We have implemented volumetric scanning in Scanbox by means of Optotune‘s electrically tunable lens, which allows for fast changes in focal plane without moving parts.
Some salient features of this particular implementation are:
- Laser power and focus can can co-vary, allowing the brightness of the images to remain approximately constant while performing volumetric imaging. This is achieved by the user setting the laser power a handful of depths and the computer interpolating for other values.
- Changes in focal plane are synchronized to the flyback time for each frame, and constant throughout each frame, making the stacking and volumetric visualization of images straightforward.
- Arbitrary waveforms can be loaded to control the lens. Standard waveforms include sinusoidal, sawtooth, triangular and square waves, but custom waveforms can be uploaded to the controller as well .
- Volumetric scanning works in both unidirectional and bidirectional scanning modes.
- We are working on a closed-loop feedback system to stabilize an optical section along the z-axis, which is particularly important in when there can be relative movement between the sample and the objective.
Below is an example of imaging with a 512-line image, 8kHz resonant mirror, bidirectional scanning, resulting in at 30 frames per second, along with a triangular scanning waveform at 1Hz. The range of the scan is about 15oum, and you can see the brightness of the images is relatively constant.[vimeo 129498546]
The slight translation of the image as depth is changed is due to coma produced by gravity deforming the Optotune lens, as it is currently mounted vertically in the microscope. A new design will provide for horizontal mounting thereby minimizing this artifact.
The latest Github release already includes these updates. A more detailed post on how to work with volumetric scanning will follow.