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

What are Application Modules?

Available modules for version 7.7

1. Angiogenesis Tube Formation2. Cell Cycle 3. Cell Health* 4. Cell Scoring5. Count Nuclei6. Granularity7. Live/Dead 8. Mitotic Index9. Monopole Detection10.Multi Wavelength Cell Scoring11.Neurite Outgrowth12.Micronuclei

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 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

Each wavelength configured independently with quick Preview of results

Consistent set of segmentation parameters: size range and staining intensity

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 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 MetaMorph 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 Data Log (Cell) ParametersNeurite Outgrowth Neurite Outgrowth

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 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

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 (a model system for angiogenesis) • Research focused on the promotion or

inhibition of blood vessel formationImportant 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• Unique ability to acquire data sets with Z series

(~1mm) 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

Top: best focus image bottom: White: tubes, green: nodes. BD BioCoat™ Angiogenesis System: Angiogenesis Endothelial Cell Tube Formation Assay. Courtesy of Min Wu, formerly BD Biosciences.

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 (6th cell classification)

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

• Vybrant #7 from Molecular Probes

• Annexin V• JC-1 with Hoechst: used to

study loss of mitochondrial potential

Cells analyzed by the module. Green: viable, blue: early apoptotic, purple: late apoptotic, red: necrotic.

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 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 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 markerTop: 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 dedicated to cancer

– Targeting cells in active mitosis using commonly available markers for M phase

Left: CHO-K1 cells treated with Nocadazole for 18 hours before staining with anti-phospho-Histone H3 (Ser28). 50 ng/ml Nocodazole. Right: green: mitotic, red: interphase.

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: 3T3-L1 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).

• 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

Multi Wavelength Cell Scoring Application Module

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• 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

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.

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 and others