oSpim

oSPIM stands for Oblique SPIM. The light sheet is generated using an objective below the sample, usually an oil objective, and is tilted partway towards TIRF. The imaging objective is a water dipping objective that is lowered into the media. The oSPIM is an ideal tool for cell biologists or other applications where the sample is in close contact with the coverslip. The same concept was independently and concurrently developed by a research group in Heidelberg and called the πSPIM (see their publication). For more details about oSPIM see the presentation given in December 2016 at ASCB.

There is a Micro-Manager plugin for the oSPIM, based heavily on the diSPIM plugin but with a few changes. In the future hopefully the oSPIM vs. diSPIM will hopefully be a setting changed by the user in the plugin, but for now there is a flag in the source code that needs to be changed and the code be rebuilt.

Start by installing a recent nightly build of Micro-Manager 1.4.x. The latest official build is quite old at this point, so make sure to grab a nightly build. For Windows the nightly builds are here.

Download the latest copy of the plugin here (last update 22-Jun-2017) or else one from the archives. Make sure Micro-Manager is not running and then copy the JAR file into C:\Program Files\Micro-Manager-1.4\mmplugins (it will take precedence over the ASIdiSPIM plugin in the Device_Control folder, or you can delete ASIdiSPIM.jar from that folder and replace it with this one).

Much like the diSPIM. “F” axis in the semi-vertical SPIM head, “G” is the horizontal SPIM head axis, and “Z” is the vertical inverted microscope motorized axis. The camera trigger goes to PLC output #1 and the laser TTLs go to PLC outputs #5-8 just like diSPIM.

Alignment of the oSPIM is significantly easier than the diSPIM though many of the same principles apply. This documentation is a work in progress so don't hesitate to ask your system integrator if you need more information.

Start by getting things aligned “to eye”, then move on to the fine alignment using the microscope cameras.

Make sure you are getting the beam out of the bottom objective. Add a sample, e.g. dish with fluorescent dye, and steer the bottom dichroic until the beam is coming out at approximately the correct angle, orthogonal to the top objective (not straight up, not tilted to TIRF, but somewhere in between tilted the correct direction).

The hardest step finding the pencil beam with the upper objective. There are 3 position adjustments: the SPIM head height, the SPIM head horizontal, and the manual linear positioner above the objective piezo for moving front to back as you view the microscope. Use these to move the upper objective to the right neighborhood judging by eye. The objective should be almost touching the coverslip, and if the glass bottom is relatively small like with a Matek 35mm dish then you should set it up so that you are imaging the left side of the coverslip to leave maximum room for the objective to dip below the plastic bottom. You can judge front to back (adjusted with the manual linear positioner) pretty well by eye. The hardest one for me is the horizontal adjustment.

Once you think you are in the right neighborhood turn on the main (top) imaging camera. Move around until you can see a line. It will start out very blurry. You can verify that you have found the beam by moving the manual linear positioner and see if the beam moves. Rotate the camera so that the beam is horizontal (later you will fine tune the camera rotation). Once you have found it, move the SPIM head height and horizontal to go down near the coverslip. You can move along the beam by adjusting the SPIM horizontal by a factor of -0.5 relative to the SPIM head height movement.

Now make sure that the beam is tilted correctly. You can do this by seeing if you move in and out of focus uniformly when you adjust the piezo, understanding that the beam (and hence sheet) is diverging as it goes into solution. Adjust the beam tilt with the dichroic of the inverted microscope. It may help to stop the iris down so that there is less of a beam waist for this. You can probably get it pretty close in dye, in the long term you also want to do this with beads in solution.

Next verify that the beam waist is right near the coverslip. The way I suggest is first make sure the iris on the scanner is relatively wide open (wider beam on the edges, narrower waist). Then make sure the bottom objective is focused on the top of the coverslip using the bottom camera with transillimunation (a flashlight or even room lights from above work well). It is convenient to set this position of the lower Z axis to 0 so you can easily return. Then switch to the SPIM camera view and on the navigation tab move the lower Z negative, so the objective is focused into the solution. You should see the beam waist move into solution. The beam waist should be very near the coverslip, if not the scanner needs adjusting. But I made this adjustment when the system was here so it should be pretty close and hopefully you won't need to adjust it.

After this it is time to put beads in solution. Check the beam tilt. Also check the rotation of the scanner. These adjustments basically are to get the plane of the swept beam exactly in the imaging plane, in both axes. If you need to slightly adjust the piezo and/or slice axis to get things in focus that is fine, but with oSPIM this usually isn't a big deal because you have probably already got the SPIM head at the right position.

The oSPIM was originally developed on ASI's modular inverted microscope and this is the recommended configuration. The two hurdles to overcome in putting the oSPIM on another inverted microscope are

1. mounting of the oSPIM head above the sample/microscope
2. getting the scanner correctly positioned to form the light sheet through the inverted microscope including
   • 4F spacing (tube lens focused at scanner c-mount on one side and objective BFP on the other)
   • get the beam coming into the BFP at the correct position (essential) and angle (some wiggle room)

ASI is designing mounting hardware to overcome the first hurdle as of June 2017; it will leverage the same adapter bracketry as used for the diSPIM. Overcoming the second hurdle will be up to the end user.