ImarisCell - Software to Analyze & Visualize Biological Cells | Imaris
 

ImarisCell

ImarisCell is an Imaris module specifically designed for the analysis of 2D, 3D, and 4D images of cells and their components. ImarisCell enables researchers to qualitatively and quantitatively examine micro relationships that exist between cells. ImarisCell also allows researchers to map organelles and other cellular components within individual cells, then examine the relationships between those components.

  • Examine relationships between cells and cellular components
  • Utilize biologically meaningful image analysis units
  • Detect cells based on cytoplasm or plasma membrane labelling
  • Detect and classify vesicular objects of various populations
  • Automatically works on 2D, 3D or 4D datasets

Making Sense Out Of Your Cell’s Relationships

All known living organisms are composed of cells. ImarisCell now allows you to use this natural and universal unit when doing your image analysis work. ImarisCell can identify individual cells in addition to their nuclei and multiple populations of intracellular vesicular objects.

You can now detect cell membranes not only when the cytoplasm is labelled but also when the plasma membrane and nucleus are labelled. Imaris Cell offers three distinct Cell detection routines; one ideal for samples with a marker that highlights all or part of the cytoplasm and two other algorithms ideal for when only plasma membrane and nuclei are labelled.

With ImarisCell, the fundamental unit of analysis has changed from a single image to clusters of cells, individual cells, or components of cells. This evolution prevents unnecessary generalizations caused by the limitations of single image analysis and facilitates specificity at the biological level of detail dictated by your research.

ImarisCell goes beyond what other image processing programs can do by using the biologist's concept of cells and applying topological constraints on the detection of organelles. It encapsulates Bitplane's powerful image analysis algorithms and simple yet high-level creation wizards to manage the detection, display and editing of multiple objects (i.e. Cells, Nuclei and multi-class, multi--sized Vesicles). As always, Bitplane provides an interactive 3D interface with real time previews, interactive sorting and object selection using: filters, graphs, statistics tables, or by selecting directly within the image.

ImarisCell provides the platform necessary for 4D analysis (tracking 3D structures over time) which allows changes in size, shape, intensity, and position to be examined between cells or between organelles within a cell. This provides researchers with the opportunity to examine communication mechanisms as well as mechanical and structural functions of cells and cell components.

Ability to utilize a biologically meaningful analysis unit

Classically, microscopists have been forced to analyze their data on a per field or per image basis. They could count, for example, the number of cells in the entire image or the number of vesicles in the entire image, but could not easily count the number of vesicles in each individual cell when an image contained more than one cell. ImarisCell allows the researcher to determine which analysis unit they would like to use.

  • Analysis can be classically performed on the entire image
  • Analysis can be performed on a per cell basis i.e statistics for components within each cell
  • Analysis can be performed on a per component basis i.e. statistics on the individual components such as nuclei and vesicles
  • Analysis can be performed on combinations of objects that meet certain statistical criteria (see object / object relationships below)

Ability to segment up to three biological components simultaneously

ImarisCell provides researchers with the ability to segment up to three different types of structures at the same time and allows those structures to be related to one another in 4D space / time.

  • Researchers segment the "cell" structure based on a cytoplasm or membrane stain. The cell component is required because if other structures are also segmented these additional structures must be related to the cell. The user can choose any color channel for this component.
  • The researcher has the option of segmenting the nucleus of each cell. Since a nucleus must belong to a cell it is essential to detect the nucleus and cell simultaneously. The nucleus can be segmented from a separate color channel.
  • The researcher has the option of segmenting vesicles. A "vesicle" for ImarisCell can be any foci-like structure within a cell. In addition to actual vesicles this ImarisCell entity could be used to identify vacuoles, lysosomes, nucleoli, centrosomes, etc. In addition to this functionality, ImarisCell offers an additional analytical capability with a new XTension. It allows for finding vesicular objects outside the cell and calculating the distance between them and the cell. Because these components are related to the cell, it is essential that they are detected at the same time as the cell. Vesicles can be segmented from the same color channel as the nucleus or cell or a different color channel.

A structured creation wizard that imposes appropriate biological constraints

ImarisCell takes biologists' knowledge about the appropriate structure of a cell and utilizes this during segmentation. This allows the program to deal with common problems associated with imaging cells and allows researchers to obtain more accurate results.

  • The Nucleus - Biologists understand that a cell is a container which is defined by a membrane. The Nucleus of the cell must reside within this container. However, during imaging it is often possible to have very limited staining of the membrane immediately surrounding the Nucleus. This would cause the Nucleus, if it were near the edge of the membrane, to appear outside of the Cell, which as biologists we know is typically not possible in living cells. ImarisCell provides several options to correct this:
    • Expand the Cell area to contain the Nucleus
    • Reduce the Nucleus to be within the area defined as the Cell
  • Many types of cells should only have one Nucleus. ImarisCell provides an option to eliminate other objects detected within a single cell as nuclei so that there is only one nuclei per cell. This is a huge benefit when nuclear staining is poor.
  • In cases where Nucleus appears to be outside a cell it can be removed from analysis by selecting this option during the Cell object creation. This ensures that only real nuclei enclosed with cells are considered for analysis that may be focused on data like nuclear envelope volume or area.
  • Closely clustered nuclei that belong to different cells may pose an analytical problem if both nuclei are wrongly allocated to one cell. You can now split such nuclei by nucleus seed growth process that ultimately separates both objects.
  • Cell Creation Wizard – Split Cells by Seed Points – Cells that are clustered together and lack a clear nuclear signal may be difficult to segment. In such case it is possible to differentiate between and ultimately produce individual cell objects by splitting cells by cell seed growth process that separates these objects.
  • Cell Creation Wizard Fill holes in Cell and Nucleus - If a Cell or Nucleus object you wish to analyze contains interior holes you can eliminate them quickly, while still retaining the existing object segmentation. The filling is accomplished by setting the voxel in each hole set to a new intensity value, threshold value, and assigning them to the surrounding object thus closing the hole inside the Cell or Nucleus object.
  • Detect and visualize vesicles that belong to different populations - sub-cellular organelles typically contain various vesicular structures that belong to different functional categories. They fulfill different roles and it often can be advantageous to discern different populations, or pools, of intracellular vesicles. ImarisCell offers this capability and allows creation of separate Spot objects corresponding to various classes of vesicles. Within each class vesicles can have a range of diameters (i.e. the diameter of each vesicle can be determined automatically by directly utilising the intensity and volume information in the raw image).

Ability to examine object / object relationships

ImarisCell gives researchers the ability to examine complex relationships between cells, between cell components, or of components within a cell. This ability is derived from the fact that ImarisCell understands that a cell is a highly complex micro environment which includes both nuclei and vesicles. Therefore statistics can be generated on a per cell basis and spatial relationships within a cell can be determined. By utilizing ImarisCell's new concept of cell's components hierarchy, the user can export key statistical information about an object bearing a unique parent object ID. Analyzed vesicles or nuclei can now be easily allocated to its parent structure, i.e. it is possible to determine whether object of interest belongs to given Cell.

Utilizing the filtering and classification of ImarisCell researchers can begin to ask a series of advanced questions and receive answers to those queries. For example, ImarisCell could easily identify all the cells in the image where the various populations of Vesicles inside the Cell were close to the membrane and the Cell itself was moving quickly relative to the other cells in the image.

Ability to use and import Imaris objects into ImarisCell

If users already have segmented datasets using existing Imaris components such as Spots and Surfaces or if users do not want to use the ImarisCell component for the segmentation because they are used to working with the other Imaris objects, they can still benefit from the power of ImarisCell. ImarisCell allows any Surface object (automatically, semi-automatically, or manually created) to be imported into ImarisCell as either a Cell or Nucleus. Likewise, any Spots object (automatically or semi-automatically created) can be imported to ImarisCell as a new vesicle type or as one of the previously detected types. This benefits users because they can utilize the wide range of segmentation functions in Imaris and still benefit from the object relationships report established in ImarisCell.

Biologically meaningful object statistics

Because ImarisCell knows that the relationship between components of the cell is important for research and because it can make associations between components, it can therefore directly provide statistics that are important to biologists.

The location of vesicles within a cell can provide many clues about cellular function or functions of those vesicles within a cell. Therefore ImarisCell provides information on vesicle distance to; the closest nuclear wall, the center of the nucleus, to the edge of the membrane as well as the relative position within the cytoplasm or the nucleus.

4D analysis (3D over time) for all components

Cells are living entities and ideally researchers want to study them in the most realistic environment possible. This includes watching them in 3D space and longitudinally evaluating behavioral changes of the cells and their cell components. Not only can researchers track the cells themselves in 3D, they can track the movement of the nucleus and vesicles within each cell. The movement of these cell components is automatically corrected for the movement of the cell itself, so that the actual movement of the cell components within the cell is measured. As an example, movement toward or away from a membrane can be measured. Likewise, changes to the count, size, shape, and intensity of the cell components over time can be measured.

Advanced visualization options

  • Each ImarisCell component can be color coded with any calculated statistical value. This allows researchers to quickly and easily show in a visual manner complex relationships between cell components or their size, shape, or behavior.
  • ImarisCell components can also be exported to Imaris where additional visualization options are available. Cells and Nuclei can be exported as surfaces thus providing access to all available options in the surpass view. Likewise, vesicles can be exported as spots.
  • The standard snapshot and animation functions of Imaris can be utilized to produce stunning visual output of the resulting analysis of ImarisCell images.

Compatibility with ImarisXT

ImarisCell is fully compatible with ImarisXT. This means that researchers can use their own applications for advanced segmentation if they choose and then can import Cells, Nuclei, and Vesicles into Imaris to visualize the results and perform the analysis. Likewise, Cells, Nuclei, and Vesicles can be exported from ImarisCell with ImarisXT if researchers wish to do the analysis in their own application. ImarisCell is consistent with the other Imaris modules in allowing open exchange of image data, derived objects, and metadata between Imaris and custom programmed code.

Selection, Classification, and Interaction

Because ImarisCell is a module of Imaris it utilizes the same selection, classification, and interaction functions that Imaris is known for. Researchers can pick cells, nuclei, or vesicles right in the 3D visual view and immediately have the associated statistics highlighted in the table and vice versa. Imaris InMotion can be utilized for selection of what otherwise might be difficult to select objects in 3D space. The same object filtering and classification methods used in Imaris MeasurementPro can be utilized to select any cell component or specific sub populations of cell components.

General

  • Requires Imaris
  • Provides statistical data for the Cells, Nuclei, and Vesicles only in combination with Imaris MeasurementPro
  • Provides statistics data for tracks only in combination with ImarisTrack

Image Input

  • One to three color channels may be used for the creation of Cells, Nuclei, and Vesicles
  • Cells and Nuclei may be imported from existing Surfaces objects
  • Vesicles may be imported from existing Spots objects
  • Vesicles imported to ImarisCell can be added as a new vesicle type or as one of the previously detected types

Segmentation - General

  • Segmentation of biological objects
    • Cells (Mandatory)
    • Nucleus (Optional)
    • Vesicles of various classes and sizes (Optional)
  • Segmentation on a per channel basis
  • Automatically works on 2D, 3D or 4D datasets
  • Wizard driven interface to guide users step by step through the segmentation process
  • Retain various sets of cell creation parameters - save and remember cell object creation process parameters to use identical settings for analysis of another data set
  • Ability to perform the segmentation on single or multiple regions of interest (ROI’s)
    • Excellent for large data sets to focus on areas of interest for fast processing
    • Ability to apply set criteria to the entire dataset after ROI processing
  • Ability to perform the segmentation on the entire dataset
  • Intelligently pre-selected (default) settings at each creation wizard step
    • Settings are automatically set on a per image / channel basis
    • Allows for processing large datasets after ROI processing without changing the parameters
  • Manually change pre-set parameters as needed
  • Filter / Classify segmentation objects as part of the creation process
  • Rebuild the segmentation once completed
    • Ability to change a few parameters once result is seen
    • All other parameters remain as previously set
    • All settings for processing are recorded
  • Drift correction for Cell objects - After the objects are tracked you can select an object that should not be moving during your time series and use it as an anchor/reference point. Images are shifted on a frame to frame basis so that the selected anchor objects no longer move and the true movement of the other objects can properly be assessed.
  • If image is over time (2D +T, or 3D +T) the resulting segmentation can be tracked with ImarisTrack

Segmentation - Cells and Nucleus

  • Segmentation based on an intensity threshold of the cytoplasm
  • Segment cells using plasma membrane staining
    • Non-Iterative (automatic)
    • Provides statistics data for tracks only in combination with ImarisTrack
    • Automatic detection based on a seeded watershed routine. This algorithm also has an option (recommended) to “fill gaps on cell membrane”, which aims to minimize the adverse effects on the cell detection caused by heterogeneous plasma membrane labeling
    • Iterative:
    • Use reiterative membrane boundary detection process
    • Depending on membrane staining's distribution, select entirely smooth or piece-wise smooth detection algorithms
    • Define, detect and isolate small membrane patches from analysis by removing tiny, detached membrane regions
    • Select shape, length and curvature of the membrane's border during detection process
  • Segmentation based on local contrast
    • Object detection does not depend purely on intensity
    • Excellent for uneven staining or unevenly illuminated fields, bleached or fading samples, and thick specimens
  • Smoothing with a Gaussian filter for noise reduction possible
  • Interactive manual selection of threshold values with real-time visual feedback
  • Biologically correct segmentation enforcement
    • Split cells by seed point growing in the absence of nuclear stain signal
    • Expansion of Cell - If the stained area for a Nucleus is outside of the stained area for the Cell the segmented area of the Cell can be expanded.
    • Erase Nucleus outside of Cell - If the stained area for a Nucleus is outside of the stained area for the Cell, the nucleus outside the cell staining can be erased.
    • Can enforce a limit of one nucleus per cell
    • Split clustered nuclei by seed point growing
  • Filling holes during creation of Cell and Nucleus objects - a hole is a voxel set that has an intensity less than the threshold value and which is completely and contiguously enclosed in all dimensions by the voxels assigned to either the Cell or Nucleus object. Such a set can be eliminated by filling its voxels during wizard-assisted creation of cells and nuclei objects.
  • Resulting objects can be filtered as part of the creation process on any calculated statistical parameter
  • Resulting objects that don’t touch in 3D space can be treated independently

Segmentation - Vesicles

  • Automatic detection of local intensity maxima in 3D
    • Excellent for independently segmenting otherwise touching objects
    • Excellent for objects that are sphere like in shape
    • Excellent for uneven staining or unevenly illuminated fields, bleached or fading samples
  • Interactive inclusion / exclusion of segmented objects with real time visual feedback
  • Works on thousands or hundreds of thousands of objects

Cell Specific Visualization

  • Show or hide the Cell membrane
  • Show or hide the Nucleus
  • Render the Nucleus and Cell as:
    • Othro Slice
    • Oblique Slice
    • MIP projection
    • Blend projection
  • Show or hide the Vesicles and render as:
    • Spheres - with user defined scale
    • Center points
  • Independently color code the appearance of Cells, Nuclei, and Vesicles based on any calculated statistical parameter.

Statistics (requires Imaris MeasurementPro)

  • Overall Statistics
    • Total Number of Cells, Nuclei, and Vesicles in image
    • Total Number of Cells, Nuclei, and Vesicles per time point
  • Object Specific Statistics - These statistics apply to each individual Cell, Nucleus and Vesicle detected in the image and are given on a per time point basis.
    • Size: # of Voxels, Area, Volume
    • Shape (Cells and Nucleus Only ): Ellipsoid Axis Lengths A, B, and C, Sphericity, Ellipticity; oblate and prolate
    • Orientation (Cells and Nucleus Only ): X, Y, Z vector components for each Ellipsoid Axis A, B, and C
    • Diameter (Vesicles Only) : Mean, Min, Max
    • Position: Center of Image Mass and Homogeneous Mass in X, Y, and Z, Time Index
    • Intensity: min, max, median, sum, std dev, and center on a per channel basis
    • Bounding Box (object oriented and axis aligned)
  • Cell and Nucleus Specific
    • Number of Nuclei per Cell
    • Nucleus to Cytoplasm Volume Ratio per Cell
    • Number of Vesicles per Cell
    • Intensity Mean, Min, and Max of all Vesicles within each cell
    • Diameter Mean, Min, and Max of all Vesicles within each cell
    • Cell Cytoplasm related (e.g. number of vesicles in cytoplasm)
    • Nucleus distance to Cell Membrane
    • Number of vesicles per Nucleus
  • Vesicle Specific
    • Distance from Vesicle to closest Nucleus border
    • Distance from Vesicle to closest Nucleus center
    • Distance from Vesicle to closest Membrane
    • Relative vesicle position within a Cell
    • Vesicle location within Cytoplasm
    • Vesicle location within Nucleus
    • Location of vesicle outside cell - distance from a spot to the closest cell (XTension - requires ImarisXT)
    • Diameter of Vesicle (Mean, Min, and Max)
  • 4D Specific Statistics (requires ImarisTrack) - These statistics are given for each Cell, Nucleus and Vesicle that has been tracked
    • For Tracks:
      • Duration, length, straightness of tracks
      • Displacement, X, Y, Z, total displacement, squared displacement of track
      • Number of branches or fusions
      • Quality of fit to autoregressive model- per-axis or mean of all axes of tracks
      • Speed Mean, Min, Max and variation within a track
    • For Objects that have been tracked:
      • Instantaneous velocity X, Y, Z, instantaneous speed, acceleration
      • Average number of Nuclei and Vesicles within the cell being tracked
      • Changes in the Object, Cell, and Vesicle specific statistics shown above over time
      • Output movement of Nuclei and Vesicles relative to Cell - movement of these cell organelles is automatically corrected for the movement of the cell itself. This means that the cell movement is not obscuring the information on the intracellular traffic.

Statistics Output

  • Generate output files as Comma Separate Value (CSV), Excel (.XML or .XLS)
  • Output files are compatible with Open Office and Macintosh
  • Output only the statistics in the selected tab (Overview, detailed, or selection)
  • Output all statistics calculated
  • Output only the statistics used in generating the selected time plot
  • Object IDs
    • Each object has a unique ID to identify it
    • Object ID’s are shown in the statistics tab and in the output file
    • Enter the ID number in the search field and search. The object is then highlighted (selected) in both the statistics list and the visual view
    • Unique higher-level parent IDs are allocated to each cell object and the lower-level IDs of individual vesicles and nuclei are subordinate to that ID. Users can group segmented object that belong to a particular cell with the same parent ID.

Image Output

  • Cells and Nuclei may be exported to Surfaces for expanded visualization
  • Vesicles may be exported to Spots
  • ImarisCell is completely compatible with ImarisXT allowing exchange of all ImarisCell components and derived data between Imaris and custom programmed code.
  • All visualization, snapshot, and animations functions of Imaris are available for ImarisCell objects.

Selection, Filtering, Grouping, Classification

  • Selection
    • Pick any single or combination of objects from the visual view by clicking on them – the associated statistics are selected in the statistics tab
    • Pick and single or combination of object statistics in the statistics tab and the associated objects are selected in the visual view
    • Selection is completely interactive and real-time
  • Utilize InMotion for smart selection and easy visualization of 3D positional information as described for Imaris
  • Filtering
    • Choose any statistical parameter from a drop down menu
    • Select in a histogram or type in values to include or exclude
    • Objects meeting the selection criteria are automatically and interactively shown in real time in the visualization of the image.
    • Combinations of statistical parameters may be used to filter on multiple criteria
  • Duplicate filtered or selected objects to an new main object to group them
    • Grouped objects can have global properties applied to them such as color and transparency
    • Statistical output can be exported only from a single group
    • See Statistics Output
  • Classify objects by using multiple filters or selection methods
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