> Hugo Dilhuydy wrote:
>
>I NEVER use the HeNe laser for confocal fluorescence imaging. I am running
a
>Zeiss LSM410 with 543 HeNe laser + 488/568/647 ArKr laser. Why would you
use
>a weak "peak mismatched" 543 laser to excite green or red fluos when you
>have powerful 488 and 568 lines? Actually i use the HeNe laser only to get
a
>transmission image to be able to superimpose morphology to fluorescence
>because the contrast image is slightly better with the 543 line.


There are a few reasons why A Green HeNe should be considered. As has been
pointed out the Green HeNe is very cheap and extremely reliable especially
in comparison Kr/Ar lasers.

But there are some other points that should be considered. I don't think it
is accurate to refer to the 543 line as being "mismatched". If we consider
the excitation and emission spectra of the commonly used green fluorescing
dye TRITC for example.

I suggest that when choosing dye and laser combinations, everyone use the
following site to look at the spectra of the dyes. This is a fantastic tool
no matter if you have a Bio-Rad system or not.

http://FLUORESCENCE.BIO-RAD.COM/

From the spectra and we can see that:
   - the 543 line will excite TRITC with an efficiency of  ~ 85%.
   - the 568 line will excite TRITC only with an efficiency of
     about 45%.

   - in addition the 568 line is well into the EMISSION spectra of
     TRITC it is actually quite close to the peak emission. Hence
      a longer barrier filter must be used than would normally used
      if the shorter 543 line was used. Therefore when exciting TRITC
      with the 568 line we can only collect the tail of the emission
      spectra.

Using Bio-Rad's filter spectra also shown on
http://FLUORESCENCE.BIO-RAD.COM/  .

Look at these examples
With 543nm excitation is ~ 85% efficient. We can use either a 570DF40 or
580DF32 band pass filter to collect the bulk of the emitted light (about
80%).

With 568nm excitation is ~45% efficient. We have to use barrier filters that
are longer to cut out the longer laser line. eg. 585LP long pass filter will
only collect about 30% of the TRITC signal.

I suggest that an Ar ion/ Green HeNe/ Yellow HeNe combination would be
better for the common FITC/ TRITC/ Texas Red combination. I've used Ar ion
and Green HeNe lasers and can recommend them. However I've never used a
Yellow HeNe (594nm) so cannot give any recommendations regarding its
reliability. I'd like to hear from anyone who has used one.

Stephen H. Cody,
Colon Molecular and Cell Biology Laboratory,
Ludwig Institute for Cancer Research,
Post Office Royal Melbourne Hospital,
Parkville, Victoria 3050, Australia.

Tel: 61 3 9341 3155   Fax: 61 3 9341 3104
email: [log in to unmask]
www.ludwig.edu.au/confocal