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.