Post by Darcy Peterka (Columbia University/Yuste lab):
We do a bunch of multiple imaging/uncaging experiments with 2+ lasers, and got tired of constantly taking apart the camera/laser ports to switch lenses and dichroics. This setup allows easy inspection of the conjugate planes to the sample and back aperture. These are just Thorlabs cubes (C4W) with the kinematic cage platforms (B4C) and dichroics holders (FFM1). They aren’t the most precise pieces, but they are easy to use and align, and are quite flexible. The upper lens off to the left is on a translator to allow easy compensation of the longitudinal chromatic aberration of the microscope for the different colors on the beam paths (~800nm and 1064nm), which would otherwise kill us during our experiments.
Depending on the objective, laser combo, we may have to move up to a centimeter. Part of the long travel required is because the divergence properties of our 1064nm fiber lasers seem to change more often than we’d like, so we end up doing compensation. I could off-load that task to another telescope, but it is easy enough to change here. Assuming everything is perfectly collimated beams, the difference in axial focus of the 60x 1.1 NA objective is two microns with 800nm and 1064nm light – large enough to screw up experiments with neuronal spines and dendrites.
