An Efficient Pipeline for Cell Fluorescence Measurements

Neuroscience, Computation · 9 August 2021

Introduction

Luke Hammond provided a comprehensive guide on the calculation of CTCF here. Hammond’s guide involves taking measurements in ImageJ, then export data to a spreadsheet application to perform calculations. This is a simple task but such repetitive procedures can be quite dull and sometimes induce mistakes. So I have created a MATLAB application to largely reduce manual procedure. A standalone version is also available for MacOS.

Pre-requisites

A valid MATLAB license is required to run the app within MATLAB. To install standalone version, MacOS and a good internet connection are required.

Instructions

Load an image by using the ‘Load Image’ button. If your image is a true colour RGB image, select the colour channel for which ROI is to be drawn from the dropdown menu, skip this step if you have loaded a greyscale image.

You may draw ROIs for cellular objects and background using the MATLAB Polygon, Circle, Freehand and Assisted Freehand tools. Begin drawing an ROI by selecting one of the drawing tools. You may cancel drawing anytime before the ROI is closed using the ‘Esc’ key. Right-click an ROI to view the “Delete” option. When you have selected all ROIs, save data and return to the main UI by clicking the ‘Apply’ button. The ROIs for cells and background should be drawn in an alternating fashion, beginning with a cell ROI. That is, Cell 1-> Background 1 -> Cell 2 -> Background 2 …. The ROI Labeller will remind you of which type you ought to delineate at all time.

Select the colour channel(s) for which CTCF is measured by ticking the corresponding checkboxes. Perform measurements by clicking on the ‘Calculate CTCF’ button. The results will be displayed as a table in pop-up windows.

Note: If you have deleted an ROI, the labeller will ask you to replace that object immediately. New ROIs can be created once it has been replaced. The default tool for replacement is Freehand.

Video Demonstration

Download

Donwload MATLAB App Donwload MacOS Installer

Updates

14th August 2021

Bugfix and improvements on ROI Labeller logistics and stability.
Allow saving of ROIs as an instance mask (PNG format) from which CTCF measurements can be reproduced.
Toggle button to show/hide ROI labels. Note: there is a current problem associated with the ‘Assisted’ ROI tool. The Assisted Freehand tool only works after the first ROI has been defined.

13th August 2021 v0.1.2 update addresses several issues in the previous versions:

The ROI Labeller will guide the user through the labelling process.
Each ROI is labelled with its corresponding object type and ordinal (e.g. “2nd Cell”), which will appear upon hover.
Selecting an ROI tool while drawing will no longer trigger a new ROI object.
The issue with overlapping of ROIs is resolved.
Delete any ROI object from the right dropdown menu and replace it with a new ROI (every deleted ROI must be replaced).
Smart workflow: the program will determine which functions are available based on the user interaction so as to prevent runtime error.

New features are added:

Save measurement data into an Excel document. Readings from each selected colour channel are stored in a separate spreadsheet.
Added ROI selection tool: assisted freehand
(Not Recommended) User can now save ROI objects as a “.ROI” file in the ROI Labeller and load at a later time.

11th August 2021 The release of v0.1.1 improves performance and stability. This update also introduced minor changes to the GUI.

10th August 2021 A MATLAB app v0.1.0 is now available and can be installed from MATLAB add-on explorer. The app is user friendly and does not require programming. A standalone version of this program is now available on MacOS. The app depends on MATLAB Runtime (~600MB). The installer will automatically download and install this from MATLAB’s server. This is a good option if you do not have MATLAB installed.

6th August 2021 Pre-release of MATLAB command-line program.