Product Overview: The M2, M4, and AIS^TM

Imaging Research makes three image analyzers. Two of these (the M2 and M4) are MicroComputer Imaging Device (MCID^TM) systems. The MCID systems use special imaging board sets for maximum speed and power, and two discrete monitors for the display of data/control and images. In contrast, the Analytical Imaging Station (AIS^TM) does not need to use an imaging board (though one is available), and displays everything on one monitor. It sacrifices some speed, image processing power, and convenience in exchange for simplicity and much lower cost.

Both images and data can be interchanged freely between networked MCID and AIS systems. The user interface is also very similar, so that a single set of skills is all you need to operate both MCID and AIS.

As MCID and AIS are designed specifically for bioscience applications, there is no need for user programming. MCID includes functions for quantitative autoradiography, grain counting, anatomical measurement, video microscopy, static and dynamic fluorescence imaging, gel and blot analysis, and many other tasks. AIS includes gel and blot analysis, and many types of calibrated densitometry and morphometry.

Summary

The Analytical Imaging Station (AIS) is a low-cost image analysis software package, that does not require special imaging hardware. AIS accepts images in many formats, via your computer disk or a network connection. It also includes very strong support for phosphor plate imagers and scanning densitometers. AIS is a cost-effective way to distribute image analysis capabilities within a networked environment. Acquire the images from a MCID core facility, and then use AIS to access these images in your own office. If you also need camera input, select AIS/C, an enhanced version which includes an imaging board and supports various cameras.

The M4 is a moderately priced image analyzer, available in two configurations. The M4/ALU performs point operations (such as frame averaging or image subtraction) in real-time. The M4/RTP includes a more powerful real-time processor, which performs color imaging and advanced image processing functions (convolutions, mathematical morphology and others) very rapidly. Both models of the M4 are suitable for most applications.

The M2 is the most capable bioscience image analyzer available. Added to the capabilities of the M4 are the additional advantages of very high spatial resolution (1280 x 1024 pixels) and large amounts of image memory. Select the M2 for applications which require large fields of view, or simultaneous display of multiple images.

MCID^TM

General

i) TWO CONFIGURATIONS

Two MCID image analyzers are available:

M2 - high resolution (1280 x 1024 pixels) and the greatest flexibility for demanding environments;

M4 - much of the M2's power in a less costly, medium resolution format.

ii) FAMILIAR COMPUTER ENVIRONMENT

MCID's advanced personal computer and Windows NT operating system have the power of an engineering work station, without the complexity. This is a standard PC and, in addition to running MCID  it will run standard Windows software.

iii) UPDATES AND SUPPORT

A one year software update contract is provided with all MCID systems, and can be extended at low cost.

iv) RELIABLE AND ACCURATE DATA ANALYSIS

MCID systems have been in the field for more than ten years, at hundreds of sites world-wide. This is a proven, mature system.

v) ACCEPTS ADDITIONAL SOFTWARE AND HARDWARE MODULES

MCID accepts the Tiled Field Mapping system, M3D reconstruction, Stereology, and High Density Grids software modules, and is the host for the Tundra ultra-low light imaging system. These additional modules can be purchased with the system, or added at any later time.

Calibration

i) SHADING CORRECTION

Shading error is density variation generated by the video camera, microscope or camera lens, and illuminator. MCID automatically corrects shading.

ii) DENSITY CALIBRATION

Calibrate to any external standards. Then, fit variable linear regression, first through fifth order polynomial, cubic spline, variable smoothed spline, exponential, linear interpolation, or other functions to the calibration standards. Goodness of fit estimates, extrapolation, and graphical display of fitted functions are provided. Calibrations may be saved from session to session, and recalled.

iii) MULTIPLE DENSITY CALIBRATIONS

MCID's image storage channels may be calibrated independently, to allow simultaneous analysis of density data from multiple animals or conditions. This feature is particularly useful in multiple isotope autoradiography. For example, calibrate channel 1 to 14C standards, and channel 2 to 3H standards. Then, align the 14C and 3H labelled images (alignment functions are provided). Finally, place a sample window on either image. MCID reads data simultaneously from both channels, and reports both 14C and 3H values for each sampled region.

iv) DIRECT REPORT OF BIOCHEMICAL VALUES

Gray level data can be passed through built-in operational equations, and reported as biochemical values. The following functions are included:

• Local cerebral glucose utilization (Sokoloff et al., 1977 or Savaki et al., 1980)
• Local cerebral protein synthesis (L-[1-14C]leucine incorporation)
• Total, nonspecific, specific, and percent specific binding (any calibration, e.g. fmol/mg protein)
• Indicator fractionation or tissue equilibration regional cerebral blood flow
• Calcium concentration, pH, and other ratiometric parameters

 

v) SPATIAL CALIBRATION

Spatial measurements are calibrated to any scale (e.g. mm, microns, relative front, molecular weight) with one simple command. MCID will then read directly in the calibration units. Calibrations may be saved and recalled.

vi) CALIBRATION SCALES

A calibration scale on the image shows the values assigned to colors or gray levels. Another scale shows the spatial calibration.

Data Gathering

i) DATA FILE CREATION

A disk file (the "log file") automatically contains a complete record of data gathered during an analysis session. At any time, the log file may be saved, re-opened, edited, added to, or parsed to create data summaries.

ii) "AUDIT TRAIL" TRACKS EVERY NUMERICAL AND IMAGE DATA POINT

MCID's automatic log file contains numerical data measured from your specimens. The image origin of each numerical data point in the log file can be tracked. Retrieve the log file and image at any time, and click on a value in that log file. The image region corresponding to that value is shown. This audit trail feature allows complete records of data origins to be kept.

iii) SAMPLE WINDOW UNDER MOUSE CONTROL

Rectangular, circular, outline (user drawn) or template (drawn and saved) regions of interest.

iv) MANUAL AND AUTOMATED DIMENSION MEASUREMENTS

MCID uses manual or automated target selection to report any combination of spatial measurements including:

• Area, proportional area, volume
• Perimeter, clipped perimeter
• Maximum chord, curved chord
• Maximum, mean, median, circular equivalent, and Feret diameters
• Form factor, point to point distance, length, angle
• Count, estimated count, X,Y location
• Stereological parameters (optional, 2D and 3D)

 

v) FLEXIBLE DEFINITION OF DENSITY OR SPATIAL DATA

Density or spatial values within sample windows are read, converted to calibrated values, passed through operational equations, and reported to the console and log file, all with one tap of a mouse button. To perform very selective automated scans, define a set of characteristics for valid targets using any combination of MCID's density and spatial measures. Targets are automatically checked against the definition before being accepted.

vi) TARGET COUNTS

A special target counting function automatically counts grains, cells, blood vessels, etc. The counting function reports target number, estimated target number (corrected for clumping), target area, target proportional area, and other parameters. Unbiased stereological counts are available as part of the Stereology module.

vii) LIST OR KEYBOARD LABELLING OF SAMPLED REGIONS

Type region names at the keyboard, or use the mouse to select names from a list.

Image Capture, Enhancement, Archiving, and Communication

i) IMAGE CAPTURE

MCID supports standard video cameras, high precision integrating cameras, and various scanners including the Fuji BAS, BioRad, and Molecular Dynamics phosphor plate imagers, and the Molecular Dynamics Personal Densitometer. Capture and work with images of any size.

ii) TRUE-COLOR IMAGING

The MCID true-color option uses color as a target definition tool. For example, cell components within a stained section could be detected using any combination of red, green, blue, hue, lightness, and saturation. User-defined color and spatial criteria may be combined to yield very selective detection of image components.

iii) VISUAL ENHANCEMENTS

There are extensive facilities for contrast enhancement, highlighting of a specific data range, and other gray scale, pseudocolor, or true color manipulations. You have complete control over image appearance.

iv) IMAGE EDITING

Image editing functions include cut and paste of arbitrary shapes, and an "eraser", which can be used to remove artifacts and background speckle. Zoom or compress any portion of an image. Create montages of multiple images. Geometric transformations allow the removal of warps and cutting artifacts.

v) TEXT GENERATION

Various fonts, sizes and colors of text, arrows, lines, and other graphics can be generated.

vi) IMAGE NORMALIZATION

Images from more than one subject must often be displayed as a single summary image, that demonstrates the conditions of an experiment. Before a valid summary image can be created, a given color or gray level must be made to represent the same data value in all of the subjects. To accomplish this, MCID transforms (normalizes) any number of images to share a common calibration.

vii) IMAGE FILE SAVING AND RESTORING

Images or parts of images are quickly saved and recalled. Multiple images may be combined into a montage, and saved as a single image. Images may be imported or exported in TIFF format, for exchange with other programs.

viii) IMAGE COMMUNICATION

Images can be transmitted to other systems (via modem, network, Internet, E-mail, floppy disk, etc.) in MCID or various other formats.

ix) IMAGE AND GRAPHICS PRINTING

Images and console graphics (e.g. density profiles) may be saved in TIFF format, for export to any graphics or desktop publishing program. For example, transfer an MCID image to a desktop publishing package, add text and other figures, and then print on any printer. MCID also prints directly to the Agfa/Matrix family of digital film recorders (ProSlide 35 and PCRII+), to Fuji Pictrography printers, and to PostScript-compatible monochrome or color printers.

x) IMAGE TILING

Tiling is the creation of large images from a number of smaller images. MCID's tiling functions (part of the optional Tiled Field Mapping System) include:

• microscope motor stage control to simplify tile acquisition;
• automated image alignment for precise positioning of the tiles with respect to each other;
• combination of any number of fields of view into large, high resolution images.

 

Image Processing Functions

i) IMAGE ARITHMETIC

One or more images are passed through a mathematical function to yield an output image. For example, IMAGE A + (2 * IMAGE B) / IMAGE C = OUTPUT IMAGE. MCID includes a flexible equation editor, which may be used to create an output image from any mathematical combination of input images.

ii) IMAGE AVERAGING

MCID averages full resolution (1280 x 1024 or 640 x 512) images in real time at pixel rates of up to 15 mHz. Even true color images can be averaged to improve performance in low-light applications.

iii) IMAGE RATIO

Display the ratio of two images to directly show proportional labelling. Dedicated functions perform ratio fluorescence imaging.

iv) IMAGE FUSION

MCID is ideal for multicolor fluorescence studies. Acquire discrete views of specimens using different excitation filters, and then fuse those views into a single image.

v) CONVOLUTIONS AND MORPHOLOGY

There are tunable filters for detail enhancement, smoothing, edge detection, erosion, dilation, and other image processing operations. A filter editor allows user-defined filters to be created. The M4/ALU performs most filters in software. The M4/RTP and M2 incorporate hardware accelerators for rapid image processing.

vi) GEOMETRIC TRANSFORMATIONS

Many images contain spatial distortions (warps) that interfere with image analysis or 3D rendering. MCID's excellent facilities for geometric transformation may be used to edit warped specimens.

vii) MACROS

An object-based "macro editor" allows the easy manipulation of program functions (eg. digitize, average, filter) into networks of sequential operations (macros). Macros may be created, saved, recalled, and assigned to a single keystroke for convenient use. The macro editor is easier to use than the scripting languages found in most other image analyzers.

Other Functions

i) THREE DIMENSIONAL RENDERING

The optional M3D software module performs 3D visualization of image sets. MCID with M3D is a fully integrated system for 2D measurement (using direct pixel counts or stereology) and 3D rendering. This combination of image analysis and visualization within the same program is unique. It provides the capabilities for quantitative rendering, absent in most visualization systems.

ii) REDIRECTED SAMPLING

MCID systems offer simultaneous storage and alignment of multiple images. This allows redirected sampling --the viewing of one section while samples are actually taken from another. Typical uses of redirection include:

• View a stained section, while MCID takes data from exactly the same regions of an autoradiograph;
• Read across images in multiple isotope autoradiography;
• Define regions on a MRI image while MCID reads data from an underlying PET image;
• Define regions of interest on a phase contrast or DIC image, while reading intracellular ion concentration from ratio fluorescence images;
• Define cells on a bright field image while MCID counts grains from a dark field or epipolarization image.

 

iii) DENSITY PROFILES

View profiles of data values under lines of any orientation, width and shape. Distances on the X (distance) axis are expressed in the current spatial units (e.g. mm). Densities on the Y axis are in the currently selected density unit (e.g. fmol/mg/protein).

iv) DATA SUMMARIZATION

MCID displays data in tabular format on the host monitor. It also records all data in log files, which are parsed to create neatly tabulated summary statistics, including regional means and standard deviations. Multiple measurements can be summarized simultaneously. For example, a single summary could contain regional areas in mm2, and densities in nCi/g and umol/100g/min. Individual data values may be expressed normalized to the subject mean, to the value for any region or to a value entered at the keyboard.

v) DATA EXPORT TO STATISTICAL OR SPREADSHEET PROGRAMS

Data can be exported in ASCII or Lotus 123 format. Data may also be cut and pasted into other programs via the clipboard. With this flexibility in data output, MCID data files can be exported directly to popular spreadsheet or statistics packages (e.g. 123/G, Excel, SPSS, BMDP, SYSTAT).

vi) FACILITIES FOR RECEPTOR AUTORADIOGRAPHY

A total binding image and nonspecific binding image can both be placed into image memory. Select either image with a single keystroke and display images of specific and percent specific binding. MCID also uses both images to report the following on each sample:

• Total binding
• Specific binding
• Nonspecific binding
• Percent specific binding

 

vii) GEL AND BLOT ANALYSIS

MCID's gel analysis software provides both graphical and numerical report of density values across single or multiple lanes in film, stained, UV, or chemiluminescent specimens. Specialized "study" modes are included, which simplify analyses of lanes, 2D gels, dot blots , and hybridization arrays. Various forms of background correction, gel unwarping, calibration to density and molecular weight standards, and peak enhancement/smoothing are available.

viii) FLUORESCENCE IMAGING

MCID is an ideal system for fluorescence imaging. It supports various types of low-light cameras, and includes many functions designed specifically for fluorescence imaging. These include multiparameter and ratio fluorescence functions. For details, see the Fluorescence Imaging pamphlet.

ix) MOTOR STAGE AND Z-AXIS CONTROL

MCID controls microscope motor stages and Z-axis drives. Motor stages are essential for image tiling, while Z-axis drives are used in extracting one plane of focus from a number of planes (deconvolution), or in combining a number of planes into a single focused plane. In both cases, MCID steps a microscope Z-axis, automatically, to acquire image stacks.

x) Images of Any size and Depth

MCID has been designed to analyze data from imaging plate readers, scanning densitometers, scanning microscopes, and other devices which provide very large (e.g. 5000 x 4000 pixels) and deep (e.g. 16-bit) images. MCID acquires, calibrates to, and analyzes these large images. It also prints them at the fullest possible resolution of the printing device.

A Host for BAS Phosphor Plate Imagers

MCID can serve as the host and analysis station for Fuji BAS phosphor imagers. That is, the BAS is connected directly to the MCID computer via a SCSI port. MCID drives all functions of the BAS (e.g. setting the dynamic range, scanning).

AIS^TM

General characteristics

i) Computers

AIS runs on PC-type computers under the Wndows NT operating system. The recommended host is a Pentium PC with PCI architecture, and at least 32 MB of RAM.

ii) Display

AIS uses a single monitor, and displays at any resolution supported by the operating system. Resolutions of 1280 x 1024 pixels x 24 bits (16 million colors) are typical.

Image Formats and Storage

i) ACQUISITION

The standard AIS package accepts data from a broad variety of scanners, including the Fuji, Molecular Dynamics, and BioRad imaging plate readers, and the Molecular Dynamics FluorImager and Storm. The camera option (AIS/C) adds the ability to accept 8 bit analog or digital data from many cameras.

ii) Images of Any size and Depth

AIS will accept very large (e.g. 5000 x 4000 pixels) and deep (e.g. 16-bit) images. AIS acquires, calibrates to, and analyzes these large images. It also gives you the ability to edit, enhance, and print large images.

iii) IMAGE FILE FORMATS

On computer disks, or via a network connection, AIS accepts images in its native (MCID) format, and in 8-16-bit monochrome, palette color, and 24-bit color TIFF formats. An image format editor is included, which imports almost any binary image format.

iv) FOUR INDEPENDENT IMAGE CHANNELS

Each of AIS' four image channels can contain a discrete, calibrated image. Use the multiple image channels to align stained sections and autoradiographs, or to display images acquired at different fluorescence wavelengths. For multiparameter fluorescence, use AIS' sophisticated image fusion functions to combine discrete fluorescence images.

Image Manipulation

i) IMAGE ENHANCEMENTS

Among the facilities for digital image enhancement are true color (24-bit) and pseudocolor display, contrast enhancement, highlighting of a specific data range, and nonlinear display mapping. A lookup table editor makes it easy to create your own lookup tables. High-quality display of deep images (10, 12, 14, or 16-bits) is assisted by a window leveling function that selectively focuses on any part of the display range.

ii) IMAGE EDITING, FILTERING AND ARITHMETIC

Cut and paste entire images or arbitrary shapes. Zoom or compress any portion of an image. Create montages of multiple images. A full library of image filters (e.g. smoothing, edge detectors) is included. An image equation editor performs almost any arithmetic functions on one or more images.

iii) TEXT GENERATION

Various fonts, sizes and colors of text, arrows, lines, and other graphics can be generated.

iv) IMAGE PRINTING

AIS prints directly to the Agfa/Matrix and Polaroid families of digital film recorders, to Fuji Pictrography page printers, to Sony digital thermal printers, and to any PostScript printer supported by Windows NT (e.g. monochrome and color laser printers, dye sublimation printers).

Calibration

i) QUANTITATIVE IMAGE ANALYSIS

AIS is a quantitative tool, and is fully calibratable for both density and spatial measurements. Distance calibrations may be linear (e.g. mm) or nonlinear (e.g. molecular weight and pH). Independent linear or nonlinear distance calibrations can be established in X and Y. Curve fitting, graphical display of calibration data, and extrapolation are provided. All calibrations are automatically saved with images.

ii) OPERATIONAL EQUATIONS

Gray level data can be passed through built-in operational equations, and are then reported as local cerebral glucose utilization, local cerebral protein synthesis (L-[1-14C]leucine incorporation), or indicator fractionation or tissue equilibration regional cerebral blood flow.

Data Gathering

i) MANUAL AND AUTOMATED DIMENSION MEASUREMENTS

AIS uses manual or automated target selection to report any combination of spatial measurements including:

• Area, proportional area, volume
• Perimeter, clipped perimeter
• Maximum chord, curved chord
• Maximum, mean, median, circular equivalent, and Feret diameters
• Form factor, point to point distance, length, angle
• X,Y location

 

ii) DATA ARCHIVING

AIS automatically maintains a disk file containing a complete record of data gathered during an analysis session.

iii) REPORT GENERATION AND DATA EXPORT

AIS generates report formats that are tailored to the requirements of specific applications. These reports can contain any combination of numerical, graphical and image data. Data and graphics can be exported to popular spreadsheet programs (e.g. Excel) via the clipboard, or in ASCII or Lotus format.

iv) SAMPLE WINDOW UNDER MOUSE CONTROL

Rectangular, circular, polygonal, outline (user drawn) or template (drawn and saved) sample windows define image data for analysis.

v) PROFILE GRAPHS

View profiles of density values under lines of any orientation, width and shape. Distances on the X (distance) axis are expressed in the current spatial units (e.g. mm, kbase pairs). Densities on the Y axis are in the currently selected density unit (e.g. fmol/mg/protein).

Camera Support

AIS/C is an enhanced version of AIS (with an imaging board) that accepts 8 bit monochrome input from analog and digital cameras. Select almost any standard video camera (e.g. the Sony XC-77), the Hamamatsu C5985 and Photometrics ImagePoint chilled digital cameras, our Midnight Sun camera, or others. Some digital color cameras are supported, including the Fujix HC1000 high resolution color camera.

A Host for BAS Phosphor Plate Imagers

AIS can serve as the host and analysis station for Fuji BAS phosphor imagers. That is, the BAS is connected directly to the AIS computer via a SCSI port. AIS drives all functions of the BAS (e.g. setting the dynamic range, scanning).

Table 1: Image Analyzer Summary

For further information, see the Imaging Research Inc. website or email berthold@berthold.com.au

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