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Why do we need to clean optical filters?

Objective: To evaluate the effect of dirty filters in a fluorescence microscope.

Materials and methods:
"3T3" Sample courtesy of Mike Davidson, Molecular Expressions ™ consisting of
Albino Swiss mouse embryo labeled with BODIPY FL – Phallacidin were imaged with Semrock FITC-3540B filter cube using an Olympus BX41 microscope.

An image (image-1) was acquired with clean filters, and another image (image-2) was acquired using the same FITC cube in which one surface of the dichroic mirror and the emission filters were smudged with finger prints. Finally, a third image (image-3) was acquired to calculate the background noise, without the sample.

In order to make sure that the results are not biased by photobleaching of the sample, the image with the dirty filters was acquired before the image acquired using clean filters.
 
Results:
Background count from image-3 = 657.8

   Clean Cube FITC
  Clean filters: Image-1 with background subtraction.
Region of interest for calculations used for calculations.
Dirty filters: Image-2 with background subtraction.
Mean Signal  3148 2833
Standard Deviation  296 272
% variation in mean signal  

((3148 – 658) – (2833 – 658))/(3148 – 658) = 12.6%

 

Conclusion:
A modestly dirty filter can lead to a significant loss of signal. Therefore, it is important to keep your filters clean.