>
> Greetings,
>        A number of people seem to be using Cy5.  An investigator here wants
> to use it in a dual labeling experiment.  My concern is in the detection of
> the emission.  Our Olympus confocal uses a Hamamatsu R928 PMT with a
> spectral response curve that suggests 30-50% detection at the Cy5 emission
> peak of 680 nm.  Has anyone else had to deal with this problem?  Is it a
> problem?  What kind of filter setups are being used with Cy5?  Thanks for
> any help.
>
I cannot give you any hard and fast data, only personal experience of using
the dye. Our confocal has the same PMT and the results with Cy5 are
outstanding. We illuminate with the 647nm line of the ArKr laser. In
thick or opaque tissue, Cy5 gives surprisingly good signals for 3D
reconstructions: 90 degree rotations seldom looked so good with other dyes.
I'm not sure why, I can only speculate. Resolution should be theoretically
poorer at longer wavelengths, but in imaging deep into thick tissue,
its certain that the theoretical limits of resolution are never approached
due to the influencenumerous other factors such as spherical aberration.
 
One important factor to consider  is certainly the variation of light
scattering (for example, Rayleigh scattering) with the wavelength of light.
There are many different types of scattering,
and their combined effect is that the longer the wavelength of light, the
less absorption and polarisation of the light takes place. What this means
for microscopy (and for that matter astronomy) is that the amount of light
both transmitted to and returning from the point of focus is greater the
longer the wavelength used. This effect is extremely well documented and
has been exploited in infra-red DIC microscopy and infra-red astronomhas y
to obtain superior resolution over visible light analogies.
 
It would be great to hear others experiences of Cy5 because I'm just
fascinated by the image quality I can achieve with it.
 
Robert England
KEBO Lab
Sweden
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