NI-IMAQ Vision and LabVIEW Automate Microscope Image Acquisition

Author(s):
Michael D. Coleman - Coleman Technologies, Inc.

Industry:
Life Science, Imaging Equipment

Products:
LabVIEW, Vision, PXI/CompactPCI

The Challenge:
Accelerating the search for cancer cells in blood samples.

The Solution:
Developing a PC-based system using an NI PCI-1424 board, LabVIEW, and NI-IMAQ Vision software to automate the acquisition and preliminary analysis of microscope images.

Introduction
Coleman Technologies, Inc., a National Instruments Alliance Program member, was contracted to develop a software program to automate the acquisition and preliminary analysis of microscope images collected by the CellSpotter system from Immunicon Corporation, which identifies and counts tumor cells in blood samples. CellSpotter required high-speed acquisition of hundreds of high-resolution, fluorescence microscope images per sample with four filters and with images acquired across very large slide areas (up to 560 images per slide). With Immunicon’s proprietary sample preparation technique, cancer cell identification is possible by overlaying the acquired images with each of the different filters at any location on the slide.

The choice of LabVIEW and the NI-IMAQ Vision software for this application proved ideal due to the frequent changes in functional requirements, the need for rapid prototyping, low software development costs, and the ability to work with a variety of vendor hardware products.

System Configuration
The CellSpotter system is based on a Pentium III 800 MHz PC equipped with LabVIEW and NI-IMAQ Vision software and an NI PCI-1424 digital image acquisition board.We use a Nikon E-400 microscope equipped with high-resolution x and y stages. We controlled these stages, the microscope focus, and the filter selection with a third party controller interfaced to the PC via RS-232. We acquire images with a Hamamatsu 12-bit, 1280 x 1024 pixel digital camera connected to the PCI-1424 board.

We place samples in a custom, 30 x 3 mm chamber. We also mounted special calibration chambers, with registration marks in the center of the chamber, in place of the sample chambers for calibration of the chamber center offset from the stage home position and the illumination source.

System Operation and Functionality
The CellSpotter acquisition program requires the operator to enter specific information about each sample prior to each acquisition sequence. When the testing of a sample commences, the software automatically:

• Verifies the correct format of the user inputs
• Determines the region of interest (ROI) over which to acquire images, the number of images to acquire, and the position of each image
• Determines the microscope focus to use at each position
• Acquires and logs to disk images from each location and each filter

The ROI is determined by moving the x and y stages to five positions that should have an edge of the chamber visible in the image. The software also:

• Determines the edge location
• Draws a line on the image display where it has found the line
• Gives the operator the ability to approve or override the selected edge location

It performs this for all four edges of the chamber and also makes two measurements on one of the long edges of the chamber to determine the angular offset of the chamber relative to the x-axis of the stage.

We required a unique procedure to guarantee that the images remain in focus across the entire area of interest. The depth of focus of the microscope was less than ±10 µm, while the custom chambers typically are “flat" to only ±50 µm. Because of the time restraints for testing each sample, it is not feasible to have the software iteratively find the focus at each image-capture location (typically 140) on a sample. We developed a novel algorithm in which the software would perform an iterative determination of the focus at five locations on the chamber. The software would then fit the empirical focus data to obtain a 2D polynomial fit that we used to determine the focus, or z adjustment, at every imaging location on the sample. The iterative focus procedure also had the unique CellSpotter System Components (courtesy of Immunicon Corp.) features of performing the focusing on only cells of a user-configurable size range (to ignore noncellular objects in the sample), and in the event no suitable particles were found, we moved it to alternate focus points in the sample.

We logged all the images from a sample into a directory unique to the specific sample ID. We then used another program for postacquisition processing of the images.

The CellSpotter Acquisition program has numerous other features, including:

• 2-level password access
• Auto log-out after a configurable idle interval
• Autocalibration after a configurable time interval
• System configuration screen, editable by supervisors for parameters such as chamber size, µm/pixel, and camera integration time for each filter
• Calibration of backlight and fluorescent sources

We also developed companion utilities with LabVIEW and IMAQ Vision for life-cycle testing and system setup and verification.

Conclusion
Thanks to the power and flexibility of LabVIEW and the IMAQ Vision software, we successfully implemented the CellSpotter system, which is now undergoing field trials at numerous facilities. The functional requirements for the CellSpotter Acquisition software underwent several major revisions during the system development. Because of the modular, graphical nature of LabVIEW and the many powerful IMAQ VIs for common image handling and analysis tasks, all the LabVIEW software development and modifications were accomplished with less than four man-months of labor.n

For more information, contact:
Michael D. Coleman, PhD
Tel (610) 459-9646
Fax (610) 459-9649
E-Mail mikecoleman@colemantech.com

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