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

6. fluorescence detecting in confocal microscopy

    Cross-talk correction 2: cross emission (emission bleeding through)

When emission spectra of two fluorophores overlaps, emission from one channel will extend to another channel. Unfortunately, many commonly used fluorophore pair have more or less overlapped emission spectra which pose the problem of emission bleeding through in most multi-labeling application. For example, FITC and TRITC, DAPI and FITC are the notorious pairs of cross-emission, like depicted below.

DAPI-FITC emission

FITC-TRITC emission

As the curve shows: considerable portion of the FITC emission is from DAPI,  and so does TRITC from FITC, and vice versa. Cross emission causes false positive co-localization and false high intensity of the emission.

If you want to eliminate the overlapped emission, it is extremely harmful for the second emission because only a small portion is truly unique.
The following images taken from specimen of FITC or TRITC single stained fluorescence beads. It shows how the data can be erroneous collected and how results can be mis-interpreted, and the final effect after cross-talking correction by adjusting laser intensity, gain and offset on PMT.

This is FITC fluorescence beads excited by 488 nm laser line, showing considerable amount of bleeding-through into red channel and present a  false co-localization in overlay image

Image after applying correction, red emission bleeding from FITC is eliminated, no false co-localization exist anymore.



    This is Rhodamine fluorescence beads excited by 568 nm laser line, showing considerable amount of bleeding-through into green channel and a  false co-localization in overlay image

          Image after applying correction, green    emission bleeding from Rhodamine is eliminated, there is no false co-localization in overlay image anymore

The correction on above image can be done easily and effectively because there is only one fluorescence staining existing. By using single-stained specimen, you know for sure any signal in other channel is false and should be removed. It serves you as reference to adjust laser intensity, gain and offset level of PMT to get rid of emission bleed-through.

 When two fluorophores co-existing in specimen and  two excitation lasers are used simultaneously, the situation become more complicated. There is no reference for you to make a clear judgment that how much is the bleeding-through from another channel. Indiscriminately reducing laser intensity or change detector gain and offset will also reduce the intensity of specific signal.

Whenever possible, in multiple labeling experiment, single stained specimen has to be prepared and used as reference for cross talking correction.

Besides adjusting laser intensity, gain and offset of PMT, there is an additional  way in Sp2 confocal microscope for cross-emission correction: moving or narrowing the detecting range of the spectrometer to avoid collecting those overlapped spectra area according to the knowledge of how much they overlapped. Here the advantage of spectrometer based system become more apparent.

If the cross-talking can not be removed successfully by those above-mentioned methods, or the signal become very weak after an aggressive suppression because your specimen do not have enough intensity for the removal, sequential scan has to be used.

In sequential scan mode, you can use single excitation at a time and collecting wider range since there is no emission from another channel. The consecutive collected data will be added together for overlay as they were collected in a single scan, although there is a short time delay.

Home |  facilities |  services |  reservation |  confocal tutorial |  other tutorial |  personnel |  link | 
  Statement about this web and tutorial
For problems or questions regarding this web contact e-mail:

This page was last updated 23.03.2004