μSPIM Toolset Calibration μSPIM Toolset provides the user with calibration tools which can be used to assess and correct the performance of the individual microscope elements. (A) and (B) show the calibration interface for X mirror with respect to the laser shutter when using masks and for Z mirror, respectively. (C) The movement of the X mirror galvanometer lags behind the supplied signal, resulting in artefacts when using laser masks. (i.) shows the laser shutter signal (green) and (ii.) shows the X mirror signal (blue) and actual mirror movement (light blue). Black bar highlights the misalignment between the shutter signal and mirror movement when the X mirror signal is not calibrated and no delay is introduced. (iii.) shows a mask used for calibration, emphasizing the difference between uncalibrated and calibrated system. (iv.) shows the corresponding light sheet produced with poor calibration for the mask signal in (i.). (D) is analogous to (C), showing a well calibrated system where the laser shutter (i.) is synchronized X mirror movement (ii.), producing overlapping masks shown in (iii.) and (iv). (E) Uncalibrated Z-Mirror results in loss of focus through the volume. (F) After calibration, the light sheet is always in focus throughout the volume scan. All figures illustrating the light sheet in both uncalibrated and calibrated scenarios were acquired by scanning the laser through a fluoroscein solution.
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