Workflow

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

Analyzing ER, PR, and Ki-67 immunohistochemistry

ImmunoRatio is an ImageJ plugin to quantify haematoxylin and DAB-stained tissue sections by measuring the percentage of positively stained nuclear area (labeling index), described in [bib]2452[/bib].

Notes for use:

• It is important to read the URL instructions and original paper to understand what is being measured. In particular, the primary measurement made is percentage of the total nuclear area, not the percentage of detected nuclei (the latter being the more common method of assessing e.g. Ki67). This may be further modified by the Result correction equation.
• Ultimately ImmunoRatio relies on thresholding (color deconvolved [bib]2451[/bib]) images to define 'nucleus' vs 'non-nucleus' regions according to staining intensity. Therefore dark artefacts, such as tissue folds, are likely to cause errors.
• The pixel size is not read automatically from the image, but rather the source image scale should be entered into the dialog box - and the image rescaled accordingly prior to analysis. This scale value is the inverse of the value normally found for pixel width and pixel height under Image -> Properties... (i.e. pixel width & height are given in microns per pixel; the dialog box asks for pixels per micron).

Web application: ImmunoRatio

Example Image: Sample ImmunoRatio results

References

1. [2452] Tuominen VJ, Ruotoistenmäki S, Viitanen A, Jumppanen M, Isola J.  2010.  ImmunoRatio: a publicly available web application for quantitative image analysis of estrogen receptor (ER), progesterone receptor (PR), and Ki-67.. Breast Cancer Res. 12(4):R56.
2. [2451] Ruifrok AC, Johnston DA.  2001.  Quantification of histochemical staining by color deconvolution.. Anal Quant Cytol Histol. 23(4):291-9.

Tracking of focal adhesions includes a number of challenges:

1. Detection of focal adhesion regions in areas of highly variable background
2. Separation of "clumped" adhesions in different objects.
3. Dynamics: Focal adhesions dynamically, grow, shrink, change their shape, they can fuse with neighboring adhesions or one adhesion can be split into multiple children.

Würflinger et al (2011) describe how to detect focal adhesion objects and how to track them over time. Interestingly, tracking results are fed back to segmentation to improve separation of clumped adhesions.

The authors implemented the workflow in Matlab, but do not provide a ready-to-use script.

This macro batch processes all the 2D images (tif and jpg files) located in a user defined folder by calling Fiji Weka trainable segmentation to classify each pixel, and reports the areas of each class in a human readable results table. The classifier to be applied to each image should be previously trained on a representative image by an expert and exported to file (Save classifier) into the image folder to be processed.

This macro segments blood vessels in a 3D stack. It is suited for well-contrasted images (low background) and works better if the width of the vessels of interest is reasonably uniform.

Sample image: 1

sample image: 2