TIRF objective overfilled in BFP

[Kyle Michael Douglass]

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Hi all,
I've got a question arising partly from my own curiosity and partly as a
need to explain a strange "reflection" I'm seeing in the lab.

If a TIRF objective (say NA >= 1.45) is illuminated with a laser beam whose
diameter is larger than the aperture at the objective's back focal plane in
an epi-fluorescence setup, is a fraction of the still light totally
internally reflected at the coverslip/sample interface? Is this true in
general, or only true if the beam is collimated or converging/diverging?

Assume that the sample refractive index is 1.33 (water) and we're using a
standard glass coverslip and oil immersion objective with index matching oil
between the objective and coverslip.

I'm curious because I usually work with wide-field laser epi-fluorescence
microscopes where the beam is focused to a point in the objective's back
focal plane. I don't usually work with beams that overfill the objective's
aperture.

Thanks in advance!
Kyle

[John Oreopoulos]

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Kyle, I think the most likely answer to your question is yes. See a recent posting on the listserver related to this topic of "accidental TIRF", and check out the paper by Thomas Burghardt:

http://lists.umn.edu/cgi-bin/wa?A2=ind1402&L=confocalmicroscopy&D=1&T=0&P=2346

However, the mysterious "reflection" you mention could be due to some other artifact. More details are required to figure that out.


Cheers,

John Oreopoulos
Staff Scientist
Spectral Applied Research Inc.
A Division of Andor Technology
Richmond Hill, Ontario
Canada
www.spectral.ca


On 2014-02-15, at 7:32 AM, Kyle Douglass wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi all,
> I've got a question arising partly from my own curiosity and partly as a
> need to explain a strange "reflection" I'm seeing in the lab.
>
> If a TIRF objective (say NA >= 1.45) is illuminated with a laser beam whose
> diameter is larger than the aperture at the objective's back focal plane in
> an epi-fluorescence setup, is a fraction of the still light totally
> internally reflected at the coverslip/sample interface? Is this true in
> general, or only true if the beam is collimated or converging/diverging?
>
> Assume that the sample refractive index is 1.33 (water) and we're using a
> standard glass coverslip and oil immersion objective with index matching oil
> between the objective and coverslip.
>
> I'm curious because I usually work with wide-field laser epi-fluorescence
> microscopes where the beam is focused to a point in the objective's back
> focal plane. I don't usually work with beams that overfill the objective's
> aperture.
>
> Thanks in advance!
> Kyle

[Kyle Michael Douglass]

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Thanks John. The JBO paper is very interesting. I'll give it a more thorough read and see if it clarifies anything in my setup.

As for my strange reflection, I'm actually using an IR beam (830 nm) in a home-built autofocus setup. The beam is introduced into the back epi-illumination port on an Olympus IX71 with a shortpass filter to combine the IR beam path with the excitation beam paths. I see a very strong reflection of the IR beam when I look at the image on the camera without an emission filter in place. The position on the camera plane can be translated by changing the angle of the incident beam.

This strong reflection also passes back through the epi-illumination port. In addition, I can also identify a portion of light that is reflected from the coverslip but not totally internally reflected. The positions of all the reflected beams in a transverse plane to the beam path on the outside of the epi-illumination port vary with the objective's axial position, and the rate of variation increases with the angle of incidence on the objective.

For these reasons, I suspect that some of the light (the very bright reflection) is totally internally reflected from somewhere around the objective.

Thanks for the help!

Kyle

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos
Sent: samedi 15 février 2014 14:05
To: [hidden email]
Subject: Re: TIRF objective overfilled in BFP

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Kyle, I think the most likely answer to your question is yes. See a recent posting on the listserver related to this topic of "accidental TIRF", and check out the paper by Thomas Burghardt:

http://lists.umn.edu/cgi-bin/wa?A2=ind1402&L=confocalmicroscopy&D=1&T=0&P=2346

However, the mysterious "reflection" you mention could be due to some other artifact. More details are required to figure that out.


Cheers,

John Oreopoulos
Staff Scientist
Spectral Applied Research Inc.
A Division of Andor Technology
Richmond Hill, Ontario
Canada
www.spectral.ca


On 2014-02-15, at 7:32 AM, Kyle Douglass wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi all,
> I've got a question arising partly from my own curiosity and partly as
> a need to explain a strange "reflection" I'm seeing in the lab.
>
> If a TIRF objective (say NA >= 1.45) is illuminated with a laser beam
> whose diameter is larger than the aperture at the objective's back
> focal plane in an epi-fluorescence setup, is a fraction of the still
> light totally internally reflected at the coverslip/sample interface?
> Is this true in general, or only true if the beam is collimated or converging/diverging?
>
> Assume that the sample refractive index is 1.33 (water) and we're
> using a standard glass coverslip and oil immersion objective with
> index matching oil between the objective and coverslip.
>
> I'm curious because I usually work with wide-field laser
> epi-fluorescence microscopes where the beam is focused to a point in
> the objective's back focal plane. I don't usually work with beams that
> overfill the objective's aperture.
>
> Thanks in advance!
> Kyle