Re: Background correction

Posted by anusuya pal on Sep 30, 2017; 6:20pm
URL: http://imagej.273.s1.nabble.com/Background-correction-tp5019468p5019471.html

Hello

Here is the sample image (raw file) attached. I am using a polarizing
microscope. And I think for my other samples, BaSiC is working good. But
for particularly this type of image, I am not getting the background
intensity and sample intensity differently. The range is more or less same.
So, if I know any plugins that will solve this correction for the current
image, It will be helpful.

Thanks
Anu

On Sat, Sep 30, 2017 at 12:17 PM, Bill Christens-Barry <[hidden email]>
wrote:

> 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 90º 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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>

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