Multiple Region of Interest (MROI) Imaging

ScanImage® allows the microscope’s full field of view to be subdivided into multiple regions of interest (ROIs).

Benefits

The use of ROIs offers the following advantages:

  1. Frame Rate Optimization

  2. Limiting tissue exposure

  3. 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.

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Full Field of View vs. ROIs

A: When scanning the full field of view, regions of low interest contribute to the frame period and therefore decrease the frame rate.

B: By defining regions of interests (ROIs) the number of scanned lines is reduced to maximize the framerate while preserving the image resolution within the ROIs

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.

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Tissue Exposure

Tissue is only exposed to the laser while image data is acquired.

The beam is attenuated in between regions of interest.

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.

../_images/mROI+Interpolation1.png

Tracing Stack

By defining multiple scanfields at different z-planes of a ROI, features can be traced through a volume.


Using MROI

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.

../_images/mROI1.png

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.

Note

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.

../_images/mROI2.png

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.

../_images/mROI3.png

For more than 2 depths for an ROI, each additional depth requires the user to click the Add ScanField at Current Z button.

../_images/mROI3rdDepth1.png

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

Powers

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 Constants

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.

Note

it is possible for ROIs to intersect.

../_images/mROI3d.png

To acquire the MROI, select the Focus, Grab, or Loop button from the Main Controls window.


Output File

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.