3D

[as of 20180524, the website is temporary not functioning do to web defacement - please check again later] This tutorial will exemplify basic rapidSTORM usage by showing how to convert an Andor SIF acquisition to a super-resoluted image with rapidSTORM.

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Fluorescence in situ hybridization (FISH) is used to study the organization and the positioning of specific DNA sequences within the cell nucleus. Analyzing the data from FISH images is a tedious process that invokes an element of subjectivity. Automated FISH image analysis offers savings in time as well as gaining the benefit of objective data analysis. While several FISH image analysis software tools have been developed, they often use a threshold-based segmentation algorithm for nucleus extraction. As fluorescence signal intensities can vary significantly from experiment to experiment, from cell to cell, and within a cell, threshold based segmentation is inflexible and often insufficient for automatic image analysis, leading to additional manual extraction and potential subjective bias. To overcome these problems, we developed a graphical software tool called FISH Finder to automatically analyze FISH images that vary significantly. By posing the nucleus extraction as a classification problem, compound Bayesian Classifier is employed so that contextual information is utilized, resulting in reliable classification and boundary extraction. This makes it possible to analyze FISH images efficiently and objectively without adjustment of input parameters.

A deconvolution component applicable to confocal and STED microscopy. The MATLAB function fo this package implements the SGP method for n-dimensional object deblurring with the option of boundary effects removal. Although this is a preliminary version, results seem to be good from their paper (Zanella et al 2013).

This Javascript works in ImageJ to measure 3D intensity profile along cylindrical space with variable radius.

These two similar KNIME workflow solutions take 3D data stacks to segment the spots first, using local thresholding with subsequent morphological operations in order to remove noise. Colocalization is then defined by overlapping or center point distance between segmented objects. Further filtering such as overlapping ratio or distance range is done through KNIME table processing.

Two different types are available. 

1. colocalization based on overlapping
2. colocalization based on distance between object centers

Sample images: Smapp_Ori files

Chapter 4 in the documentation.