Alignment of the detection path

The detection path might require two types of alignment:

• Alignment of the tube lens & camera relative to the MVX-10 zoom body to compensate for a lateral drift of the image when zooming.
• Tip & tilt & rotation alignment of the immersion cuvette.

Alignment of the zoom system

• Disconnect the zoom servo from the MVX-10 Zoom axis - you can use the mesoSPIM zoom controls, but changing the zoom manually is much faster.
• Use the camera software (HCImage for Hamamatsu cameras) in live mode. If necessary, change the trigger settings so that no external trigger is required. You can use the mesoSPIM software, but then the frame rate is limited to typical mesoSPIM acquisition rates.
• When using HCImage: Be aware that the camera is rotated by 90 degrees! The mesoSPIM-control software rotates the image back, HCImage does not.
• If necessary, dismount the tube lens from the filter wheel. Depending on the results of the alignment steps described down below, it might be better to leave it permanently disconnected. The camera mount is stable enough to keep the camera & tube lens position relative to the zoom body over time.
• Use the Thorlabs R2L2S3P2 250 µm grid distortion target as a test sample. Use a spare kinematic mount and a FFM1 Filter mount to suspend the test target in front of the objective in air.
• Focus on the test target and change the zoom manually. Use crosshairs in HCImage (enable the intensity profile layer and make the cross-hair by setting the X and Y line positions with the pop-up box that appears such that the cross hairs centered on the middle of the image) and observe the lateral shift of the grid pattern. You do not need to refocus (the Olympus MVX-10 has a pronounced focus drift with zoom), just observe the drift.
• The drift will be easier to see at higher frame rates (20 FPS will do). Alter the exposure and lighting levels if necessary to achieve this. You might need to place a white card somewhere behind the dot target if the image is too dim at shorter exposure times.
• If you see a drift in the horizontal plane (parallel to the optical table), loosen the screws connecting the X95 base plate of the camera mount to the X95-carrier and gently move the whole camera. Choose the mounting flange of the filter wheel as the pivot point.
• If you see a drift in the vertical direction (perpendicular to the optical table), change the height of the camera using the Thorlabs PSHA height-adjustable collar
• Be aware that any movement of the camera will induce a shift of the image - you might have to reset the crosshairs or move the sample to judge the lateral shift accurately.
• Constantly move the zoom position back and forth to check the alignment. In the final steps of the alignment process, it might be advisable to tighten the locking screws a bit and move the camera using "percussive alignment" - by gently hitting the baseplate with a lightweight rubber hammer or comparable items.
• If you find that along one axis you can't fix the drift by translating the camera then try small changes in tilt. e.g. by loosening the screws that bolt the camera to the right angle bracket. Change the tilt slightly then repeat the translation steps.
• You may find that tightening the clamp for the vertical alignment causes the camera angle to change and the alignment to alter. You either need to compensate for that before tightening or leave the clamp a little looser.
• A lateral shift of 50 - 100 µm (approximately the size of a single dot on the grid pattern) is usually the optimum. Some MVX-10 zoom bodies show a "spiraling" movement of the optical axis when the zoom is changed -- if this becomes apparent, it cannot be compensated with this alignment procedure.

Alignment of the outer cuvette

The wall of outer cuvette facing detection objective must be orthogonal to the detection optical axis. Misalignment of the outer cuvette can introduce astigmatism in the images.

Optical method

To do the alignment you will need a Thorlabs R2L2S3P2 or similar distortion target. Put a drop of immersion liquid on the cuvette wall to make it wet, and press the target against it. It will stick thanks to surface tension of the thin layer of immersion liquid.

Make an even illumination on the background (user side), e.g. by using a bright lamp and a paper sheet. Image the target at various zooms, and adjust tip and rotation of the cuvette until all edges of the target are equally in focus across the entire FOV.

Mechanical method

If you do not have a distortion target, you can bring the objective so close to the cuvette that they nearly touch (with a very small gap), and make the gap uniform across the contact surfaces by sliding in a piece of paper and adjusting the cuvette tilt and rotation. This trick is commonly used to adjust the distance between 3D printer nozzle and the heatbed. It gives extremely fine alignment, because it measures the gap (~100 micron, paper thickness) across a very large field, 45 mm (size of the cuvette wall). This method assumes that objective can be brought very close to the cuvette, the lens metal rim is smooth and orthogonal to the detection optical axis.