We like to build things. All our custom solutions start with sketches on a note pad and a lot of discussion. After we have a good sense of the best approach, we develop the design in Zemax, optimize, and bring the optical layout into SolidWorks where we do the mechanical design and part drawings. Then its ordering, building, aligning and testing.
Two Photon Pulse Splitter
When using a Ti:Sapphire oscillator with a repetition rate of 80MHz for two photon excitation there is 12.5 ns of dead time between each pulse. A number of studies have shown that splitting and delaying the excitation pulses to reduce the time between pulses can reduce photobleaching resulting in better two photon images. For example, see the work by Ji and colleagues (Nature Methods 5(2):197-202).
Inspired by the two photon pulse splitter design reported by Mutze and colleagues (Biophysical Journal 102(4):934-944) we wanted to create a compact inline unit that would be easy to align and slot into our existing beam path. The result is pulse splitter that measures 245 x 245 mm and splits our two photon excitation pulses 16 times. This requires the longest delay path inside this box to be 2.2 meters.
Deformable Mirror Module
A number of setups in our lab use deformable mirrors. We wanted to package our deformable mirrors in a module that included light paths to bypass the mirror for larger fields of view and allowed for easy calibration via interference with a flat reference mirror.
We created a module that holds a deformable mirror in a five-axis mount and includes a light path to bypass the mirror. It also includes a port to input a laser beam which is split between the DM and a reference mirror acting as an interferometer for calibration.
For multi-color imaging we wanted a flexible splitter that could be configured to split color channels onto two cameras or split the color channels onto a single camera chip. We also wanted a setup that would maintain telecentricity when adjusting the image position on the camera.
We created a dual view module that allows the use of one or two cameras and can be easily configured for a left or right handed microscope stand. The dual view has vertical mirror adjustments so its easy to adjust the image positions once everything is setup and the box is closed. The dichroic mirrors are inserted from the top and held in by magnets so no tools are needed. Depending on dichroic choice, up to three colors can be imaged simultaneously.
Line Scanning Confocal Module
For imaging off the cover glass and inside tissue we needed a solution that had speeds comparable to wide-field / epi-fluorescence but with the optical sectioning / background suppression of confocal. Further, we needed this in a light tight module that could be bolted on the back of our Olympus IX83 base with minimal moving parts.
By taking advantage of the rolling shutter / light sheet mode on our Hamamatsu Orca Flash sCMOS camera we can electronically generate an effective confocal slit. We then developed a module that includes a cylindrical lens to create an excitation line of light at the sample and a glavo mirror to scan the excitation line synchronously with the sCMOS camera shutter. This gives us background suppression closer to that of confocal with imaging speeds approaching wide-field illumination.
4Pi Sample Holder
In 4Pi microscopy, two opposing objective lenses view the specimen from two sides simultaneously. With high NA, low working distance objective lenses, this means there is only a small ~400 µm gap for the sample and two cover glass. Further, because the sample must be within a few hundres micrometers of both lenses, there are mechanical restrictions on how far you can move the sample laterally. We needed a compact, easy to assemble, sample holder that would allow for maximum lateral sample movement and included the possibility of removing and reinstalling the sample without losing the imaging position.
We designed a custom sample holder based around 50 mm Willco dishes with cover glass bottoms. The cover glass bottom of the dish holds the sample while a 30 mm round cover glass is placed on top and either mechanical held in place or glued onto the dish. The holder includes registers on the bottom so the sample can first be imaging using a 10X objective on a conventional microscope, positions of interest marked, and then transferred to the 4Pi system and the marked positions found.
With limited space on our optics tables we wanted to put our excitations lasers somewhere else. Thus, we needed compact solution that would fit under our optics tables, hold multiple lasers lines, and allow the lasers to be shared between systems.
Taking advantage of a 19" rack mount drawer from Thorlabs, we put together a solution that holder 5 excitation lines (405, 488, 546, 560, and 642 nm) with all their controllers, an AOTF, and has three fiber outputs (with electronic flip mirrors for selection) to share the excitation lines between multiple setups. The solution includes a 14 port rack mount USB hub so the entire unit only has two cables that leave the box: the power cord and a single USB cable. It fits under the optics table and out of the way of users.
STED Laser Box
While setting up our custom STED microscope, the cable management situation became a bit of a problem. After a few years, we added new hardware and moved some laser heads and then things became unbearable.
After our positive experience with our laser box (above), we decided it was time to consolidate equipment into a single 19" rack and get things organized. The top of the unit has a small honeycomb breadboard with our STED laser heads where we couple both depletion beams into single mode fibers. Next is a 19" rack mount drawer from Thorlabs with our excitation laser heads, AOTF, and fiber coupling. After that, we have a power switch box so the whole system can be powered up without having to hunt for individual device on/off switches. Then a 14 port rack mount USB hub and the rest of the unit holds the timing electronics and laser power supplies. Overall, this saves a lot of space on our optics table and consolidates a lot of equipment and cables into one compact well organized unit.