I would like to use laser illumination for a widefield fluorescence
microscope. I am wondering how to handle the polarization and/or
coherence effects.

I have read that it is better to have circular polarization on the
sample, to avoid any preferential excitation of fluorophores with a
specific orientation. How much of this is true and how much is superstition?

Secondly I am wondering how to combat laser speckle to get uniform
illumination of the sample. I might focus the laser in the back focal
plane of the objective and then move the spot around in the BFP by
adjusting the tip/tilt of the mirror. There are probably other ways.
What is generally used for this?

Thanks in advance

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On 15/07/16 09:47, Fitzgerald wrote:

I would like to use laser illumination for a widefield fluorescence microscope. I am wondering how to handle the polarization and/or
coherence effects.

I have read that it is better to have circular polarization on the
sample, to avoid any preferential excitation of fluorophores with a
specific orientation. How much of this is true and how much is
superstition?

Secondly I am wondering how to combat laser speckle to get uniform illumination of the sample. I might focus the laser in the back focal
plane of the objective and then move the spot around in the BFP by
adjusting the tip/tilt of the mirror. There are probably other ways.
What is generally used for this?

Thanks in advance

I find Kohler illumination does the biz.

--
Rusty Hinge
To err is human. To really foul things up requires a computer and the BOFH.

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