Spot detection

Quote: *A GUI-based program which manually detects spots and places them into previously detected meshes. Currently the program runs from MATLAB only. *

In this case study, MATLAB, the Image Processing and Signal Processing toolboxes were used to determine the green intensities from a small portion of a microarray image containing 4,800 spots. A 10x10 pattern of spots was detected by averaging rows and columns to produce horizontal and vertical profiles. Periodicity was determined automatically by autocorrelation and used to form an optimal length filter for morphological background removal. A rectangular grid of bounding boxes was defined. Each spot was individually addressed and segmented by thresholding to form a mask. The mask was used to isolate each spot from surrounding background. Individual spot intensity was determined by integrating pixel intensities. Finally, integrated intensities were tabulated and saved to a data file for subsequent statistical analysis to determine which genes matter most.

Matlab toolbox to analyze single molecule mRNA FISH data. Allows counting the number of mature and nascent transcripts in 3D images. See 2513. Following toolboxes are required: - Optimization toolbox - Statistics toolbox - Image processing toolbox - (Optional) Parallel processing toolbox

Input data type: 3D image

Output data type: CSV

Quote:

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.

These two KNIME workflow solutions are similar: first one detects nuclei and spots inside the nuclei without taking care of surrounding regions, i.e. mitochondria. The second one provides the full solution including spots in mitochondria.

see section 2.4 for KNIME workflow. Section 2.3 is also available, using Fiji. 

Sample image: hela-cells.tif (674k x 3)