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MetaMorph Analysis

Once images have been acquired, researchers can use MetaMorph's extensive analysis tools to measure areas, intensities, distances, volumes, graph intensity levels over time, segment and quantify objects, and more. All measurements can be directly logged to a text file or Microsoft® Excel. MetaMorph gives users complete control over the type of data logged, as well as row and column label locations.

A Complement to Your Confocal System

MetaMorph is a great addition to a core facility's confocal station. With the MetaMorph Offline package, users can measure, analyze, and display multi-dimensional data acquired from a confocal system.

Morphometry Analysis

The Integrated Morphometry Analysis module allows objects to be measured and separated into user-definable classes, based on any combination of measured morphometric parameters, such as shape, size, or optical density. A point-and-click interface, linking images and spreadsheet results, makes it easy to choose representative objects for classification.

Intensity Measurements

MetaMorph is capable of measuring intensity values from selected regions over time, plane number, Z-axis distance, or wavelength from a stack of images.

Intensity data measured, graphed, and exported include: average intensity, standard deviation of the intensity, integrated intensity (summed over all pixels in the region), maximum and minimum grayscale levels, thresholded area (expressed as either numbers of pixels or as a percent of total), and more.

Colocalization

Colocalization in MetaMorph provides quantitative data regarding regions of overlap of two fluorescent probes. The area, average intensity, and integrated intensity in the region of overlap can be measured and saved to a log file.

Time Analysis

Using region tools, making intensity-over-time measurements can be simplified with MetaMorph. Such measurements are important to studies of protein motility or stability, fluorescence recovery after photobleaching (FRAP), protein-protein interactions, fluorescence resonance energy transfer (FRET), gene translation (luciferase assays), calcium transients (aequorin or Fluo-3), and other types of studies.

3D Measurements

The 4D Viewer/3D Measurement module facilitates the visualization of multidimensional data sets, stacks and sequential images. Users can simultaneously view multiple z sections, wavelengths, time points and stage positions in a single intuitive viewing window, as well as binarize multidimensional image data into discrete objects, for 3D isosurface viewing and rotation. Isosurfaced objects selected by mouse are linked to an interactive spreadsheet of 3D object measurements, including volume, intensity, and several other measurement parameters

Figure 1. A Z-series of a nerve stack is thresholded and isosurfaced to obtain volumetric measurements.

Motion Analysis and Kymograph

MetaMorph provides automated and semi-automated analysis of movement-over-time for individual tagged particles, such as fluorescently-labeled cell surface molecules, microtubules, nucleic acids, or lipids.

One or more selected objects can be tracked through each image in a stack or sequential series of images. Measurements of the paths, positions, and velocities of the points can be obtained. Once tracking parameters are configured, MetaMorph determines intensity centroids for the defined target regions, and tracks their displacements automatically through the planes in the source stack.

To measure linear motility, such as distance, time, and velocity, MetaMorph features a kymograph function that creates a cross-sectional view of the intensity values of a user-defined line region through the planes in a stack.

Fluorescence Resonance Energy Transfer (FRET)

FRET involves observing the energy transfer between an excited donor fluorophore and a nearby acceptor fluorophore. This energy transfer is dependent on the overlap of the acceptor excitation spectrum with the donor emission spectrum, as well as the distance between the fluorophores. Energy transfers can only occur when a donor and acceptor are very close together (within nanometers) and their spectra sufficiently overlap. Due to the overlap in spectra of the donor and acceptor, bleed through between observed wavelength channels (filter sets) influence FRET observations.

MetaMorph features a dedicated FRET dialog box to correct for background and bleed through between fluorophore filter sets.

Application Modules

MetaMorph offers eleven user-friendly Application Modules for biology-specific analysis. Each Application Module features a dedicated dialog box with intuitive setting selections, improved segmentation through adaptation to local content, and both field and cell-by-cell data logging. After analysis is complete, users can interactively view tabular and image results side by side. Application Modules may be incorporated into macros for increased customization and automation of analysis. All Application Modules are validated in-house and with third-party collaborators.

Available Application Modules are: A number of different Application Modules can be used to analyze data from common experiments such as:
  • Endothelial tube formation (Angiogenesis)
  • Promotion or inhibition of blood vessel formation (Angiogenesis)
  • Cell viability (Live/Dead, Cell Cycle, Cell Health, Multi Wavelength Cell Scoring)
  • Cell cycle (Mitotic Index, Cell Cycle)
  • Membrane potential (Cell Health)
  • Cytotoxicity and apoptosis (Live/Dead, Cell Health)
  • Cell proliferation (Count Nuclei)
  • Cell migration (Count Nuclei)
  • Cell counting (Count Nuclei)
  • Kinase activation (Cell Scoring)
  • Fatty acid uptake (Cell Scoring, Multi Wavelength Cell Scoring)
  • Mitosis (Mitotic Index, Cell Cycle)
  • Adipogenesis (Cell Scoring, Multi Wavelength Cell Scoring)
  • Examining transfection efficiencies (Cell Scoring, Multi Wavelength Cell Scoring)
  • Studying intracellular structures (Granularity)
  • Receptor internalization (Granularity)
  • Clustering target molecules(Granularity)
  • Process extension (Neurite Outgrowth)
  • Neurodegenerative or neuroregenerative diseases (Neurite Outgrowth)
  • Cell differentiation (Stem cell research) (Neurite Outgrowth)
  • Protein expression & modification (Cell Scoring, Multi Wavelength Cell Scoring)
  • Transient transfection (Cell Scoring, Multi Wavelength Cell Scoring)
  • Detect monopolar spindles (Monopole Detection)
  • Cell signaling (Cell Health, Cell Cycle)

Angiogenesis Tube Formation Application Module

  • Better quantitation by creating a single in-focus composite image from multiple Z-series images
  • Multi-parameter analysis measurements include tube length, number of branch points, number of nodes and more

Figure 1. Left: a best focus image is obtained from a Z series, right: The application module identifies tubes (white) and nodes (green).

Cell Cycle Application Module

  • Classification and quantification of cells in different stages of the cell cycle
  • Option to use specific apoptosis and mitotics stains, for increased classification accuracy

Figure 2. Cell cycle classification can be performed with a single or multi wavelength assay. Left: CHO-K1 cells stained with Hoechst 33342. Right: The Cell Cycle module identifies cell cycle phases: G0/G1 (dark blue), S (light blue), G2 (green), Early M (orange) and Late M (red).

Cell Health Application Module

  • Analysis of up to three fluorescent probes for cell-based apoptosis and necrosis assays
  • Classification and quantification of viable, early and late apoptotic cells, necrotic cells

Figure 3. Adherent Chinese Hamster Ovary (CHO-K1) cells were incubated with various concentrations of staurosporine for 6-12 hours. Top left: control, top right: 0.1 µM staurosporine, bottom left: 3 µM staurosporine, bottom right: measured (Green: viable, blue: early apoptotic, purple: late apoptotic, red: necrotic).

Cell Scoring Application Module

  • Identification of two sub-population of cells
  • Ideal for counting and logging measurements of cells in two-wavelength experiments

Figure 4. The module can be used as a segmentation tool to identify two different stains or to score cells.

Count Nuclei Application Module

  • Automatically counts nuclei and captures intensity measurements
  • Accurate segmentation of touching cells

Figure 5. The Count Nuclei module identifies cells as unique objects.

Granularity Application Module

  • Count punctuate objects
  • Designed for receptor internalization or clustering target molecules
  • Choice of six granularity indices
  • Measurements include count, total and mean area, and intensities of granules and nuclei

Figure 6. U2OS cells, top: overlay, bottom: the module identifies granules and nuclei, even in cells with high background (red arrow).

Live/Dead Application Module

  • Classification and quantification of live and dead cells
  • Measurements include counts and percentages of live and dead cells, per-wavelength measurements and more

Figure 7. CHO-K1 cells incubated with DMSO for 6-12 hours prior to staining. Cells were labeled with H33342 and PI diluted in PBS for 30-60 minutes before image acquisition. Top left: control, top right: 20% DMSO, bottom: measured (red: dead, green: live).

Mitotic Index Application Module

  • Classification and quantification of mitotic and interphase cells
  • Measurements include count, percentage and wavelength-specific intensities of mitotic and non-mitotic nuclei, and more

Figure 8. Left: CHO-K1 cells treated with Nocadazole for 18 hours before staining with anti-phospho-Histone H3 (Ser28). Right: mitotic (green) and interphase (red) cells.

Monopole Detection Application Module

  • Classification and quantification of mitotic cells with monopolar or bipolar spindles
  • Measurements include count and percentage of monopoles, bipoles and interphase cells, DNA structures area, cell classification and more

Figure 9. 3T3-L1 mouse fibroblast cells treated with monastrol and stained with mouse anti-beta tubulin primary antibody detected with a FITC conjugated goat anti-mouse secondary antibody. Nuclei are stained with Hoeschst 33342. Left: monastrol, right: segmented image shows interphase cells (red), bipolar spindles (blue) and monopole (green).

Multi Wavelength Cell Scoring Application Module

  • Multi-parametric analysis of up to seven wavelengths
  • Identification of sub-populations of cells
  • Measurements include scoring profiles, wavelength-specific count and percentage of negative and positive cells, cell-by-cell wavelength-specific stained area, integrated and average intensities.

Figure 10. Top: two wavelength images, bottom left: overlay, bottom right: segmentation.

Neurite Outgrowth Application Module

  • Designed for the measurement and analysis of neurite outgrowths
  • Works with or without nuclear stain
  • Measurements include total neurite outgrowth, total branches and cell bodies, straightness and more

Figure 11. Left: PC-12 cells, middle: the module identifies cell bodies and outgrowths, right: segmentation image.

Read more about MetaMorph: acquisition, processing, customization.