MetaMorph  for Olympus

MetaMorph for Olympus is an advanced digital imaging software for capture, display, and analysis of biological images. Built on the market-proven MetaMorph platform, this powerful package offers functionality, flexibility, and stability that meet the most demanding image acquisition and analysis needs. With over 10,000 publications citing MetaMorph, you can rest assured that your imaging needs will be met now and in the future.
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MetaMorph Applications

Developed in conjunction with researchers, MetaMorph® for Olympus offers a range of tools for a variety of applications such as:



3D Deconvolution

Out-of-focus intensities are present in all acquired images. These intensities can be accounted for by observing the behavior of light originating in a point source and passing through the microscope optics. This behavior is described by the Point Spread Function (PSF). It can be used to quantitatively compensate for the blurring of images due to out-of-focus information. This process is called deconvolution.

MetaMorph's 3D Deconvolution module from AutoQuant helps improve images by reassigning out-of-focus intensities back to the spatial locations to where they originated. The results are images with sharper definition and lower background, better contrast, and improved signal-to-noise ratio.

3D Reconstruction

The 4D Viewer enables the visualization of multi dimensional data sets, time lapse or Z stacks. Users can simultaneously view multiple Z sections as a 3D reconstruction, with multiple , wavelengths, time points, and positions in a single intuitive viewing window. Users can interactively rotate the 3D view and obtain volumetric measurements.

A 3D model consisting of rotated views from a stack of images can also be created. Using a stack of planes from a Z-series, users may configure the angle, orientation, Z-axis distance, and reconstruction type for the model.

Angiogenesis

The Angiogenesis Tube Formation Application Module facilitates the acquisition and analysis of tube formation experiments. To capture the three-dimensional behavior of tubes, MetaMorph allows for Z-series acquisition. A best focus image is then obtained from the stack of images, and the analysis is run on that image, identifying tubes and nodes.

Intensity Measurements

Users can obtain intensity measurements from selected regions over time, plane number, Z-axis distance, or wavelength from a stack of images.

Cell Counting

The Count Nuclei Application Module is designed to automate accurate counting of nuclei for most types of cells. The module counts nuclei even when the background is uneven, providing superior segmentation compared to simple thresholding. This module can be used to count nuclei across large data sets and log the results to a spreadsheet.

Cell Cycle

The Cell Cycle Application Module is designed for the classification and quantification of cells in various stages of the cell cycle using a DNA stain. Additionally, a mitosis-specific probe may be used to better identify M-phase cells and an apoptosis-specific probe may be used to identify cells undergoing apoptosis.

Cell Migration/Cell Proliferation

The Count Nuclei Application Module is designed to automate accurate counting of nuclei for most types of cells. The module counts nuclei even when the background is uneven, providing superior segmentation compared to simple thresholding. This module can be used to count nuclei across large data sets and log the results to a spreadsheet.

Cell Viability

Researchers conducting cell viability studies can use a number of Application Modules to analyze their results, such as Live/Dead, Cell Cycle and Multi Wavelength Cell Scoring.

The Live Dead Application Module is designed to automate accurate classification of cells after staining with a variety of live/dead staining kits.

The Multi Wavelength Cell Scoring Application Module is designed for the scoring of cells with up to seven fluorescent stains. This flexible module is ideal for segmenting and providing measurements of cells in multiple wavelength experiments.

Colocalization

Fluorescence imaging can be used to follow a protein through development, over time, or after stimulation. Colocalization tools provide intensity measurements of the region overlap between two fluorescent probes.

Cytotoxicity and Apoptosis

A number of Application Modules can be used to analyze and quantify cytotoxicity and apoptosis, such as Live/Dead and Cell Health.

The Cell Health Application Module is designed for the classification and quantification of apoptotics and necrotic cells using three different dyes.

The Live/Dead Application Module is designed to automate accurate classification and quantification of cells after staining with a variety of live/dead staining kits.

Deconvolution

For 2D deconvolution, MetaMorph offers No Neighbors and Nearest Neighbors algorithms to remove out-of-focus information from individual planes or a Z-stack. Both modules use an estimated three-dimensional Point Spread Function (PSF).

The 3D deconvolution module can be used for analysis of Z-stacks and may be configured to use either a Blind algorithm which estimates a Point Spread Function (PSF) or an algorithm where a measured PSF which characterizes the objective lens is provided.

Densitometry

MetaMorph can perform quantitative densitometry. The software will display the optical densities of a brightfield source image in a scaled 8-bit or 16-bit image.

Fluorescence Overlay

To better visualize events, up to six fluorescence images can be overlaid over a background Differential Interference Contrast (DIC) or phase contrast image.

Figure 1. Sample overlay of two images.

sample overlay of two images

Fluorescent in situ Hybridization (FISH)

FISH is used for the detection of target DNA or RNA molecules with a system of coupled fluorochromes. The detection of nucleotidic sequences on a combed DNA molecule is performed indirectly, by first hybridizing the sought nucleotidic sequences with the combed DNA (also called the matrix DNA or target). If the probes are synthesized with incorporated fluorescent molecules or antigenic sites which can be recognized with fluorescent antibodies, the direct visualization of the relative position of the probes is possible.

MetaMorph easily automates the process of acquiring, color combining, and visualizing multiple wavelengths from FISH and immunocytochemistry experiments.

Figure 2. Chromosome Fluorescence In Situ Hybridization. Acquired at the Cold Spring Harbor In Situ Hybridization Course.

Chromosome Fluorescence In Situ Hybridization. Acquired at the Cold Spring Harbor In Situ Hybridization Course.

Fluorescence Immunocytochemistry

Immunocytochemistry is the in situ detection and demonstration of cellular constituents using specific antigen-antibody reactions. It has evolved to become an integral aid in modern histopathology. Crucial to its success has been the development of technologies that have allowed the highly sensitive and reliable detection of cellular markers within routinely fixed and processed samples. MetaMorph for Olympus can acquire, combine, and measure images of one or more wavelengths from immunocytochemistry and in situ hybridization experiments.

The morphological measurement tools in MetaMorph are ideal for processing immunohistological tissue samples. MetaMorph for Olympus can acquire, combine, and measure images of one or more wavelengths from immunocytochemistry and in situ hybridization experiments.

Fluorescence Recovery After Photobleaching (FRAP)

FRAP is a live-cell imaging technique used to study the mobility of fluorescent molecules. Both MetaMorph for Olympus and MetaFluor® provide mechanisms for triggering photobleaching, background subtraction, and shading correction. Most importantly, both support high speed acquisition from low light level cooled CCD cameras. MetaFluor specifically provides real time measurements and graphing of DeltaF/F ratios from multiple regions of interest as the experiment progresses.

MetaMorph for Olympus offers laser control and supports rapid shuttering of illumination to minimize photobleaching before exposure to the laser light and while monitoring recovery. Maximal temporal resolution can be achieved with cameras that support streaming subsequent to laser illumination. MetaMorph for Olympus also controls the MicroPoint® Laser System from Photonic Instruments to perform photobleaching routines.

Figure 3. Left: pre-bleach, right: post-bleach (yellow circle).

Left: pre-bleach, right: post-bleach (yellow circle)

Fluorescence Resonance Energy Transfer (FRET)

FRET involves the non-radiative transfer of energy from a fluorophore in an excited state to a nearby acceptor fluorophore. FRET may occur when fluorophores are within angstroms of one another. This technique is used to infer protein-protein interaction and colocalization.

Several key features make MetaMorph for Olympus a powerful platform for FRET imaging. First, FRET takes place at extremely low light levels and depends on the detector for most signal amplification, so dark current noise must be minimized. MetaMorph for Olympus supports highly sensitive cooled CCD cameras with high quantum efficiency (less noise) and fast readout rates-ideal tools for this application.

Second, FRET images are taken at different wavelengths.MetaMorph for Olympus can acquire multiple dimensions (XY, Z, wavelengths, stage positions, time), which makes it easy to handle automated wavelength devices and automatically align multiple images.

Third, speed is key to FRET experiments. MetaMorph for Olympus and MetaFluor meet this challenge with its support for multi-wavelength streaming using appropriate devices. Finally, a FRET-specific dialog box automates the complex image arithmetic needed to account for and correct fluorescent background and bleedthrough in your images.

Figure 4. Monitor interactions between two proteins. Top row: Both YFP-PP1gamma are expressed. NIPP1 binds and retargets PP1 to nuclear speckles outside of nucleolus. Bottom row: Mutant form of CFP-NIPP1. It does not bind PP1, so cannot retarget speckles from nucleolus. After bleed-through correction, minimal FRET can be observed (right). Images acquired during the FISH course, Cold Spring Harbor Lab, NY.

Monitor interactions between two proteins. Top row: Both YFP-PP1gamma are expressed. NIPP1 binds and retargets PP1 to nuclear speckles outside of nucleolus. Bottom row: Mutant form of CFP-NIPP1. It does not bind PP1, so cannot retarget speckles from nucleolus. After bleed-through correction, minimal FRET can be observed (right). Images acquired during the FISH course, Cold Spring Harbor Lab, NY.

Fluorescence

MetaMorph for Olympus can acquire multiple wavelengths using either Multi Dimensional Acquisition or Acquire Multiwavelengths. Each wavelength image can then be segmented and analyzed, and the images can be color combined together for better visualization.

Live/ Dead

With MetaMorph for Olympus, users can count cells that have been labeled with two different fluorescent dyes to specifically study cell viability using the Live/Dead or Cell Scoring Application Modules.

The Cell Scoring Application Module is designed for the identification of two subpopulations of cells. Wavelength-specific parameters can be set and intensity and area measurements are included on a wavelength-by-wavelength and cell-by-cell basis.

Manual cell counting options are also available.

Morphometry

Integrated Morphometry Analysis (IMA) is one of the powerful tools MetaMorph for Olympus provides for counting cells and measuring cell shapes, sizes, intensities, and other parameters. IMA can be used for analysis of both monochrome and color images, or regions of interest of an image. Users can perform numerous different morphometric measurements of cells. Researchers can also select parameters for measurement or define filters, which restrict measurements to objects that meet defined criteria.

Integrated Morphometry Analysis

Motion Analysis & Particle Tracking

MetaMorph for Olympus provides tools for motion analysis and particle tracking, enabling users to follow the movement of tagged particles over time, such as fluorescent-labeled cell surface molecules, microtubules, nucleic acids, lipids, and other objects with sub-pixel resolution.

Motion Analysis & Particle Tracking

Mitosis

The Mitotic Index Application Module is designed for the quantitative discrimination of mitotic and interphase cells. Cells are labeled with a DNA stain and a mitosis-specific marker, such as immunofluorescence staining for Histone 3 S10 phosphorylation. Two different wavelengths are acquired and the images are analyzed with the module.

Monopole Detection

In some serious diseases such as cancer, cells proliferate uncontrollably, such as cancer. Progression through mitosis may be stopped by simply disrupting the formation of normal bipolar spindles. Recently, a new compound named monastrol was found to disrupt spindle formation by affecting centrosome separation. In comparison with microtubule drugs, this effect was specific to mitosis. When the two centrosomes fail to replicate or separate, a monopolar spindle forms instead of a normal bipolar spindle.

The Monopole Detection Application Module is designed for the quantifictation of mitotic cells with monopolar or bipolar spindles where cells are labeled with a DNA stain and a second probe for microtubules.

Multi Dimensional Imaging

Multi dimensional imaging tools in MetaMorph can automatically acquire and display images in X and Y (XY dimension), Z or multiple focus series (Z dimension), multiple fluorochromes (wavelength dimension), time lapse (time dimension), and multiple stage positions (stage dimension). For any multi-dimensional experiment, more than 200 different combinations of features can be selected. Users can also produce movies, montages, color-combine images, measure images, and perform 3D reconstruction on all or any part of the data sets.

Neurite Outgrowth

Inhibition or stimulation of neurite outgrowth is implicated in a broad range of CNS disorders or injuries including stroke, Parkinson''''s disease, Alzheimer''''s disease, and spinal cord injuries. The Neurite Outgrowth Application Module is designed to facilitate the analysis of neurite outgrowth experiments.

Ratio and Calcium Imaging

MetaFluor is designed for dual-wavelength intracellular ion measurements, making it an ideal tool for ratiometric imaging. The system provides simultaneous display of the raw data, ratio image, graphs of intensities, ratios and ion concentrations, and a non-ratiometric image such as a brightfield or phase-contrast image. Two different ratiometric indicators can be imaged and measured simultaneously.

Ratio and Calcium Imaging

Receptor Internalization

The Granularity Application Module is designed to facilitate the segmentation and analysis of punctate staining.

Stitching

Image stitching is a convenient option to use when working with large samples, high-resolution objectives and a scanning stage. Using the Stitch Stack command in MetaMorph, related fluorescence or transmitted light images in a calibrated stack can be smoothly knitted together into one complete image.

The Scan Slide module can be configured to automatically scan an area larger than the field of view, by acquiring multiple images and stitching them together-ideal for large tissue samples. Users simply choose the top left corner and bottom right corner of the desired area to acquire, and MetaMorph does the rest. Once MetaMorph has scanned the area and tiled the images on the fly, users can select a region of interest and view a high-resolution image that is automatically stitched. Module includes a step-by-step calibration wizard.

Time Lapse Experiments

Easy-to-use tools in MetaMorph for Olympus simplify taking measurements involving intensity changes over time. Such measurements are important to studies of protein motility or stability, wound healing, cell division, microtubules dynamics, FRAP, protein-protein interactions, FRET, gene translation (luciferase assays), calcium transients (aequorin or Fluo-3), and other types of studies.

Tissue Imaging

The Scan Slide module can be configured to automatically scan an area larger than the field of view, by acquiring multiple images and stitching them together-ideal for large tissue samples. Users simply choose the top left corner and bottom right corner of the desired area to acquire, and MetaMorph does the rest. Once MetaMorph for Olympus has scanned the area and tiled the images on the fly, users can select a region of interest and view a high-resolution image that is automatically stitched. Module includes a step-by-step calibration wizard. Find out more.

Volume 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.

Volume Measurements

Z Series

MetaMorph for Olympus has the ability to automatically acquire a series of frames at specified Z positions (focal planes) using Z motors and high-speed focus changers. Z-series acquisition can be used with device streaming and time lapse to take 3D data-sets of living cells. Z-series can also be combined with auto-focusing, acquisition of multiple wavelengths, stage positions, and time points.

MetaMorph and MetaFluor are trademarks of Molecular Devices, Inc.

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