Telomere length was assessed on TMA sections containing areas of adenocarcinoma using a telomere-specific fluorescence in situ hybridization (FISH) probe and 4’,6-diamidino-2-phenylindole (DAPI) for labeling total nuclear DNA as previously described (6 (link),14 (link)). Each individual TMA spot on each TMA slide was imaged using the TissueFAXS Plus (Tissue Gnostics) automated microscopy workstation and Zeiss Z2 Axioimager microscope. The digitized fluorescent telomere FISH signals were quantified using the TissueQuest software module to analyze the fluorescent images with precise nuclear segmentation. For each case, we evaluated prostate cancer cells and prostate CAS cells. Based on their unique morphologic features, other cell types (e.g. infiltrating lymphocytes) were excluded from the digital image analysis. Men without a valid telomere measurement were excluded (PCBN lower-grade N=4; PCBN higher-grade N=8).
Z2 axioimager microscope
Manufactured by Zeiss
The Z2 Axioimager microscope is a high-performance optical microscope designed for advanced research and analysis. It features a modular design that allows for the integration of various imaging techniques, such as brightfield, darkfield, phase contrast, and fluorescence. The Z2 Axioimager is capable of delivering high-resolution images and data for a wide range of applications, including materials science, life science research, and industrial inspection.
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3 protocols using z2 axioimager microscope
1
Quantifying Telomere Length in Prostate Cancer
2
Automated Whole-Slide Imaging and Analysis
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All chromogenic slides were scanned using a Roche-Ventana DP200 (Roche) or a Hamamatsu Nano-zoomer XR whole-slide scanner (Hamamatsu Photonics), and uploaded to Concentriq (Proscia) for whole-slide image viewing and micrograph documentation. For Telo-CISH images, HALO software (Indica Labs) and its ISH v2.2 and area quantification v1.0 algorithms were used to analyze the images. All fluorescent slides were scanned using the TissueFAXS Plus (Tissue Gnostics) automated microscopy workstation, which contains an 8-slide ultra-precise motorized stage and utilizes a Zeiss Z2 Axioimager microscope. For image analysis, we used the accompanying TissueQuest software module to analyze the fluorescent images with precise nuclear segmentation.
3
Quantifying Telomere-PML Colocalization
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Combined telomere FISH and PML immunofluorescence was performed on cytospins as described above. The TissueFAXS Plus (Tissue Gnostics) automated microscopy workstation was utilized for slide-based imaging. The platform contains an 8-slide ultra-precise motorized stage for high-throughput scanning and utilizes a Zeiss Z2 Axioimager microscope with high quality optics. A separate high-performance workstation with TissueQuest software (Tissue Gnostics) was used to analyze the fluorescent images. For each cell line, automated, precise nuclear segmentation was performed to generate total cell number. Images containing the segmentation masks, along with telomere and PML signals, were exported for analysis using using ImageJ software [60 (link)]. Background subtraction was performed for PML images using a rolling ball algorithm, and a threshold was applied to the remaining spots. Rolling ball and threshold values were empirically determined for each set of images. Colocalization between telomere foci and PML bodies was identified using the Colocalization plugin for ImageJ [46 (link)]. All analyses were limited to segmented nuclei.
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