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

1. Introduction

In practical, almost all specimen for confocal microscope are fluorescent specimen, but don't assume confocal microscopy as a "high quality image fluorescence microscopy". Confocal microscope is not always the best tool for fluorescence detecting.

Confocal microscope is capable of detecting multiple fluorescence channel simultaneously or sequentially, but do not take confocal microscope simply as a tool for co-localization study.

These are two common misconception about confocal microscopy.

As discussed in the first part of this tutorial, the true power of confocal microscopy lies on its strong suppression of out-of-focal-plane signals, namely, the depth discrimination or optical sectioning. The major applications of confocal microscopy rooted from this feature including:

  • Tomographic view of the specimen (microscopic CT)

  • 3-D reconstruction from z-section series.

  • Resolve thick specimen.

The feature of multiple channel simultaneous detecting without any time delay benefits applications including:

  • Physiological studies such as monitoring intracellular ion concentration change where timing really make sense.

  • Living cell imaging of multi-labeled marker for highly dynamic morphological events.

The effect of suppressing stray light improves confocal image quality,  but this effect is not always apparent on every case, and can be compromised by its vulnerability to SNR in case of weak, noisy signal.

Other advantages of confocal come from the use of laser for its mono-chromatism, coherent and high intensity, use of PMT for its high sensitivity, high dynamic range and wide adjustable gain and offset control, use of powerful digital image processor and post-acquisition manipulation of raw image data. All these have nothing to do with confocal effects, but they are indispensable parts of confocal system and add extra power to confocal microscopy.

Simply taking, confocal microscopy is not the first choice for ordinary fluorescence detecting nor for simple co-localization of multi fluorescence labeling. These tasks can be easily done by digital camera based conventional fluorescent microscopy without the bother of setting up loads of parameters and go through lengthy image optimization, and bleach signal away before getting a satisfactory image: a frustrating process if the specimen does not have a high SNR required by confocal microscopy, as discussed in part 1: optical sectioning.

The frequently-heard notion "the fluorescence in my specimen is weak and noisy, I want to get a good picture from confocal microscope" is a common misconception. You might get a even worse picture in this case. For details, please refer the following section: fluorescence detecting in confocal system.

To help users understanding how these features are in practical use, examples are given below in different categories. As you can see, most of the specimen are stained by fluorescence dyes, but keep in mind, the purpose here is not simply for detecting fluorescence.

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This page was last updated 23.03.2004