Why You Might Want Fluorescence Dimming

 

Why would you want fluorescence dimming? In general you want as much fluorescence as you can get! Many naturally fluorescent subjects do not glow very brightly, and the fluorescent proteins and stains used in the life sciences can range from dim to bright but tend to be localized in small areas. The more fluoresced photons you can get out the better, and at times we are even asked how to get more intensity out of the system (for example, by using the Dual Light Head Hanger Kit).

But there are some instances in which there can be too much of a good thing, and for these fluorescence dimming with the analog DIM option or PWM dimming with the PULSE option for our Model SFA Stereo Microscope Fluorescence Adapter may be a good choice:

• If your fluorophore is sensitive to photobleaching you want to use the minimum excitation needed to see the subject: any more just hastens the degradation;
• Fluorescent indicators used in industry can be extremely bright, to the point that they are easier to observe if the intensity is reduced;
• If you want to balance fluorescence and reflected white light in an image to provide context you may need better control of the excitation intensity;
• There are situations in which a reduction in excitation intensity can result in an improvement in viewing contrast.

The first three points above are fairly obvious. The last is more interesting. We encountered this while working with a customer interested in finding defects (gel) in nylon granules (read our article for more information on this application). The entire granule fluoresces and the small defect shows up as a brighter spot. The customer found that decreasing the excitation intensity produced an increase in viewing contrast. This is nicely illustrated in the two images below.

(Click image for larger view)

Dimming decreases the excitation intensity over the full image area, but the fluorescence intensity of the background appears to decrease faster than that from the areas of interest. There is a ‘sweet spot’ that the operator could set for optimum viewing contrast. This certainly relates to non-linear responses of human visual perception to changes in intensity, and differences in those responses in areas of low and high intensity.

The ability to dim excitation is not for everyone, but in some cases it can be a very useful capability. Please contact us if you have any questions about this.