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.

Long Stokes Shift Dyes

I’ve recently had a request from a group trying to do five-color imaging on our four-laser confocal system. On a widefield system, one way to do this would be to add an infrared dye to the usual combination of DAPI / FITC / Cy3 / Cy5 (or equivalents) but on a confocal system the expense of adding a new laser is considerable. So instead, I’ve recommended to them to try using a long Stokes shift dye. This is a dye whose emission wavelength is unusually far red-shifted from its excitation wavelength, and potentially allows reuse of the existing excitation lasers and emission filters on the microscope.  For example, a dye that is excited at 488 nm and emits at 700 nm could be excited with the 488 nm laser and detected with the Cy5 emission filter, or one that is excited at 488 nm and emits at 610 nm could be excited with the 488 nm laser and detected with the Cy3 emission filter. Assuming there isn’t too much excitation of the Cy3- or Cy5-like dyes at 488 nm, adding either of these dyes would allow imaging a fifth channel. Continue reading

Laser Launches and Fiber Combiners

A key component of any laser based microscopy system (say, a TIRF or confocal microscope) is the laser source itself. Since typically we want multiple laser lines for imaging different fluorophores, we typically use a laser launch – an item that combines the output of several different lasers and launches them into a single mode optical fiber that is attached to the microscope.  These come in a wide variety of types, from integrated units that are completely turn key, to homebuilt launches that mount the lasers on a table, combine the beams using dichroics, and then focus the combined beams into the fiber. The homebuilt launches have been cheaper and more flexible, but tricky to align and prone to drifting out of alignment.

Now with advances in fiber optics, and directly modulatable lasers, launches are starting to appear that combine multiple fiber inputs into a single fiber output. For instance, the OBIS Galaxy from Coherent, or simpler products from other companies. I haven’t yet seen any of these used for microscopy, but the prospect of purchasing three or four fiber coupled lasers, attaching the fibers to a combiner, and attaching the output fiber to the microscope is pretty appealing, and may offer cost savings as well as increased flexibility.