Multiple Region of Interest (MROI) Imaging¶
ScanImage® allows the microscope’s full field of view to be subdivided into multiple regions of interest (ROIs).
The use of ROIs offers the following advantages:
Frame Rate Optimization
Limiting tissue exposure
Tracing features through a volume
Frame Rate Optimization¶
In a traditional laser scanning microscope, the frame rate is calculated as follows:
frame rate [Hz] = 1 / (lines per frame * line period [s])
To increase the frame rate, the line period and/or the number of lines per frame can be decreased. Since the number of lines per frame directly translates to the vertical resolution of the image and the line period is either hardware constrained (resonant scanning) or would result in a decreased horizontal resolution (galvometric scanning), this method is often unacceptable for scenarios where a high image quality is desirable. For small objects that are distributed over a large range of the microscope’s field of view, a compromise between frame rate and detail resolution needs to be found.
To overcome this limitation, ScanImage® supports the definition of regions of interest (ROIs). ROIs allow to optimize the frame period by skipping image regions of low interest, while preserving the image resolution within the ROI.
Limiting Tissue Exposure¶
ScanImage® allows to control the laser power by using Pockels cell in the optical path. By fully attenuating the beam power while traversing between ROIs, the exposure of tissue is minimized. Additionally, ScanImage® allows beam power to be set differently for each ROI. This allow you to limit power differently to certain regions if some are more sensitive than others.
Tracing of features through a volume¶
A ScanImage® volume is the collection of multiple image planes at different z-planes. Typically, the volume is scanned by using a piezo actuator to axially sweep the microscope’s objective while continuously acquiring images. This way each acquired frame represents one slice of the volume.
In ScanImage’s multi-ROI feature, an ROI is the generalization of a standard volume. To define a ROI a user can define ‘scanfields’ at multiple cross sections. A scanfield is defined by x,y position, x,y extent, rotation and resolution. It is sufficient to define scanfields at a ROI’s top and bottom cross sections since ScanImage® interpolates scanfields within the volume as needed.
Before starting an MROI acquisition, start a Focus acquisition to tune the image quality, then commence a grab acquisition for one frame of the whole scanfield (Set zoom multiplier to 1x). Save the Tiff with a known filename and location.
To do a MROI acquisition, check Enable MROI in the Main Controls window, and click the Edit ROIs button beneath it.
This will summon the ROI Group Editor. In the Layers/Legend panel of the window, scroll down to find the Add Image from File Button. Use the image acquired at the beginning. Locate ranges of interest within the scanfield.
Under the context of image acquisition with MROI, clicking the Add ROI… button allows the user to draw a Rotated Rectangle ROI over the range of interest. Shown below are two ROIs on the same scanfield.
The location, width, height, and rotation of drawn ROIs are limited by the selected imaging system’s scanner configuration. See the Scanner Configurations page for more details.
This tutorial is done with an RGG scanhead configuration, which yields the highest frame rates with flexibility in specifying the Center X of the ROI.
If it is desired to scan at various depths to capture a 3-dimensional ROI, the Z-slider can be used to set the depth before resizing an ROI drawn at a different depth.
For more than 2 depths for an ROI, each additional depth requires the user to click the Add ScanField at Current Z button.
To view the volumetric ROIs in 3D, click the 3D button.
The ROIs can be configured to use ROI specific power controls, i.e. a unique beam power vs depth relationship per ROI. This is input in the bottom of the selected ROI Properties panel. See the below table for an explanation of the parameters.
ROI Specific Power Controls
These parameters allow specifically controlling the power of an ROI. If any of the values are unset, the parameters from the POWER CONTROLS interface are used like normal
Laser power. If there are multiple pockels cells, a value for each should be entered separated by commas
Enable P/z Adjust
Enter 1 or 0 (to indicate on or off) to control whether power/depth adjustment is enabled for this ROI
Length constant for each pockels cell for power/depth adjustment
Once the ROIs have been completely defined, the user can save the ROI Group for later acquisitions.
it is possible for ROIs to intersect.
To acquire the MROI, select the Focus, Grab, or Loop button from the Main Controls window.
The output of an MROI acquisition is a TIFF with metadata concerning each of the ROIs contained.
See the ScanImage® BigTiff Specification page for the big Tiff specification.