Stereo Investigator Features

Four Principle Aspects in the
Performance of a Stereological Analysis:

Cavalieri on cortex
Example: the Cavalieri estimator can be used to determine the volume of anatomical regions.

  • Strict adherence to sampling protocols that lead to bias-free estimates
  • Use of graphic overlay test systems that facilitate rapid unbiased sampling of the sections
  • Automatic control of microscope stage movement to facilitate measurement of areas, volumes cell counts throughout a tissue section
  • Calculation of reliable results from complicated stereological formulas

Stereo Investigator utilizes a carefully designed user interface that allows you to create a sampling protocol that is specific to your research requirements. Interactive graphic overlays for the probe measurements are then superimposed over the specimen. The use of motorized microscope stages or stage encoders allows fast and accurate navigation through the tissue preparations in the X, Y, and Z axes. Finally, Stereo Investigator computes the results of each probe using the appropriate mathematical estimation formulas.

Stereo Investigator also includes the most sophisticated tools available for anatomical mapping and morphometry. These tools are based on the technology we originally developed in our acclaimed Neurolucida software for neuroanatomical mapping, neuron tracing, and 3D serial section reconstruction.

Features for Systematic Random Scans

Space Balls on dendrites
Example: the Space Balls estimator can be used to estimate the length of fibers.

  • Delineate a region to perform a scan – it can be any shape, any size, any number of fields of view, using any objective lens – the scan will visit sites only in the region that you define
  • Set an unlimited number of exclusion regions
  • The distance between scan sites can be specified separately for the X and Y axes
  • The distance between scan sites can be manually entered, or automatically calculated by specifying the desired number of scan sites
  • Scan at any angle in relation to the region – this angle may be specified by the user or randomly selected
  • Preview feature allows you to see the defined region and the locations of each site that will be sampled
  • Overview feature allows you to see the position of the current site in relation to the other sites
  • The motorized stage automatically moves to each site by pressing a toolbar button or a key
  • Move backwards in the scan to validate results
  • Stop a scan before it is completed, then resume the scan at a later date
  • Full audit capability allows replays of scans at a later time to check results for quality control

Features for Counting Frames, Disectors, and Optical Disectors

Counting Frame on Nissl stained cells
The counting frame is used in the
Optical Fractionator to count cells.

  • Define the size of the counting frame numerically or graphically
  • Define custom colors for the counting frame’s inclusion and exclusion borders
  • The counting frame automatically rotates to be aligned with the systematic random scan
  • Define multiple counting frames for counting objects of different sizes
  • Specify a guard zone (distance from the top or bottom of section to the start of the counting block)
  • The stage automatically focuses through the guard zone to the start of the counting block
  • Focus through the counting block automatically or manually to identify and count objects
  • Visual indicators let you know if you are viewing an object within the Z axis bounds of the counting block
  • Focus position meter allows you to easily see the exact focal position within the section
  • Perform local estimator probes (i.e., nucleator, spatial rotator, etc.) within a counting frame
  • Physical disector estimator easily accommodate differential orientation of section pairs

Features for Serial Section Management

  • Software assists in the selection of a systematically random sequence of serial sections or allows manual entry of section sequence
  • Random number generator is used to automatically select the starting section for analysis
  • Display the tracings of all sections in a sequence, or just the section currently being evaluated
  • Assign a meaningful name to each section
  • Graphically rotate a traced section to align it with succeeding sections, or to re-analyze the section at a later time
  • The program automatically keeps track of the sections that are needed and prompts you to analyze the correct sections in the sequence
  • Any section may contain multiple regions of interest for analysis

Features for Calculation and Display of Stereological Results

  • Results of each estimator are calculated and displayed for each individual probe, or for a group of probes
  • Results may be viewed immediately after completing a probe, or at any later time
  • Coefficients of error and variance levels (CE and CV) are estimated and displayed
  • Sampling parameters used during data collection are displayed for future reference
  • The equations that are used for all calculations are clearly displayed and documented
  • Interactive dialog boxes allow entry of variables (i.e., shape factor) used in results calculations
  • Results may be saved to a file, printed, or copied to the Windows clipboard for use with other programs

Parameters Calculated for Morphometric Analysis

  • Perimeter
  • Length
  • Area
  • Form Factor
  • Roundness
  • Aspect Ratio
  • Convexity
  • Solidity
  • Compactness
  • Shape Factor
  • Optical Density

Anatomical Mapping – Complete Solution for Tracing Regions and Marking Distinct Objects

Anatomical mapping and serial section reconstruction
Create 3D publication-quality anatomical maps of regions and cells. Courtesy L. Zaborszky

  • One hundred distinguishable types of contours (user nameable)
  • Fifty marker types for specifying distinctive objects (user nameable)
  • Unlimited number of contours and marks permitted per tracing
  • Add text labels to any position in the tracing space using any font, size, and color
  • Automatic depth measurement within section
  • Automatic computer controlled XYZ stage movement facilitates tracing objects across multiple fields of view
  • Erase function allows immediate correction of errors
  • Complete set of editing capabilities allows correction of any errors and insertion of overlooked data
  • Tracing may contain an unlimited number of data points
  • View a single section or a single structure at a time
  • Correct spatial shrinkage to handle sequences of serial sections that are processed with different histological techniques and have different shrinkage characteristics
  • Customize the color settings, thickness, and line style for each type of line
  • Customize display color settings and size of each type of marker symbol
  • Tracing modes supported: continuous, discreet, and rubber-band
  • Create publication quality output using any Windows output device – including color printers and slide makers
  • Export the tracing to a number of different graphics file formats or use the Windows clipboard
  • Macro View window continuously displays a complete overview of the tracing
  • Step through tracing: point by point or object by object
  • Dynamically select an object, or a group of objects for editing
  • Re-name erroneously named contours or markers
  • Change the color of any traced contours or markers
  • Dynamically insert missed points in contours
  • Dynamically move or rotate any object or group of objects
  • Dynamically reposition any vertex of a contour
  • Delete points from any contour
  • Delete any marker

Instant Measurement Functions Always Available

  • Measure the distance between two points with a rubberband line
  • Create a rubberband circle of any size to determine distance of multiple objects
  • Determine the angle formed by any two lines
  • Determine the Cartesian coordinates of any point
  • All measurements are instantly displayed, and may be easily transferred to other programs
  • All instant measurement functions can be called at any time, even while performing a tracing or a probe

Image Processing Features

  • Save and read image file formats: Binary Image Format (BIF), Windows Bitmap (BMP), CompuServe (GIF), JPEG, PCX, Targa (TGA), Tagged Image Format (TIF)
  • Make densitometric measurements of image luminescence
  • Display a dynamic histogram of pixel luminance values for the current image
  • Display the pixel luminance values for the image under the cursor
  • Manipulate the image brightness and contrast with the following algorithms: Histogram Equalize, Histogram Brighten, Increase Contrast, Brighten Midrange
  • Background correction function compensates for image illumination irregularities
  • Dynamically adjust the contrast and brightness of images
  • Interactively switch between live and stored video images.
  • Collect a Z series stack of images

Motorized Stage Movement

  • Integrated stage movement is synchronized with the graphics overlay movement
  • Automatic stage movement during tracing greatly facilitates tracing large, complicated structures
  • Meander Scan automatically scans through a user defined region of any size and shape
  • Linear Scan automatically scans along a vector defined by any two points
  • Automatic tracking of Z axis (depth) while tracing
  • Movement of stage only needs to be performed when object extends beyond a field of view
  • Specify a new Z axis position for motorized stages and stage encoders
  • Adjust automatic-move field size
  • Additional user controlled movement of stage accomplished through menu commands
  • Move to the reference point
  • Move to the last point traced
  • Move to any selected point
  • Move stage one field of view: left, right, up, or down
  • Move stage a specified distance: left, right, up, or down
  • Move stage manually with joystick
  • Option to disable automatic computer controlled stage movement
  • Align graphics overlay with true optical image
  • Automatic motorized stage backlash correction
  • Optional support for the Leica DMRBE and Zeiss Axioplan 2 integrated microscope motorized focus drives

Complete Lens Magnification Calibration

  • Calibrate any lens magnification – there are an unlimited number of user definable magnifications available
  • Change objective lenses any time while tracing and maintain proper position and size scaling
  • Automatically corrects for parcentric and parfocal lens aberrations
  • Graphics tracing overlay automatically rescales and redraws when a lens change is made
  • Lens calibration procedure allows you to define lens magnifications for all objectives on your microscope, or from other scopes
  • Automatic correction for Snell’s law of optical refraction to allow accurate Z measurements for all type of objective lenses
  • Differentiate between graphics overlay calibration and data tablet calibration (for photomicrographs) and MRIs

Miscellaneous Features

  • Enter free form text comments and information for each file
  • Store sampling parameters for each study – quickly use them at any time
  • Instantly view the total number of each type of marker counted
  • Choose to view or hide the text labels, or display their location with a special symbol
  • Set unique user configuration for multi-user systems
  • Temporarily hide overlay from screen to see objects that may be obscured
  • Determine relationship of the current field of view to the entire tracing
  • Display current information about lens size, data file in use, structure in progress, and stage position
  • Customize numerous display features to your preference
  • Browse files and view comments without opening a data file
  • Browse files and view a graphic thumbnail without opening the file