Spot detection

3D spot detection using the Determinant of Hessian (DoH) and the detection of 3D minima.

Spot detection in 3D images by Wavelet Adaptive Threshold in Icy.

This workflow detects spots in a 2D image by filtering the image by Laplacian of Gaussian (user defined radius), thresholding (user defined threshold) and finding local intensity maxima in mask distance map (Dmap).

This workflow uses ICY wavelet based spot detector to detect spots in 2D images.

This workflow detects spots from a 3D image by using straightforward set of ImageJ components. It receives the Laplacian Radius and the Threshold  value s input.

This workflow detects spots in a 2D image by filtering the image by Laplacian of Gaussian (user defined radius) and detecting regional intensity minima (user defined noise tolerance).

CLIJ2 is a GPU-accelerated image processing library for ImageJ/Fiji, Icy, Matlab and Java. It comes with hundreds of operations for filtering, binarizing, labeling, measuring in images, projections, transformations and mathematical operations for images. While most of these are classical image processing operations, CLIJ2 also allows performing operations on matrices potentially representing neighborhood relationships between cells and pixels.

CLIJ2 was developed to process images from fluorescence microscopy data of developing cells, tissues, organoids and organisms.

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GaussFit_OnSpot is an ImageJ plugin for fitting Gaussian profiles onto selected positions in diffraction-limited images (e.g. single molecules, protein clusters, vesicles, or stars).

The plugin performs a function fit in regions of interest (ROI) around spots marked by point selections in grayscale images. Single or multiple spots can be either selected manually with the Multi-point tool or automatically with the Find Maxima function.

There is a PDF with more information, and also an example image.

Quote " finding and/or analyzing colocalization of bright intensity spots (cells, particles, vesicles, comets, dots, etc) in images with heterogeneous background (microscopy, astronomy, engineering, etc). "

Uses Gaussian-Mexican hat convolution for preprocessing.

h-Dome transformation, useful for spot detection.

Jython code example:

from de.unihalle.informatik.MiToBo.core.datatypes.images import MTBImage
from de.unihalle.informatik.MiToBo.morphology import HDomeTransform3D
from ij import IJ

imp = IJ.getImage()
mtb = MTBImage.createMTBImage( imp.duplicate() )
hdome = HDomeTransform3D(mtb, 10.0)
hdome.runOp()
mtbdone = hdome.getResultImage()
imp2 = mtbdone.getImagePlus()
imp2.show()

This is a classical workflow for spot detection or blob like structures (vesicules, melanosomes,...)

Step 1 Laplacian of Gaussian to enhance spots . Paraeters= radius, about the average spot radius

Step 2 Detect minima (using Find Maxima with light background option to get minima). Parameter : Tolerance to Noise: to be tested, hard to predict. About the height of the enhanced feautures peaks

The interactive Watershed Fiji plugin provides an interactive way to explore local maxima and threshold values while a resulting label map is updated on the fly.

After the user has found a reliable parameter configuration, it is possible to apply the same parameters to other images in a headless mode, for example via ImageJ macro scripting.

Acquiarium is open source software (GPL) for carrying out the common pipeline of many spatial cell studies using fluorescence microscopy. It addresses image capture, raw image correction, image segmentation, quantification of segmented objects and their spatial arrangement, volume rendering, and statistical evaluation.

It is focused on quantification of spatial properties of many objects and their mutual spatial relations in a collection of many 3D images. It can be used for analysis of a collection of 2D images or time lapse series of 2D or 3D images as well. It has a modular design and is extensible via plug-ins. It is a stand-alone, easy to install application written in C++ language. The GUI is written using cross-platform wxWidgets library.

The freely available software module below is a 3D LoG filter. It applies a LoG (Laplacian of Gaussian or Mexican Hat) filter to a 2D image or to 3D volume. Here, we have a fast implementation. It is a perfect tool to enhance spots, like spherical particles, in noisy images. This module is easy to tune, only by selecting the standard deviations in X, Y and Z directions.

run("LoG 3D", "sigmax=1 sigmay=1 sigmaz=13 displaykernel=0 volume=1");

An ImageJ plugin for DEFCoN, the fluorescence spot counter based on fully convolutional neural networks

Part of ATLAS software

Comment / Instructions: 

You can upload your image at the Mobyle@SERPICO portal and download the result. The workflow is only available online, i.e. no download possible.

Quote: "The GDSC ImageJ plugins are a collection of analysis programs for microscopy images including colocalisation analysis and peak finding (FindFoci)."

Many types of analysis besides simply finding foci detection (spot detection) is bundled in this plugin. One prominent function is "FindFoci Optimizer". This allows feeding images with spot annotation by the user (multi-point selection tool) and scans through various parameter combinations to find the best parameter set that gives the results similar to the annotation. This is almost like machine learning... but with well-established parameter types that allows you to fully understand what is going on.

This is an example workflow of how to perform automatic registration by

- first detecting spots in both images using wavelet segmentation (with different scale according to the image scale)

- second using Ec-Clem autofinder to register both images

Click on a block to know more about a tool. Non referenced tools are non clickable.

Spot detector detects and counts spots, based on wavelet transform.

- Detects spots in noisy images 2D/3D.
- Depending on objective, spots can be nuclei, nucleus or cell
- Versatile input: sequence or batch of file.
- Detects spot in specific band/channel.
- Multi band labeling: automaticaly creates ROIs from one band and count in the same or an other band.
- Filters detection by size.
- Sort detection by ROIs
- Output data in XLS Excel files: number of detection by ROIs, and each detection location and size.
- Outputs withness image with ROIs and detection painted on it.
- Outputs binary detection image.
- Displays detections
- Displays tags

Image-processing algorithms developed at the MOSAIC Group for fluorescence microscopy. Tools included:

• 2D/3D single-particle tracking tool which can be used to track bright spots in 2D/3D movies over time.
• Optimal filament segmentation of 2D images. 
• Curvature filters for image filtering, denoising, and restoration. 
• Image naturalization for image enhancement based on gradient statistics of natural-scence images. 
• Tool for automatically send and distribute jobs on clusters and get back the results.
• Multi-region image segmentation of 2D and 3D images without needing to know the number of regions beforehand. 
• Squassh for globally optimal segmentation of piecewise constant regions in 2D and 3D images and for object-based co-localization analysis. 
• Tool for inferring spatial interactions between patterns of objects in images or between coordinates read from a file.
• Tool for robust, histogram-based background subtraction well suited to correct for inhomogeneous illumination artifacts.
• A tool to estimate the Point-Spread Function of the microscopy out of 2D fluorescence images.
• A tool to measure the 3D Point-Spread Function of a confocal microscope from an image stack.
• Addition of synthetic Poisson-distributed noise to an image in order to simulate shot noise of various signal-to-noise ratios. 
• Convolution of an image with a Bessel function in order to simulate imaging with a microscope. 
• A utility to detect bright spots in images and estimate their center. 
• A utility to create manual segmentations to be used as ground truth to test and benchmark automatic segmentation algorithms.
• A tool for replacing one color in an image with another color.

The FindFoci plugins allow the identification of peak intensity regions within 2D and 3D images. The algorithm is highly configurable and parameters can be optimised using reference images and then applied to multiple images using the batch mode. Details of the benefits of training an algorithm on multiple images can be found in the FindFoci paper: 2591

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

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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.

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)

Download the protocol,use and modify in Icy. It permits to detect spot with wavelet spot detector block. Input : loop on a folder Outputs: excel, binary, and detection screenshot

Oufti (previously named MicrobeTracker) is a MATLAB application / suite of tools for analysing fluorescent spots inside microbes. MicrobeTracker can identify cell outlines and fluorescent foci, and generate plots and statistics based on positions and intensity (kymographs, histograms etc.) The MATLAB code is easy to modify and extend to add additional plots and statistics: see e.g. Lesterlin et al. (2014).

The Outfi Forum is quite active.

The macro segments and classifies human spermatozoids nuclei (DAPI) based on the number of FISH signals (spots) they contain. It reports the percentage of occurrences of user defined classes (combinations of spot multiplicity in the FISH channels) as well as the position (point selections) of the detected nuclei falling in these classes. The input image should be an hyperstack with 4 channels: DAPI (first channel) and three FISH channels. The images are typically obtained as a maximum intensity projection of few channels (confocal) or a single z slice acquisition (widefield).

Example image available in the linked page. 

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This pipeline shows how to identify smaller objects (foci) within larger objects (nuclei) and how to use the Relate module to establish a relationship between the two as well as perform per-object aggregate measurements (such as number of foci per nucleus). Sample images are included in the download package.

WASH, Exo84, and cortactin spot detection and codistribution analysis To detect endosomes, an automatic Otsu threshold is applied to the Gaussian-filtered MT1-MMP–positive endosome image (= 1.5 pixels for the sample image). Statistics about each endosome are then saved, for example random positioning of spots can be compared to actual positioning. For each endosome, WASH and Exo84 (or WASH and cortactin) spots are searched for in a neighboring of x pixels in their respective channel. Their number and position are saved per endosome (**see the macro in Text file S2 downloadable from here**).

From the position of WASH and Exo84 (or WASH and cortactin) spots around each endosomes, each WASH spot is paired with its closest Exo84 (or cortactin) spot neighbor, optimized over all spots around this endosome.

This allowed measuring of the distribution of distance between WASH-Exo84 (or WASH-cortactin) spots (**for the co-distribution analysis, see matlab scripts in Zip file S3 downloadable).

This workflow estimates (densely distributed) object counts by the density of objects in the image without performing segmentation or object detection. Current version only works for 2D images of roundish objects with similar sizes on relatively homogeneous background. Users should provide a few labels of background and objects (especially on clustered objects), and the tool predicts the density of objects on the entire image. Counting is then estimated by integrating the density values on the whole image or specified rectangular regions of interests.

A set of classes and functions which can be used by plugins performing spot detection and spot tracking.

An ImageJ plugin for evaluate intra-nuclear 3D cross-distances between fluorescent spots in multi-channel images 

Automatic finding of image features are very convenient for registering two images to align them in proper orientation. Two image plugins are implemented for extracting image features and are placed as menu items at: [Plugins > Feature Extraction > Extract SIFT Correspondences] and [Plugins > Feature Extraction > Extract MOPS Correspondences].

For more details, see the linked page in Fiji wiki. For details about SIFT algorithm, see 2569. For more details about MOPS algorithm, see 2570.

TrackMate provides the tools to perform single particle tracking (SPT). SPT is an image analysis challenge where the goal is to segment and follow over time some labeled, spot-like structures. Each spot is segmented in multiple frames and its trajectory is reconstructed by assigning it an identity over these frames, in the shape of a track. These tracks can then be either visualized or yield further analysis results such as velocity, total displacement, diffusion characteristics, division events, etc...