Full Field of View Fluorescence Performance

I finally got around to doing something I’ve wanted to do for a while: inspecting the point spread function of our new wide field of view microscope that uses the Andor Zyla camera. If you look back at some of my early posts, you can see that I’ve been wondering for a while what limits the effective field of view we can image through the microscope. As it’s clear that a much bigger field of view is accessible from the objective than makes it to the camera, why is it so hard to access that larger field of view? Once possibility that I’ve suspected is that the image quality is poor at the edges of the field of view.

To test this, I’ve measured point-spread functions (PSFs) for a Nikon Plan Apo VC 100x/1.4 objective using beads distributed across the field of view.  The PSF is an excellent way to see aberrations in your image (a colleague once compared measuring a PSF of your microscope to being naked; both are excellent at spotting imperfections that might otherwise be hidden). These images were recored on our Andor Zyla camera, which captures nearly the full field of view of the eyepieces.  As this is a new lens, the PSF in the center of the field of view is excellent, aside from some modest spherical aberration (see below).

CentralPSF

Z-series montage of the point-spread function of a 100nm bead in the center of the field of view. Sections are space 200 nm apart.

If we look at one of the corners of the image, however, the PSF appears very different. Below is the PSF from the upper left corner of the image. Here we can see that as we go out of focus there is a pronounced elongation of the PSF. The PSF is elongated perpendicular to the vector connecting the location of the PSF to the center of the image.

CornerPSF

The point-spread function from the upper left corner of the image. Otherwise identical to above.

We see similar aberrations elsewhere in the image – at the edges of the field of view the PSF becomes elongated. Fortunately, the aberration is only pronounced at the very edges of the field so that by reducing our image size modestly, we throw away most of the worst parts of the image. For high-resolution work on this microscope, I’m now recommending using the 2048 x 2048 ROI on the camera so that the worst aberrations are eliminated.

2 thoughts on “Full Field of View Fluorescence Performance

  1. Do you use water-cooling on your Zyla? If so, which cooler did you get and how do you like it?

    • We do not water cool. It’s on my to-do list but we just haven’t had a chance to do it yet.

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