Confocal microscopy

The research goal of this paper was to provide unbiased counts of labeled astrocytes and to estimate the area they cover, further to develop tools for defining the orientation of coupling within astrocyte networks under different stimuli.

In order to count the astrocytes and estimate the area they cover the following steps were used in this software.

Pre-processing: z-project (using max intensity); split channels; subtract background; remove outliers.

Segmentation: adjust threshold and convert to a binary file; Watershed.

Cell counting: Analyze particles

Measure Astrocytic network area: select a ROI using the polygon tool; set measurements (area); ROI manager -> add the traced polygon; measure.

Nessys: Nuclear Envelope Segmentation System

Nessys is a software written in Java for the automated identification of cell nuclei in biological images (3D + time). It is designed to perform well in complex samples, i.e when cells are particularly crowded and heterogeneous such as in embryos or in 3D cell cultures. Nessys is also fast and will work on large images which do not fit in memory.

Nessys also offers an interactive user interface for the curation and validation of segmentation results. Think of this as a 3D painter / editor. This editor can also be used to generate manually segmented images to use as ground truth for testing the accuracy of the automated segmentation method.

Finally Nessys, contains a utility for assessing the accuracy of the automated segmentation method. It works by comparing the result of the automated method to a manually generated ground truth. This utility will provide two types of output: a table with a number of metrics about the accuracy and an image representing a map of the mismatch between the result of the automated method and the ground truth.

FluoRender is an interactive rendering tool for confocal microscopy data visualization. It combines the rendering of multi-channel volume data and polygon mesh data, where the properties of each dataset can be adjusted independently and quickly. The tool is designed especially for neurobiologists, allowing them to better visualize confocal data from fluorescently-stained brains, but it is also useful for other biological samples.

3-D density kernel estimation (DKE-3-D) method, utilises an ensemble of random decision trees for counting objects in 3D images. DKE-3-D avoids the problem of discrete object identification and segmentation, common to many existing 3-D counting techniques, and outperforms other methods when quantification of densely packed and heterogeneous objects is desired. 

Calculates and corrects for lens-distortion models including chromatic abberation from confocal stacks.