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. Find out more.
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. Find out more.
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. Find out more.
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. Find out more.
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. Find out more.
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. Find out more.
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.
Find out more.
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.
Find out more.
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.
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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.
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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).
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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.
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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. Find out
more.
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.
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.
Find out more.
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. Find out
more.
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. Find out more.
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. Find
out more.
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. Find out more.
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. Find out more.
Receptor Internalization
The Granularity Application Module is designed to facilitate the segmentation and
analysis of punctate staining. Find out more.
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. Find out more.
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.
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 MDS Analytical Technologies Inc.
MetaFluor® for Olympus
Fluorescence ratio imaging is the monitoring of live cells in which a fluorescent indicator
of intracellular ions is introduced. Indicator dyes have been designed to shift their fluorescence
excitation or emission spectrum when binding with specific ions. Images are obtained at two
different wavelengths, typically matching the absorption bands at the high and low binding conditions.
By rationing the intensities in the images, it is possible to construct a map showing the
local ion concentrations throughout the field of view. Since the monitoring process is nondestructive,
image acquisition can be repeated frequently to trace and monitor the time course of cellular responses.
MetaFluor® for Olympus is designed for dual-wavelength intracellular ion measurements. 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.
Custom Configuration
Toolbars, menus, wizards and dialog boxes help move you through the image
processing steps quickly. Features such as multiple image windows, flexible
device control, synchronization and timing, and journals allow for automated
image acquisition and analysis unlike any other system.
With MetaFluor for Olympus, you customize the set-up once, then let the experiment run
by itself. You are able to collect a large amount of data online and process
it with either MetaFluor® for Olympus or an analysis-only copy of the software.
Monitoring of Live Cells
Regions of interest can be generated automatically or manually placed on
your image to monitor intensity, ratio value or ion concentration.
Measurements are then made simultaneously on all the regions of interest
and update continuously on a scrolling graph, allowing you to follow
dynamic changes as they occur in your living samples.
A display of multiple graphs gives flexibility in the presentation of your
experiment's data. MetaFluor enables you to click on graph traces to
display a readout of the time and data value for the region nearest to
the click.
The Event Mark function is useful to record when drugs or solutions were
added, experimental conditions changed, triggers were received or sent or
other events occurred. You have the option to associate a timer and an
alarm bell to each event. Additionally, for perfused samples, ambient
conditions can be logged and tracked. Each image has an annotation that
is saved within the TIFF file format. The annotation will record
wavelength-dependent settings. Additional information can be stored
in a protocol file.
Export Data for Analysis
If needed, MetaFluor for Olympus can log and export all measurements to either a text
file or to a spreadsheet program such as Microsoft® Excel.
Compatible with MetaMorph for Olympus
Because MetaFluor for Olympus saves images in TIFF file format, you can import them into
MetaMorph for Olympus for further processing and analysis.
Presentation and Publication
Images in MetaFluor for Olympus can be displayed in monochrome, pseudocolor, or using a
variety of user-defined set of values. Ratio images can also be displayed
using a special display mode called Intensity Modulated Display, or IMD.
With the IMD mode, color is used to represent the relative ratio value,
while the intensity or brightness of the color is used to represent whether
the brightness of the source images. This technique helps automate the
process of extracting spatial information from the background, by automatically
eliminating background fluorescence from the scene.
MetaMorph and MetaFluor are trademarks of MDS Analytical Technologies Inc.
