Imaging Applications

• Immunology

  NF-kB translocation in T:APC conjugates

  

  Measurement of NFkB translocation in transgenic T cells which are in contact with antigen presenting cells (APC) and specific peptide. Specific T cell:APC conjugates are identified and translocation of NFkB from the cytoplasm to the nucleus specifically within the T cells is measured in the presence (shown) or absence of peptide.

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• Drug Discovery

  Co-localization of an antibody-drug conjugate to endosomes or lysosomes

  

  In this experiment we use the capabilities of the ImageStream system to measure the transit of a fluorescently labeled antibody-drug conjugate (ADC) through the cellular endocytic pathway. The high spatial resolution afforded by the ImageStream system allowed accurate measurement of ADC co-localization to endosomes and lysosomes.

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• Oncology

  Apoptotic index using the ImageStream

  

  DNA condensation and fragmentation are hallmarks of apoptosis. By measuring the area and the intensities of the brightest portions of the nuclear image, the bright, punctate nuclear imagery of apoptotic cells can be distinguished from the evenly stained nuclear imagery of a normal, healthy nucleus. This makes possible the automated identification of apoptoticWhen cells begin to dye by apoptosis there is fragmentation and condensation of the DNA. By measuring the area and the intensities of the brightest portions of the nuclear image, the bright, punctate nuclear imagery of apoptotic cells can be distinguished from the evenly stained nuclear imagery of a normal, healthy nucleus. This makes possible the automated identification of apoptotic nuclear morphology.

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• Parasitology

  Trypanosomes

  

  Trypanosoma brucei is a parasite which causes African sleeping sickness. Shown in the figure is a study in which nuclear localization of a GFP tagged molecule was quantitated in two samples. The histogram shows the Similarity score for a cross correlation between the GFP image and the DRAQ5 nuclear image for each cell. A high Similarity score indicates that the GFP image and the DRAQ5 image are highly similar (GFP has translocated to the nucleus). A low Similarity score is obtained for untranslocated events.

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• Biochemistry

  FRET using the ImageStream for detection of protein-protein interactions

  

  In fluorescence microscopy, light at one wavelength is absorbed by a fluorophore and emitted at a longer wavelength. FRET (Fluorescence or Forster Resonance Energy Transfer) can occur when a second fluorophore is in very close contact such that it can accept the energy from the first fluorophore and emit the light at an even longer wavelength. The efficiency of the transfer is extremely sensitive to the separation distance between the fluorophores and therefore when the emission is detected at the longer wavelength the conclusion is that the fluorophores were within approximately 10 nanometers of each other. When two fluorophores (a donor and acceptor pair) are used to label two different proteins, the close proximity of the two proteins are inferred by the measurement of the FRET from the donor to the acceptor fluorophore. The combination of high speed image acquisition and automated quantitative image analysis with FRET on the ImageStream allows measurement of the spatial location of the protein interaction within the cell or between cell conjugates even for rare subpopulations. Distinguishing intracelluar location of FRET vs. location of the proteins when not exhibiting FRET behavior offers a major advancement in understanding signaling pathways.

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• Hematology

  Erythroid differentiation

  

  As hematopoeitc stem cells differentiate through the erythroid lineage, they shrink and eventually enucleate as they progress towards becoming RBC. They also progressively gain expression of glycophorinA and lose CD71 expression. The extruded nuclei express low levels of glycophorinA on their membranes. This experiment demonstrates the unique ability of the ImageStream to classify cells using a combination of intensity, morphology, and location based parameters.

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• Microbiology

  Identification of Harmful Bacteria and Analysis for Filamentation

  

  Species of bacteria from culture or from contaminated food supplies can be identified and quantitated by analyzing for fluorescence intensity of species specific FISH probes, size, and shape. Filamentation, either due to defective replication or as a response to evade host innate immunity can be measured.

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• Toxicology

  Micronuclei testing

  

  The micronucleus assay is the preferred method for evaluating the genotoxicity of drugs and other chemical agents. This experiment demonstrates the ability of the ImageStream system to rapidly classify apoptotic, bi-nucleated, and micronucleated events within cell populations simply using brightfield and nuclear imagery.Traditionally, cell biologists performing micronucleus assays have had to choose between manual microscopy, which is time-consuming and tedious, and flow cytometry, which can rapidly process large cell populations but lacks the ability to image the nucleus in intact cells. The Amnis^® ImageStream system solves both problems by combining flow cytometry with sophisticated digital imaging and analysis capabilities.

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• Virology

  HIV-specific translocation of NFAT

  

  HIV-specific nuclear localization of NFAT was measured in HIV-experienced T cells from peripheral blood of HIV+ patients. HIVgag peptide specifically stimulates NFAT (green) to move to the nucleus (red) in HIV-tetramer positive cells (orange). The Similarity score correlates DRAQ5 nuclear stain with the NFAT signal. The higher the Similarity score, the more translocation is visualized in the example images from each quadrant.

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• Oceanography

  Analysis of organisms in wharf tows by shape and size

  

  Unique species of diatoms and other plankton species from culture or from seawater can be identified and quantitated by analyzing for size, shape, fluorescence intensity, texture, and location/co-location of fluorescent labels and/or chloroplasts.

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