Re: [pgfocus] A note about alignment and scattering of the TIR beam

[Karl Bellve]

Kyle, thanks for the great descriptive write up. This is helpful to many, including me. :D 

Cheers 

Karl Bellvé

Biomedical Imaging Group

Molecular Medicine

University of Massachusetts Medical School

On Thu, Dec 3, 2015 at 3:45 AM Kyle Douglass <kyle.m....@gmail.com> wrote:

Hi all,
I noticed something important recently about the alignment of the optics with regards to the pgFocus performance. I thought I would share what I learned on the forum.

In previous posts I noted that the pgFocus performance can be hindered if cells on the coverslip scatter the laser beam and create a messy beam profile. This essentially turns a nice, Gaussian beam into a beam with a lot of fringes, which is difficult for the pgFocus to lock onto. What I didn't realize at the time--though now it seems obvious--is that the strength of the scattering by the sample varies with the penetration depth of the evanescent field into the sample space. This penetration depth is essentially controlled by the location of the finite-sized, focused beam is in back exit pupil of the objective. I attached an illustration below to highlight this.

To get optimal alignment for pgFocus operation, I start with the IR beam in widefield, i.e. it goes straight up from the objective (be careful about looking into the objective when you do this). I then turn the screw on the mirror that translates the beam across the exit pupil of the objective. This translates the focused beam across the exit pupil, as seen in the left-most part of the figure. Once the beam enters the annular region where TIR occurs, and you will see a faint spot on the IR viewer if everything is well aligned. Translating slightly further, the beam goes fully into TIR, producing a bright spot on the IR viewer. This is not optimal, however, because the evanescent light is not strongly confined to the coverslip.

The ideal location is to have a very small amount of light from the focused beam passing near the edge of the aperture, with the rest of the light being cutoff from the finited-sized aperture. This ensures that most of the light is hitting the coverslip at the maximum angles allowed by the objective, which means the evanescent field penetration is small. This reduces scattering of the beam and should give a nice, clean profile. (Note that I also use a zero-aperture iris immediately in front of the laser to clean up the beam before it enters the microscope.)

By consciously placing most of the beam outside the aperture, I have managed to get ~2 to 3 nm std. deviation locking with an NA 1.4 objective and the sample in water.

I hope this helps everyone with their alignments!

Kyle

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