| Tissue Section Analysis |
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| Introduction |
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| The instruments are ideally suited for tissue analysis. Quantitation of tissue constituents can be determined using appropriate fluorescent markers. Analysis of tissue (and other samples) is accomplished using two approaches which may be used independently or as a combined analysis: |
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- Cellular segmentation—Identifying cells either by identifying nuclei and then measuring associated fluorescence or by identifying cellular constituents. Due to the complex nature of some tissues, it may be impossible to identify single cells.
- Phantom contouring—Estimating tissue constituents using a stereological random-sampling approach. This approach may be combined with cell-based analysis.
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| Highlights of Analysis with iGeneration Imaging Cytometers |
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| The images below demonstrate the problem of identifying cells using a nuclear DNA marker. |
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Tissues vary with respect to nuclear density. The liver section has well defined, isolated nuclei with areas of cytoplasm separating adjacent nuclei. Identifying these nuclei in an automated system is easily done, and the fluorescence associated with each nucleus may be measured. The example on the right has closely packed nuclei with little cytoplasm separating the nuclei. The liver sample can be analyzed either by nuclear identification or using a random sampling approach. The spleen, shown on the left, must be analyzed by either identifying a subset of cells or using a random-sampling approach.
Tissue analysis by nuclear identification and measurement of associated fluorescence is shown in the examples below.
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| BCL Staining in Mini-Pig Dermal Sections – Provided by Dr. Danielle Roman, Novartis, Basel, Switzerland |
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In the example above, mini-pigskin was treated with an Adenovirus designed to transfect cells with BCL-2. LSC was used to measure the expression level of BCL-2 in a dermal section. The left scattergram resolves DNA content (X axis) vs. BCL-2 expression level (Y axis). Events (or cells) expressing BCL-2 fall into the red-colored region. The center image is a scattergram that plots the X and Y position of events. The red identifies events that fall in the red region in the first scattergram. Note that the highest expression of BCL-2 is seen near the skin’s surface. This observation is confirmed by the epi-fluorescence image on the right, where nuclei appear as red and BCL-2 appears green.
The color-composite laser-scanned image below shows a tissue analyzed for DNA breaks. The scatter image of the tissue is colored gray, nuclei are shown in blue and apoptotic cells are green.
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| The apoptotic population may be quantified using histogram plots or obtaining measured values such as the number of apoptotic cells or the amount of green fluorescence associated with these cells (red and green circles in the table below, respectively). |
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| Using Phantom Contours (Random Sampling) |
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| The example shown below is a tissue where the nuclei are closely spaced. Phantom contours are randomly placed by iCyte or iCys to measure fluorescence in this sample. Phantom contours in this sample may fall in areas where nuclear staining co-localizes with a cell-specific marker (A, yellow), areas of DNA staining in nuclei only (B, red) or in areas with no fluorescence (C). |
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| Plotting the fluorescence in a scattergram comparing nuclear vs. cytoplasmic marker fluorescence will resolve these three types of phantoms. Applying a color code allows these different phantoms to be seen in a plot of their X and Y position, showing their position in the tissue. |
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| In this scattergram and expression map: |
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- A-type phantoms are green.
- B-type phantoms are red.
- C-type phantoms are blue.
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| The green areas indicate areas expressing the tissue constituent of interest, allowing comparison of quantitatively different tissue samples and correlation of expression levels with structural characteristics in each tissue. |
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| Automated tissue analysis |
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| iCyte can use an optional robot loaded with up to 45 4-slide holders. Each slide can contain any number of tissue pieces. |
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In the example shown above, twelve slides were analyzed, each with two tissue sections. iCyte, using the optional iNovator Application Development Toolkit, can automatically identify these tissue sections and determine a scan area for high-resolution analysis (seen as a red rectangle).
Once the tissue sections have been scanned and data generated, reports can be created, using the iBrowser™ Data Integration Software, to combine images and quantitative data.
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| Benefits of Using iGeneration Technology |
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| Quantitative information for tissue constituents may be correlated with tissue structure because of the high-quality imaging capabilities of the laser scanning cytometer. Multiple lasers and detectors allow for complex assays requiring multiple markers. Analysis may be done using identification of individual cells or using a random-sampling approach. iGeneration systems allow you to perform automated analysis of multiple slides with multiple tissue elements. With iCyte, the optional robot feature automates the analysis of up to 45 4-slide plates. |
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| More Information |
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For more information, review the CompuCyte Published Materials and CompuCyte Bibliography.
Request the Tissue Analysis Application Note.
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