Automated imaging studies speed human-cell research

At the National Institutes of Health (Frederick, MD), Dr. Martin Brown and his colleagues are investigating the adhesive interactions of human T-cells with other cells. In his experiments, Brown uses a Zeiss LSM410 laser scanning confocal microscope to examine the redistribution of signaling and structural molecules in T-cells after membrane receptor stimulation.

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Automated imaging studies speed human-cell research

At the National Institutes of Health (Frederick, MD), Dr. Martin Brown and his colleagues are investigating the adhesive interactions of human T-cells with other cells. In his experiments, Brown uses a Zeiss LSM410 laser scanning confocal microscope to examine the redistribution of signaling and structural molecules in T-cells after membrane receptor stimulation.

"The experiments involve cross-linking the T-cell antigen-receptor complex with antibody-coated, cell-size latex beads to partially mimic the interactions of T-cells with antigen-presenting cells (APCs) that occur during the initiation of an immune response," explains Brown.

The binding of T-cells to the APCs triggers rapid and dramatic changes in cell shape. To perform this analysis, Brown is using Image-Pro Plus software from Media Cybernetics (Silver Spring, MD) to examine a three-dimensional confocal image series of fluorescently labeled cell-bead conjugates. By quantifying the spatial changes of F-actin and other molecules in the T-cells after bead stimulation, Brown hopes to understand the signal-transduction pathways controlling cell responses.

To study these effects, T-cells are first bound to a 6-mm latex bead on a glass slide (see Fig. 1). Fluorescent agents are used to mark cell features such as actin and signaling molecules in red and green, respectively. After acquiring a series of z-scan images through the cell and bead, Image-Pro is used to draw two rectangular regions of interest (ROIs) around the bead and cell-bead combination. A personal computer is then used to determine the geometric centers of gravity for the two ROIs and to perform a series of 11 equally spaced z-scans centered about the line that intersects the two x-y centers of gravity.

"A typical experiment involves six samples and 11 z-scans from 10 cells in each sample for a total of 60 eleven-image sequences," says Brown. To process these data, Brown uses three Image-Pro macros for formatting, setup, and automation. During operation, the images from the Zeiss confocal microscope are first merged into Image-Pro files. Each of the 60 sequences is then selected manually, and an ROI is drawn around the cell and bead. Then, the processing macro is activated. This macro automatically opens the sequence of images, crops them to the size of the prechosen ROI, performs low-pass filtering, extracts the red and green information, and exports the information to a set of 60 Microsoft Excel spreadsheets.

"By using macros especially written for Excel," says Brown, "the eleven z-scans of a single cell (see Fig. 2) are converted to a 10 ¥ 11 matrix of either average or integrated pixel intensities." This procedure allows data from different size cells to be merged and averaged, so that differences between groups can be statistically analyzed (see Fig. 3).

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FIGURE 1. Human T-cell line interacts with a 6-µm latex bead. The F-actin cell feature is stained in green and the tubulin feature is stained in red. Each image is an x-y plane acquired by a Zeiss confocal microscope. The transmitted light image (lower left) shows the orientation of the acquired z-scans.

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FIGURE 2. Series of z-scans is generated from the human T-cell shown in Fig. 1. The red-green-blue series is displayed in the left column. The extracted red in the middle column represents tubulin, and the green column at the far right represents F-actin.

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FIGURE 3. Three-dimensional intensity profile of the cell in Fig. 1 can be generated from the z-scans of Fig. 2. The red area indicates the position of the bead`s geometric center of gravity.

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