Month: June 2015

Visualizing Scanbox stacks with Vaa3D

Some scanbox functions allow you to export *.sbx files to *.tif files that can be then visualized using other software like Vaa3d or ImageJ.  For example, after collecting a z-stack you can use sbxtifstack() to generate a tif file that can be read directly using Vaa3d.  If your stack has the hypothetical file name abc_010_nnn.sbx (where nnn represent the different slices of the stack), all you have to do is to type

sbxtifstack('abc_010')

This will align all the images for each of the individual slices and generate a tif file named abc_010_stack.tif. Opening this file in Vaa3D and visualizing the volume will give you something like this:

v3a

You can easily rotate and visualize your data and Vaa3D provides a number of plug-ins for further analysis. A similar process can be followed if you are more familiar with ImageJ.

 

Fast volumetric scanning with an electrically tunable lens

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.