Fluorescence microscopy

CellDetector can detect cells (or other objects) in microscopy images such as histopathology, fluorescence, phase contrast, bright field, etc. It uses a machine learning-based method where a cell model is learned from simple dot annotations on a few images for training and predict on test sets. The installation requires some efforts but the instruction is well explained. Training parameters should be tuned for different datasets, but the default settings could be a good starting point.

Very simple application that lets you load your time-lapse intensity data to generate the normalized FRAP recovery curve and perform exponential curve fitting.

Quote: The user can handle simultaneously large data sets of raw data, visualize fluorescence recovery curves, exclude low quality data, perform data normalization, extract quantitative parameters, perform batch analysis and save the resulting data and figures for further use. Our tool is implemented as a single-screen Graphical User Interface (GUI) and is highly interactive, as it permits parameterization and visual data quality assessment at various points during the analysis.

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.

In this human cytoplasm-nucleus translocation assay, learn how to load a previously calculated illumination correction function for two separate channels, measure protein content in the nucleus and cytoplasm, and calculate the ratio as a measure of translocation. This is a clumpy cell type, so studying the settings in primary object identification may be helpful for users interested in the more advanced options that module offers. More about these images can be found at the BBBC.

Task

Quantify the length of microtubules (MT) and the MT average density per cell.

Workflow descriptions

Simple two step workflow, allowing visual & manual correction of microtubule between the 2 steps. Batch measurement of microtubule lengths for multiple images is achieved by segmenting the MTs and then their skeletonizations. The number of pixels in the microtubule is proportional to their length, so the length can be estimated.

Script

Workflow is written as an ImageJ macro (Fiji) with following steps:

1. The enhancement of tubular structure by computing eigenvalues of the hessian matrix on a Gaussian filtered version of the image ( sigma 1 pixel), as implemented in the tubeness plugin.

2. The tubules were then thresholded , and structures containing less than 3 pixels were discarded.

3. If needed, a visual check and correction of segmented microtubule is then performed.

4. After correction, segmented MTs were then reduced to a 1-pixel thick line using the skeletonize plugin of Fiji. The length of the skeletonized microtubules was then directly proportional to their length.

5. Data were grouped by condition and converted back to micrometers units under Matlab for the statistical tests.

Pitfalls

Commented but not that general without editing some fields in the macros.

Sample Data

Sample data and workflow (see above URL) can be accessed by - login: biii - password Biii!

Misc

3D version also available here. Use of components Skeletonize and Tubeness Filter