David,

Unfortunately is the intensity of your signal (and the sensitivity of your camera) that dictate your exposure time.

I agree 2 seconds is a pretty long time and, together with the bleaching you mention, I would be also concerned with some bleed-through  of the green in your red (of course the 594 is far from the 488, but if the signal in green is strong this is possible).

I imagine that you already have the sections so increasing the concentration of the fluorophore is not possible. Can you bin the pixels in your camera? A 2x2 binning would reduce 4 time your exposure time, off course decreasing the resolution of your images.

Another possibility is to go higher with the magnification, ideally using a oil objective, this should give you a better signal, but I am not sure is pratical for your tissue sections.

hope this help.

Good Luck

Max

 Hi Max,

Thanks for your reply.  As I say, its only for a small minority of images. The only thing I can think of is that the high signal at 594 occurs on some of my sections when have been slightly creased or folded over during the mounting and coverslipping procedure.

I already went through the exposure time settings with an experienced user of this scope and he was happy with what I have.  I'm only taking single images, not a stack and I'm already imaging at x40 objective with oil immersion.  The microscope I'm using is used by the whole school I'm in and I don't want to play around with settings too much in case I compromise someone's else's experiments.  

Re the bleedthrough, I'm highly confident I'm not getting any as the staining at 488 is very specific and when it is strong, the signal doesn't appear in the 594 channel. \

At this stage, I have over 1500 czi files so to go back and re-image everything would take me forever!

Thanks for your suggestions,

Dave

Objective with higher magnification does not give better signals. The numeric aperture of an objective however does affect the amount of light collected by the objective. Oil immersion objective already has a numeric aperture surpassing water immersion and air objective. Changing the objective will not improve the signals as long as the objective has no obvious defects. Optimizing the correction ring of the oil immersion objective to better match the refraction index of material in which the sample is embedded however can improve the signals.  If the contrast between signals and noises is high enough, one can decrease the exposure time or decrease the excitation intensity to minimize photo-bleaching.  

By the way. for most of research grade fluorescence microscopes, it is possible to save and reuse customized settings for every user, so one does not need to worry so much about compromising the performance of the microscope for other users. It can be a problem of course for less advanced microscopes as such an option may not exist.