaps on the focal plane, thus the image sharpness is compromised. To be worse,  weak signals from a given plane of the specimen can be totally un-detectable because they are buried within the mixture. This is  more dramatic in thick specimen where a sharp focus can not be achieved at all.

In another configuration, a plate with a small hole called pinhole is placed before the image detecting device like below:

In this configuration, light from under-focal-plane will be focused at a plane behind the pinhole such is blocked away by the pinhole plate. The light from above-focal-plane will be focused before the pinhole and is blocked away by the pinhole too. Only the light from focal plane is just focused at the pinhole thus can reach the image detector. This process simulates what you do with a microtome to cut some unwanted tissue away, but you do it here optically, this is so called "optical sectioning".

The size of pinhole determines how thick an optical slice will be. The smaller the pinhole, the thinner the slice. But the thickness will not go down indefinitely. It is also limited  by all those factors affecting resolution of the lens: the wave length of light, Numerical aperture of the lens, reflecting index of media, together with pinhole size, the z-resolution is usually 2 times worse than lateral resolution of an objective. For a lens of 1.4 NA, blue light at 488 nm, the lateral resolution is 200 nm, the achievable optical section thickness is about 400 nm.

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This page was last updated 23.03.2004