Do I need to use all three channels when observing through a broad bandpass barrier filter?
If I am exciting at a given wavelength and observing through a broad bandpass barrier filter do I need to use all three channels?
If you are using a monochrome camera you have one channel to which you assign the peak of the fluorescence emission spectrum. If that peak is cut off by the bandfilter, the effective emission wavelength at the detector will be close the the filter cutoff wavelength.
If on the other hand you are using an RGB camera, the situation is a bit different. Each of the camera components has it's own, probably not so sharp band filter. The camera filters are in cascade with the microscope bandfilter. Suppose the microscope filter passes all wavelengths longer than 500nm, and the blue camera filter passes all shorter than 500nm, then the Blue channel will be dark. In a different case, suppose the microscope filter passes all wavelength longer than 570nm, the green camera filter from 500-600nm, and the red camera filter from 580nm and longer. Then both Green and Red channel contain an image. With little loss of accuracy these could be added and deconvolved at 570nm emission wavelength (you need Huygens Pro for that).
In the most likely case two of the three R, G, B channels will contain only a very dim image. You might as well discard these and concentrate on the brightest image.
Keywords: RGB color monochrome camera barrier filter
Categories: Faq Microscopy, Huygens Faq, Imported Faqs
Platforms: Irix Linux Windows Mac AIX
Related products: Hu Ess Hu Pro
