newCAST fact-sheet
Understanding complex relationships in biology often requires the ability to quantify microstructural properties in tissue. To obtain trustworthy data, it is typically required to sample of the structures of interest adhering to fundamental statistical principles of randomness. newCAST is a computer based stereology system controlling hardware and software, implementing a wide range of probes and other tools for obtaining unbiased statistical estimates of number, length, area, and volume. newCAST, combined with advanced image analysis, automated imaging and fully integrated data management capabilities is probably the most sophisticated histoinformatics platform available today.



Figure 1
newCAST overview showing an example of the Optical Disector performed on a 30 µm thick section of a human cerebellum


Stereological principles allow estimation based on small samples of tissue. newCAST provide efficient tools for unbiased estimates of structural properties, minimizing working time. Automated systematic random sampling of image fields can be performed on-line while working at the microscope, or unattended, allowing for off-line estimation.

Common applications of stereological methods include:

Counting: Cells, morphological structures (e.g. glomeruli, Alveoli)

Morphometry: Structure areas, surface areas (e.g. Alveoli, blood vessels), circumference areas, structure length (e.g. Neurons, blood vessels, filaments), circumference length, volumes,

Densities: Cell, length, surface

Volumes: Cell, organ, structure (e.g. Glomeruli, alveoli, islets of Langerhans)

In some cases, stereological principles are used for determining connectivity of certain tissues (e.g. cancelleous bone).

Why stereology?
Whereas image analysis is extremely useful for determining the presence of certain structures in tissue sections, this approach has obvious limitations when it comes to quantifying structural properties in an organ or tissue volume.

With Stereology, users readily obtain unbiased estimates, based on a few but correctly sampled tissue sections, by counting events in just a few but correctly sampled fields of view.

With such methods it has now become possible to accurately estimate the number of neurons in the human neo-cortex (~20-25x10E9), the number of synapses (~164x10E12), the number of alveoli in a rodent lung (20x10E6), the number of beta cells in a mouse pancreas (1x10E6), and much more.

Such estimates cannot be obtained using exhaustive counting, and requires application of the efficient estimation methods made available to life-science researchers by stereology. With modern stereological tools, such estimates are readily obtained with only limited manual labor involved.

Today, stereological methods are widely recognized and accepted as a quantitative tool of colossal importance in life-science.

Why newCAST?
CAST was developed based on scientifically recognized principles, and in close collaboration with world leading stereologists. Scientifically, CAST has been the leading stereology package for the last decade.

With newCAST, this work is continued, and Visiopharm has developed a new generation of tools for design-based stereology with still more efficient probes and an increasing level of automation.

The newCAST software is based on a software platform offering unattended image acquisition, making fast off-line analysis an option, and with image analysis software which is probably the strongest software currently available for analysis of digital microscope images of tissue.

Finally, newCAST is providing comprehensive data management capabilities combined with automated computation and visualization of results.

Comprehensive, still easy to use
An important topic when working many hours in front of a microscope and computer is the flexibility and ease of use of the system.



Figure 2
In all dialogue windows it is possible to save configurations and attach information about the configurations. The information becomes visible when the cursor is brought over the configuration name.


newCAST introduces a new flexible way to ease every day work. From installation of hardware, to sampling, and data storage, the same straightforward procedure is used to save user specific configurations and data.

With configurations that are easily saved and loaded you are never more than one mouse click away from your settings or a change to the probes.

Overview
Using stereology overview is an essential and important part of the workflow. To ensure that underlying statistics and sampling settings are transparent to the user, sampling, probe, and other important information is available at a glance at all times during the work.



Figure 3
In the top image, a point grid is setup by use of rulers. A right click in the input box reveals a ruler that is used to change the values in the box. Alternatively, numbers can be keyed directly into the entry field.

In the lower image, the counting frame size is setup using the ruler.

A new setup of the probes or a change to existing probes is seamlessly saved in a new or an already existing configuration.


Ensuring flexible setup, different ways of adjusting the important stereological probes are present in newCAST.



Figure 4
Illustration of the information area in the Control Bar. In the information area the current information about the sampling in progress is visualized.


Z-axis navigation is visualized in a bar to ensure the much needed overview of depth through the specimen. Active control of the z-axis movements are easily accessed in the navigation bar, giving the user the possibility to operate the z-axis using the mouse to navigate through the thickness of the specimen, or simply to step through the thickness in predefined step sizes.
A few easy steps
newCAST uses the principles of stereology and the control of hardware to facilitate work. The philosophies of newCAST are:

Loyalty to stereological sampling protocols leading to unbiased estimates.

Test systems implemented as graphical overlays make unbiased sampling of sections easy.

Automated control of the microscopes, cameras, stages and microcator facilitates measurements of area, volume, length, surface area and cell counting throughout the tissue sections.

Seamless storage and work with acquired data facilitates calculation of accurate results.

A typical path through an experiment is:
  1. The user places the specimen under the microscope and circumscribes the area to be sampled.

  2. The selected probe is activated and shown as a graphical overlay over the image. The probe setup is refined to the application’s need and eventually saved if the user wants to use the setup in a later experiment.

  3. Systematic random sampling is performed with marking of the cells, points, or probe intercepts using the count-tool.

  4. The data is transferred to the database either automatically without user interaction, or manually by the user, depending on user preferences.


Figure 6
Above is an example of a point grid overlay on a human cerebellum.

On the top right is the grid setup with an illustration of the point appearance. Lower right is the Appearance dialogue, demonstrating the ease of changing the appearance of a probe.

The setup is easily saved under the tab Config.




Figure 7
Count-tool setup and work. Up to 255 different marks are available and size, color, shape of the marks is easily changed and saved. Follow the progress of counts in the work tab.


Data management
For seamless workflow it is of great importance that the acquired data is stored in an easily accessible database.

newCAST records all raw data generated during sampling ensuring no data is lost. A unique filtering of the acquired raw data, presents the data in a clear and hierarchy way.

Furthermore data can be presented as calculated stereological values for a verity of estimators.

Custom solutions
Cutting-edge research is constantly challenging the research tools. With newCAST research will never be limited by limitations of the software. Visiopharm offers custom development tailored to the requirements of the research application.

Custom development, including new probes, automation, hardware integration, and much more is carried out in close collaboration with our users.

Technical specifications
Supported hardware

Stages
Prior
Märzhäuser
Ludl (on request)
Microscopes (Automated)
Nikon N90i
Olympus BX series
Leica confocal microscopes
Microcator
Heidenhain MT1201
Heidenhain MT12
Heidenhain ND281B
Heidenhain IK220
Digital cameras
Nikon DS5M
Nikon DXM1200
Olympus DP70
SIS ColorView
PixeLink
uEye
DeltaPix
Frame grabber (Analogue cameras)
Matrox
Image import
Digital still images
Confocal image stacks
MRI image stacks

newCAST Features

Combination of multiple probes
Measures different aspects of a specimen
Isotropic design
Vertical design
Multiple sampling regions
Regions can be grouped to utilize the same sampling conditions on numerous regions or a region can be kept as individual with different sampling setting
Region definition at low magnification or on SuperLens
Sampling and counting at high magnification.
Systematic random sampling
Fractionator sampling
Optical
Physical*
Meander sampling
Random angle option
SuperLens*
Specimen overview for region outline, specimen navigation and count overview
2D measurements
Lengths, areas, angles, circumference etc



Figure 8
Examples of 2D measurements in an image from mouse lung tissue.

Besides length, circumference and radius it is possible to measure angels and diameters.


Seamless change of measurement units
µm, nm, mm, cm, m, inch and pixel



Figure 9
A combo box with the measurements units makes fast and simple change of unit possible.


Visible rulers in images
Image acquisition
Local enhancement of the image
Visualization in all color channels, the enhancement can be made global, covering the entire image.



Figure 10
Local enhancement of a live image in newCAST. During sampling it can sometimes be difficult to se contours of for example a cell. Using local enhancement on the live image outlines the contour of the cell.

Local enhancement is alterable in color channels, can be refined in level and brightness etc.


Z-axis navigation with visual boundary indicator
Visualize specimen cut thickness, measured thickness and Disector height

Stereological probes

Counting frame
3 individual counting frames sizes are available

Multiple counting frames including group setup
Optical Disector
Physical Disector*
Point grid
Area and volume estimation, Cavalieri’s estimations

3 individual point grid systems are available all with point group
Planar Rotator
Nucleator
2D Nucleator
Lines*
Cycloides*
PSI*

Database storage

Multilevel database
Raw data storage
Data filtering
Data protection
CFR 21 part 11*
Image storage
Search functions

Image analysis

VIS image analysis
Image segmentation
Image montage
2D and 3D
Advanced image actuation
Morphometric analysis
Densitometric measurements
Batch processing of images
Comprehensive database
Morphometric measurements
Automatic features such as segment area, segment numbers, densitometry measurements etc
Automatic 2D counting
Cells, colonies etc

* Available from March 2006. Some will be available in automated versions.
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