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Application Modules Application Modules

What are Application Modules? • Intuitive, dedicated dialog boxes for biology-specific applications, consistent basic What are Application Modules? • Intuitive, dedicated dialog boxes for biology-specific applications, consistent basic set of input parameters, interactive data and graphics • Adaptive Background Correction™: Improved segmentation through adaptation to local content • Field and cell-by-cell data logging • “Canned, ” walk-away automation • Validated results in-house and with third-party • Can be incorporated into a journal for increased customization and further automation of your analysis

Available modules for version 7. 7 1. Angiogenesis Tube Formation 2. Cell Cycle 3. Available modules for version 7. 7 1. Angiogenesis Tube Formation 2. Cell Cycle 3. Cell Health* 4. Cell Scoring 5. Count Nuclei 6. Granularity 7. Live/Dead 8. Mitotic Index 9. Monopole Detection 10. Multi Wavelength Cell Scoring 11. Neurite Outgrowth 12. Micronuclei

Adaptive Background Correction: robust algorithms built-in • Splitting of touching cells • Detection even Adaptive Background Correction: robust algorithms built-in • Splitting of touching cells • Detection even in noisy and poorly stained images • Stable parameters perform consistently across entire experiment set Analysis automatically adjusts for uneven image background

Running the Application Modules • Modules require grayscale 16 bit fluorescent* images (one for Running the Application Modules • Modules require grayscale 16 bit fluorescent* images (one for each wavelength analyzed) • Modules require a set of size and contrast parameters to adjust the processing the image(s) • Approximate object size range • Intensity above local background levels • The measurements typically include • Object counts and normalized counts • Areas • Intensity, average and total • Cell classifications *some modules can be used with transmitted light also

Running the Application Modules: preview each wavelength Consistent set of segmentation parameters: size range Running the Application Modules: preview each wavelength Consistent set of segmentation parameters: size range and staining intensity Each wavelength configured independently with quick Preview of results

Running the Application Modules: determining size parameters • • • Select the Line tool Running the Application Modules: determining size parameters • • • Select the Line tool from the Region toolbar Move the mouse pointer the edge of a small object and click A tool-tip appears showing the current X and Y values of the pointer, as well as the length Move the mouse pointer to the opposite edge of the object and note the Length value • This number represents the cell width in pixels • If the image is calibrated, the length is in both pixels and calibrated units Type or select the value in microns as “Approximate min width” Repeat steps for a large object—values are approximate to control filtering during segmentation

Running the Application Modules: intensity parameters • The “Intensity above local background” field is Running the Application Modules: intensity parameters • The “Intensity above local background” field is common for all application modules • Part of automatic Adaptive Background Correction system • Specifies the intensity threshold relative to nearby background values • Controls the sensitivity of object detection • Estimate the difference between the object’s signal and background • Hint: gray-level values of image pixels under the mouse cursor are shown in the main Meta. Morph status bar (bottom of main window) • Enter this number in the “Intensity above local background” field

Running the Application Modules: logging measurements Summary Log Parameters Neurite Outgrowth Data Log (Cell) Running the Application Modules: logging measurements Summary Log Parameters Neurite Outgrowth Data Log (Cell) Parameters Neurite Outgrowth

Running the Application Modules: interactive graphics Graphic overlays of segmentation results interact with cell-by-cell Running the Application Modules: interactive graphics Graphic overlays of segmentation results interact with cell-by-cell spreadsheet views of measurement data

Running the Application Modules: interactive data table Cellular Results Table: click on objects in Running the Application Modules: interactive data table Cellular Results Table: click on objects in the table and the corresponding cells in the source images are highlighted in yellow. Click on a cell in the source images and the corresponding data is highlighted in the Cellular Results Table.

Count Nuclei Application Module • Single-wavelength segmentation and counting • Building block for multi-wavelength Count Nuclei Application Module • Single-wavelength segmentation and counting • Building block for multi-wavelength applications • Applications • Cell Proliferation • Cell Counting • Cell Migration • Note: Grey value in result image = cell assigned label #

Angiogenesis Tube Formation Application Module • Applications • Measuring and characterizing endothelial tube formation Angiogenesis Tube Formation Application Module • Applications • Measuring and characterizing endothelial tube formation (a model system for angiogenesis) • Research focused on the promotion or inhibition of blood vessel formation Important in cancer, diabetes and other vascular disease research • Specific kits available from leading manufacturers • Area of research increasing in popularity with success of drugs such as Genentech’s Avastin which blocks VEGF activity – Reference and list available of other similar drugs in clinical trials • Features Top: best focus image bottom: White: tubes, green: nodes. BD Bio. Coat™ Angiogenesis System: Angiogenesis Endothelial Cell Tube Formation Assay. Courtesy of Min Wu, formerly BD Biosciences. • Unique ability to acquire data sets with Z series (~1 mm) to as tubes do not grow in a flat plane in the Matrigel™ matrix • In vitro models in combination with automated image acquistion and analysis are cost effective

Cell Cycle Application Module • Applications • Cancer research • Features • Interactive color Cell Cycle Application Module • Applications • Cancer research • Features • Interactive color coded graphs for data display and setting classification cutoffs • Flexible configuration – Single wavelength mode: Nuclear stain provides DNA content and average intensity indicates mitosis— 5 classifications of cell cycle stage – Optional Mitosis-specific staining – Optional Apoptosis staining (6 th cell classification)

Cell Health Application Module • Three wavelength analysis • • Nuclear stain, apoptotic stain, Cell Health Application Module • Three wavelength analysis • • Nuclear stain, apoptotic stain, dead stain Applications • Apoptosis – Classification of cells into four classes: viable, early apoptotic, late apoptotic, necrotic • Validated with common flow cytometry kits • Cells analyzed by the module. Green: viable, blue: early apoptotic, purple: late apoptotic, red: necrotic. • • Vybrant #7 from Molecular Probes Annexin V JC-1 with Hoechst: used to study loss of mitochondrial potential

Cell Scoring Application Module • Applications: 2 wavelengths • Identifying subpopulations of cells tagged Cell Scoring Application Module • Applications: 2 wavelengths • Identifying subpopulations of cells tagged with a second fluorescent probe – Probe 1: nuclear marker – Probe 2: anything • General and flexible – Examining transfection efficiencies – Kinase activation – pathway analysis – Adipogenesis Top: Acquired image, two wavelengths overlay. Bottom: Analyzed image shows the identification of two probes.

Granularity Application Module • Identification of punctate staining • Robust detection of granules even Granularity Application Module • Identification of punctate staining • Robust detection of granules even in noisy and uneven image backgrounds • Optional nuclear marker for normalized counts • Applications • GPCR internalization • Assays of clustering target molecules

Live/Dead Application Module • Applications • Cell Proliferation • Cell Death – Lack of Live/Dead Application Module • Applications • Cell Proliferation • Cell Death – Lack of cell death associated with cancer – Premature cell death involved in neuromuscular disease (Alzheimer's, Parkinson’s) – Cytotoxicity and Apoptosis • Features • Works well with multiple kits and assays available from a variety of vendors • Flexible 2 wavelength choices – Probes can target any part of cell, does not require a nuclear marker Top: 1 µM Staurosporine. CHO cells in one 96 -well plate at ~10, 000 cells per well. Cells were incubated for ~24 hours and apoptosis was induced by incubating different concentrations of Staurosporine for 6 hours. Assay kit used: Molecular Probes, Vybrant Assay Kit #7. Bottom: Live/dead cells are identified simultaneously.

Mitotic Index Application Module • Applications • Cell Cycle – Active body of research Mitotic Index Application Module • Applications • Cell Cycle – Active body of research dedicated to cancer – Targeting cells in active mitosis using commonly available markers for M phase Left: CHO-K 1 cells treated with Nocadazole for 18 hours before staining with anti-phospho-Histone H 3 (Ser 28). 50 ng/ml Nocodazole. Right: green: mitotic, red: interphase.

Monopole Detection Application Module • Applications • Cancer research – Disruption of normal bipolar Monopole Detection Application Module • Applications • Cancer research – Disruption of normal bipolar spindle formation – Disruption of centrosome separation (e. g. monastrol) • Features • Classification of cells as interphase, bipole or monopole Left: 3 T 3 -L 1 mouse fibroblast cells treated with monastrol and stained with mouse anti-beta tubulin primary antibody detected with a FITC conjugated goat antimouse secondary antibody. Nuclei are stained with Hoechst 33342. Right: segmented image shows interphase cells (red), bipolar spindles (blue) and monopole (green).

Multi Wavelength Cell Scoring Application Module • Applications • • Your research—configure your own Multi Wavelength Cell Scoring Application Module • Applications • • Your research—configure your own custom module easily without the need for programming or macros Configurable from 1 to 7 wavelengths Cell-by-cell and summary scoring profiles reported across wavelengths Features • • • Independently preview individual wavelength settings Customize wavelength and profile naming to match your research Interactive graphics link scoring data between individual wavelengths and full multi-wavelength profiles

Neurite Outgrowth Application Module • Applications • Measuring and characterizing outgrowths (length and branching) Neurite Outgrowth Application Module • Applications • Measuring and characterizing outgrowths (length and branching) • Used in the study of – Neurodegenerative disease such as Alzheimer’s and Parkinson’s – Neuroregenerative research: Spinal Cord Repair – Cell Differentiation (Stem Cell Research) • Features Top: image acquisition. Right: Each filament is assigned to a cell body. All the filaments and cell bodies are then measured. Images courtesy of Kris Poulsen and Davide Foletti, Rinat Neuroscience Corporation. • Complements imaging system -this assay is impossible to perform without imaging techniques • Consistent and faster results over manual tracing which is a prevalent technique in academia

Micronuclei Application Module • Applications • Counting of micronuclei • Counting of mono-, bi- Micronuclei Application Module • Applications • Counting of micronuclei • Counting of mono-, bi- and multinucleated cells • Classification of cells by health, interphase, number of nuclei, presence of micronuclei • Used in the study of – Genotoxicity and chromosomal damage studies – Mitotic abnormalities • Features • Complements imaging system -this assay is impossible to perform without imaging techniques • Faster results over manual scoring which is a prevalent technique in industry

Running the Application Modules: Micronuclei Nuclei and Micronuclei settings Additional probes for cell health Running the Application Modules: Micronuclei Nuclei and Micronuclei settings Additional probes for cell health and others