This guide walks through the full workflow for installing and operating the super-resolution microscope, from basic optical alignment to acquisition.

Basic System Adjustments

  1. Make sure the microscope does not contain the lens that focuses light at the objective back aperture, nor any lens in the excitation light path. If present (as is the case with the IX-81), remove those components.
  2. Attach the optical fiber from the laser box to the optical table using a fiber mount.
  3. Place an achromatic doublet on the optical path at a distance equal to its focal length. Fine-tune the lens position along the optical axis until the beam is collimated — verify this by confirming the beam size stays constant when measured at two or more distances from the lens.
  4. Place two mirrors on kinematic mounts to allow beam alignment.
  5. Verify the light is correctly aligned using two alignment disks at each end, connected by lens tubes, making sure the beam passes through the iris at both ends. Use the kinematic mirror mounts for fine adjustment.

Installation and Operation of the TIRF Module

  1. Secure the linear translation stage to the optical table at the back of the microscope and mount the aluminum breadboard on top of it.
  2. Place two mirrors on kinematic mounts (see Supplementary Figure 1b), directing the beam toward the microscope.
  3. Remove the objective from the microscope and adjust the beam output position so it exits centered, using the kinematic mounts of the TIRF module mirrors.
  4. Place an achromatic doublet lens of appropriate focal length on the aluminum breadboard of the TIRF module so that the beam focuses at the back aperture of the objective.
  5. Mount the piezo on the microscope, establishing the final height at which the objective will sit.
  6. Verify the light is effectively focusing where the objective back aperture would be, using translucent paper. If not, move the TIRF module lens forward or backward relative to the microscope until it does.
  7. Mount a low-magnification objective and verify the light exits correctly aligned — if observed on the ceiling, it should go straight up. If not, adjust with the TIRF module kinematic mirrors. A bead sample (or similar) can also help confirm the light is centered during alignment. Once verified with the low-magnification objective, mount the objective intended for SMLM (e.g., 100×, NA 1.45) and re-verify alignment and centering.
  8. Verify that rotating the linear translation stage screw makes the light exit the objective at an angle rather than straight, with the inclination occurring laterally. Fine adjustments can always be made with the kinematic mounts.
  9. Place a sample and verify the system works correctly: acquire images under straight illumination and under TIRF (no light exits the sample — total reflection), then calculate the SBR for each case. You should see a significant SBR increase under TIRF illumination compared to straight illumination.

Installation and Operation of the Focus Lock Module

  1. When illuminating in TIRF or HiLo mode, a back-reflected beam is visible on the TIRF module mirrors and moves laterally as focus is adjusted. Focus a sample using TIRF and, while watching the back-reflected beam, place a pick-off mirror on the optical table that diverts this light without interfering with the excitation path.
  2. Redirect the light with the pick-off mirror toward an area of the optical table with enough space to install the focus lock module.
  3. Place an achromatic doublet of known focal length to focus the back-reflected beam onto a diaphragm. Leave space between the doublet and the diaphragm in case neutral density filters need to be added later to prevent saturating the webcam.
  4. Place another achromatic doublet after the diaphragm, at a distance equal to its focal length, so the beam exits collimated.
  5. Place a further achromatic doublet in the optical path to focus the beam at the position where the webcam will sit.
  6. Make sure the webcam/camera used has no built-in lenses (only the detector is needed), and secure it to the optical table with an appropriate mount (in our case, a 3D-printed holder) at a distance equal to the focal length of the last lens placed.
  7. Verify the webcam correctly detects the reflected light. If it saturates, add neutral density filters to the focus lock module path. Moving the focus should produce a visible displacement of the light on the webcam.
  8. Open the focus lock program (Python) and confirm in the GUI that the webcam is correctly detecting the light. The sigma and gain parameters can be tuned to improve detection — ideally you want as circular a beam as possible with few spurious reflections. Unwanted reflections can be reduced by adjusting the diaphragm or the detection parameters. The contour centroid detection mode (mask-based) is also available, and its threshold can be tuned to exclude these reflections, which typically have lower intensity.
  9. Once parameters are set, click Calibrate to verify a linear relationship between objective position and back-reflected beam position. If the relationship isn’t linear, check the detection parameters and whether additional neutral density filters are needed (this varies by detector).
  10. Once linearity is confirmed, tune the Ki and Kp values for your system:
    • Start with Ki = 0 and test Kp values until engaging the focus lock produces no overcorrection (no abrupt movements), while still correcting focus in response to a small perturbation (e.g., slightly moving the micrometer). In our system, the optimal value was Kp = −0.01.
    • Then adjust Ki, typically to a value smaller than Kp, and verify that with the focus lock engaged, focus is maintained over time. Test several Ki values until you reach optimal performance (in our system, Ki = −0.001).

Installation and Operation of the 3D Astigmatism Module

  1. Add an element to the emission path that lets you insert optical components without changing the distance between the camera and the microscope. In our case, this was a 3D-printed element with slots for the astigmatic lens.
  2. Place the plano-convex cylindrical lens in the emission path.
  3. Using a bead or gold nanoparticle sample, verify the astigmatic lens is correctly positioned: you should see an ellipse elongated along the lateral axis when defocusing in one direction, and an ellipse elongated along the vertical axis when defocusing in the other. You may need to slightly rotate the cylindrical lens until this deformation is properly oriented.
  4. Using a bead or gold nanoparticle sample, acquire a z-stack with 5 nm steps to capture the PSF at different z positions. Then, using Picasso Localize (Calibrate), verify the calibration curves behave as expected — lateral widths (σx and σy) should vary smoothly, in opposite directions, as a function of z, consistent with the astigmatic PSF shape reported by Huang et al. 2008.

Operating Recommendations Once Each Module Is Installed and Verified

  • Before every 3D experiment: insert the astigmatic lens and acquire a z-stack of beads/gold nanoparticles as described above, every time the lens is removed and reinserted. Confirm a correct calibration before imaging your sample of interest.
  • Illumination angle: place your sample of interest and adjust the beam inclination angle with the linear translation stage screw until no light exits upward from the sample (TIRF illumination). For deeper imaging beyond the coverslip vicinity, back the screw off slightly to switch to HiLo illumination. Note the linear translation stage screw values so the same inclination angles can be reproduced in later acquisitions.
  • Focus lock check before acquisition: focus the sample and confirm in the focus lock GUI that the webcam is correctly detecting the light. Adjust detection parameters, the diaphragm, or add neutral density filters if needed. Once set, press Calibrate to confirm a linear relationship between focus position and light position under these conditions.
    • Re-calibration is required every time the field of view changes or focus is adjusted, to ensure correct focus lock performance under the current acquisition conditions. This step is fast — usually under a minute.
    • If the relationship is non-linear before imaging, this points to suboptimal optical conditions. Try restoring linearity by adjusting acquisition/detection parameters (digital gain, gamma correction, binarization threshold if using the contour centroid method) or, in atypical cases, by partially closing the iris. If that doesn’t work, try varying the TIRF/HiLo illumination angle to optimize the back-reflection geometry, check that you’re within the objective’s axial working range, or attenuate the reflected beam with neutral density filters to prevent detector saturation.
  • Stability check: with the focus lock engaged, press Calculate Std Dev to verify in the GUI that the z-position standard deviation is within an acceptable range before starting acquisition. We routinely achieve ~10 nm or better stability for both 2D and 3D SMLM, and recommend confirming this level of performance before proceeding.
  • Acquisition: run the acquisition for your experiment, selecting exposure time and number of frames in Micro-Manager.

Found an error or have a suggestion? You can open an issue in the project’s GitHub repository, or reach out — see Contact.