Launch setup for optogenetics and optopharmacology on a two-photon laser scanning microscope

[Benjamin Smith]

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Hey Microscopists,
    I recently designed and built a launch optic setup to allow for
simultaneous two-photon imaging and optogenetic epi-stimulation on a laser
scanning microscope.  The setup worked perfectly and was easy to align, so
I wanted to share it with anyone who may be trying to do something
similar. The CAD file can be found here: https://goo.gl/JHhK5h and an image
of the setup can be found here: https://goo.gl/7yEkga The CAD file can be
opened using FreeCAD: https://www.freecadweb.org/

   The goal of the setup was to ensure that all the lasers pointed to
exactly the same point in 3D space on the sample plane, and that the
two-photon lasers were diffraction limited at the sample plane.  Some
additional goals were to minimize the number of kinematic parts to reduce
thermal drift, make the footprint compact, and to be able to fully fill the
back aperture of a 20x/1.0NA objective.

   In detail, the mode-locked lasers are joined via a polarizing beam
splitter and then pass through a 4x beam expander, while the optical lasers
are joined via a dichroic mirror and then pass through a 6x beam expander
so that all beams are the same diameter going into the microscope.  Both
expanders were optimized to use the shortest focal length lenses possible
without introducing significant aberrations.

    To ensure the lasers stay laterally aligned in the sample, all four
combined beams then pass through a pinhole that is one Airy unit in
diameter for the mode-locked lasers.  The focus of each optical laser can
then be adjusted to bring them into alignment with the mode-locked lasers
along the optical axis at the sample plane.  In our setup, the mode-locked
lasers have a >99% transmission through the pinhole, while the optical
lasers have a >80% transmission.

     One side note, the polarizing beam splitter has a pretty high GVD, but
thanks to the narrow bandwidth of the 140 fs lasers in our setup, the cube
results in only a 5% broadening of the pulse.  However, with a 70 fs laser
this will result in a 61% pulse broadening, so using the 5 mm beamsplitter
cube and/or a prism compressor may be necessary.  All the other optics in
the path have a relatively insignificant contribution to GVD compared to
the beam splitter cube (other than the objective, of course).

Hope this helps,
   Ben Smith

--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Life Sciences Addition
Berkeley, CA  94720-3200
Tel  [phone]
Fax [phone]
e-mail: [hidden email]
http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>