OPenT

(detail of sample block [with specimen embedded] submerged in the cuvette filled with BABB and illuminated with blue incident light)

The camera should also be leveled, and the front wall of the sample chamber (cuvette) should be perfectly parallel to the objective front. Even after these adjustments, minor corrections may be necessary a posteriori as explained in the Supplementary Materials. When the specimen is longer than wider (eg an embryo) the camera should be rotated 90º to fill the camera chip (in the screen, the embryo will be rotating horizontally; before back-projection reconstruction, images need to be rotated again so that the axis of rotation is vertical!).

Adjusting the illumination:

Transmitted light source: center the light-bulb with the middle of the back wall of the cuvette and ensure that the FOV is evenly illuminated in the image. To hold the diffuser glass upright, glue it to a metal plate. If FOV is uneven, acquire an image without sample and use it later to subtract the background.

Incident light source (fluorescence): the source should be placed as close to the sample as possible ensuring that it is fully illuminated. Try to illuminate from an angle as close to the detection axis as possible (without blocking the field view) and avoid the cuvette corners. Larger samples will require moving the LED source away as possible to ensure full illumination and will require longer exposure times.

Note: There are in-axis options commercially available for incident light; it requires collimation of the LED light and additional optics which are quite expensive, and in our experience the light losses are considerable in some cases. All images we have produced or published so far were done with off-axis illumination as described above, for samples from a few mm to cm thick.

Positioning the sample:

After embedding the sample in agarose, dehydrating and clearing (see Supplementary Materials) glue the agarose block to a 3cm diameter metal washer using high-quality cyanocrylate glue, press gently and wait a few seconds. If block is smaller than hole in the washer, first glue a coverglass to the metal washer to serve as base. (Attention, make sure you use metal washers of a larger diameter than the magnet, otherwise centripetal magnetic forces will make it difficult to center the sample to the axis of rotation!).

Lift the motor as far as possible using the Y translation of the XYZ stage holder and, using forceps, invert the block and bring the metal washer part to contact with the magnet, immediately submerging the block to prevent the formation of bubbles. Using transmitted light, move the XYZ stage controls to bring the sample close to the front wall of the sample chamber (cuvette). Bring camera to focus and lock the rail carrier. Using forceps try to slide the blocks so specimen is as close to the axis of rotation as possible, first by looking from the front, then sideways. Minor focus adjustments can then be done either with the Infinitube standard focusing knob, or by moving the Z translation of the XYZ sample stage. While the motor rotates (use the function "lines" in the OPEN SPIN plugin window, and rotate the sample 360º), gently push the metal washer using forceps so that the center of the specimen coincides as much as possible with the yellow cross-hairs in the middle of the FOV. This is often a tedious trial-and-error task, but necessary! If the specimen is off-axis it will orbit and fall off the field of view occasionally. Also, because low magnification lenses are not perfectly flat-field corrected, image quality degrades away from the center of the field of view. When the specimen is finally aligned with the axis of rotation proceed to acquiring a full projection dataset and then reconstruct, as per instructions in Supplementary Materials of our original 2013 article.