Light Beads
ScanImage® accommodates systems utilizing light beads microscopy via specialized alignment and display features so that image data is represented correctly in 3D.
Light beads microscopy is explained in depth here.
MBF Biosciences has collaborated with the Vaziri Lab at Rockefeller University to provide a light beads microscopy solution called vCAm.
Configuration
In the resource configuration editor under the devices tab, press the + button.
Under the Vidrio tab, you will find the vCAm device.
In the configuration page for this this device, you can set the depth offsets of the slices
via editing the table. The values used here will be loaded between sessions of ScanImage.
Under the ScanImage tab of the resource configuration editor, select the imaging scanner
that is on the path of the light beads beam. Under the advanced tab, select the newly configured
vCAm device from the vCAm dropdown and press Apply. When the imaging system is selected for
acquisition from the Scanner Configuration Gui, the light beads visualization
will be applied while imaging.
When using light beads microscopy, it is necessary to synchronize acquisition to the laser clock.
Follow the instructions on the Laser Clock Synchronization page to set this up.
Then the temporal windows of the individual planes can be set. For this, a thick fluorescent sample is recommended.
First bypass the vCAM so that the temporal offset to the first plane can be measured in ticks. We can set
this number of ticks in the Filter Ticks parameter from the signal conditioning controls to align the first bead
with the first few samples following the zeroeth tick.
From there, temporally, the planes are
interlaced with beads from cavity A and cavity B alternating in time both imaging progressively deeper into the sample up
until the next laser pulse. The general ordering of windows for vCAM can be set by clicking the Configure Default Virtual Channel Settings
button in the vCAm device configuration page. From there, the windows can be fine-tuned by left-click and dragging the windows to overay
their respective spikes in the PMT signal graph.
Use
When imaging, the image planes will be rendered in their correct 3D locations in the viewport.
For each image plane you want acquired, its corresponding channel must be checked to be received from the channels
GUI in the Display tab. To quickly enable several channels at once, double click the Receive table header.
From there, you can enter simple MATLAB expressions to select which channels to receive.
For example, to receive channels 1, 3, 5, and 7, enter 1:2:7 into the user dialog.
When defining the scanning spatially, it is recommended to use a single large ROI or few ROIs of fewer acquired lines (i.e. pixel resolution in Y) using MROI.
To see multiple planes simultaneously, select Tiled Z View from the dropdown at the top-left of the viewport.
To view a selected plane in more detail in the left pane, you can click on the image of the desired plane in the right pane.
From the left pane, you can use the mouse scroll wheel to zoom in and out of the image display.
For all acquisition planning, processing planning, and alignment, the Planar viewport display mode should be used.
Alignment
Alignment allows ScanImage to place image data in the correct 3D locations in the viewport. This is especially important for those utilizing a separate photostimulation arm of the micrscope to target based off of the imaged data.
Typically, the implementation of light beads will exhibit a slant to the optical axis post-objective. To correct for this, the light beads alignment tool can be used. The tool is also equipped to handle differences in scaling, rotation, or shear along Z.
A thin beads sample should be used to perform the alignment. measure out a volume of beads solution that when sandwiched between slide and coverslip is approximately 5 microns.
Find the plane of the beads by adjusting the Z position of the sample via z-stage while the vCAm cavities are bypassed. Once the beads are in focus, zero the sample coordinate system and take a reference image.
For each plane that is calibrated, the live image will be compared to the reference image and the z-stage will be adjusted to bring the beads back into focus to determine the true depths of bead planes.
Additionally, the XY offset of each plane will be accommodated via a point-adding-and-dragging fitting tool.
Planes calibrated in this way are interpolated between Zs, so it is possible to calibrate fewer than the full number of planes. It is recommended, however, to align laterally for all planes to ensure the highest accuracy.
Output File
Images are saved in a BigTIFF file format when using light beads microscopy. The depths are separated by channel index as determined by the virtual channel ordering.
See the ScanImage® BigTiff Specification page for the big Tiff specification.