Image Analysis Software

The cutting-edge tool for cell-based staining intensity analysis of immunohistochemical experiments

HistoQuest - Features

• HistoQuest can load complete HistoFAXS projects or import any series of .tif or .jpg images for analysis.
• Complete section overviews for ideal orientation
• Tunable color separation algorithms for optimal differentiation of colors
• Color picking tool for adding shades
• No limitation as to the number and types of cromogenes used
• Powerful image processing algorithms for the identification of single cells in tissue based on nuclear staining
• Automatic generation of cell body masks for intensity measurements
• Complete set of manual modification and correction tools (splitting, merging, deleting and creating nuclei)
• Powerful DotPlot operations
• Forward Gating of cells to the DotPlots and Backward Gating of dots, gates or cutoff quadrants to the images
• Unlimited custom DotPlot and histogram generation, also based on gates in existing DotPlots, allow for near-unlimited new analyses and refinement of existing ones.
• Extensive printed report and data export options

Contrary to immunofluorescence, the marker information in immunohistochemistry images is contained in one image – in order to analyse it, a color separation has to be done. Information for the optimization of the picked color values can be gained by very rapid interim result calculation.

In HistoQuest, this is done semiautomatically. RGB color values are manually picked with a color picker tool.

The automatic segmentation which follows produces a gray value channel image for each marker, allowing treatment with the TissueGnostics algorithms.

HistoQuest - color separation

The color shades acquired in this way can be adjusted and optimised for automatic nuclear segmentation by manipulating gain, the color space and blur. Information for the adjustment of the picked color values can be gained by very rapid interim result calculation.

HistoQuest - Shades optimization

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Nuclear segmentation in HistoQuest is completely automatic after the input of a few starting values:

• Average nuclear size
• Discrimination area -Exclusion of smaller nuclear sections)
• Discrimination gray (Exclusion of weakly stained nuclei)
• Background threshold (Default setting is automatic)

Automatic nuclear segmentation is achieved via TissueGnostics patented set of algorithms.

Once rapid test calculations done on representative images have established that a good segmentation has been achieved, nuclear segmentation as well as the measurement masks can be automatically calculated for virtually any number of corresponding images.

HistoQuest - Cell detection and data interpretation

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HistoQuest - Cell nuclei and cytoplasm detection

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Data produced by segmentation and measurement is immediately plotted in default DotPlots automatically created according to the experiment setup.

These DotPlots give a visualisation roughly equivalent to forward and side scatter analysis in FACS as well as the cells double positive and single positive for the markers used.

DotPlot operations are mostly analogous to those used in FACS (setting of cutoffs, gating), however, there are special features.

New DotPlots can be generated for any combination of the available measurement parameters (currently nine). The most important feature is that DotPlots can be generated based on any number of gates. This allows the user to define cell subpopulations as the basis for new measurement combinations, offering near-unlimited possibilities for re-evaluating and refining data.

Backward Gating allows connecting backwards from the DotPlot to the images for any dot or group of dots as defined by a gate or for entire cutoff quadrants or combinations thereof.

Forward Gating shows the position of any cell in the images in all DotPlots by simply double clicking on the cell in the image.