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Re: Background correction

Posted by Bill Christens-Barry-2 on Sep 30, 2017; 4:17pm
URL: http://imagej.273.s1.nabble.com/Background-correction-tp5019468p5019470.html

Anu,

You mention wanting to reject reflected light; am I right in thinking that the light you do want to capture in the image is due to fluorescence? If so, two approaches come to mind:

It might be possible to use linear polarizers. Reflected light often retains the same linear polarization as that of the illumination, while fluorescence emission is often highly depolarized. If you place a linear polarizer in front of the light source and place another in front of the camera, with its polarization axis perpendicular to that of the illumination polarizer, the reflected light will be greatly attenuated while the fluorescence emission will be decreased to a lesser extent (~ 2x for full depolarization). This is especially true if the reflected light has been reflected from the surface alone; translucent materials will allow more penetration and consequent depolarization due to internal scattering, which reduces the effectiveness of this approach. Try rotating the illumination polarizer and the camera polarizer about their polarization axes together, i.e. by maintaining the 90ยบ difference between the orientations of their polarization axes as you rotate both. There will likely be a best orientation for the pair of polarizers.

Alternatively, you might employ a long pass filter in front of the camera so that only the longer emissions are passed while the shorter illumination wavelengths are blocked. This presumes that you are illuminating with a range of wavelengths shorter than those you wish to capture, otherwise some of the illumination may be passed by the filter.

Hope this helps,

Bill Christens-Barry

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