Jul 25, 2025
Version 2
Bioloc3D (B3D) : Protocol for 3D automated analysis of colocalization in microscopy V.2
- Thibault Dhellemmes1,
- Rémi inet1,
- Fabrice Cordelières2,
- Marc Landry1
- 1Université de Bordeaux - Institut des Maladies Neurodégénératives (IMN) / UMR 5293;
- 2Bordeaux Imaging Center
- Bioloc3D (B3D)
External link: https://github.com/Bioloc3D
Protocol Citation: Thibault Dhellemmes, Rémi inet, Fabrice Cordelières, Marc Landry 2025. Bioloc3D (B3D) : Protocol for 3D automated analysis of colocalization in microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx4p5zl8j/v2Version created by Thibault Dhellemmes
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: July 25, 2025
Last Modified: July 29, 2025
Protocol Integer ID: 223277
Keywords: Software, Microscopy, Automatization, Colocalization, Fluorescent, Quantification, Automated analysis, Statistics, bioloc3d, tools capable of colocalization quantification, microscopy this protocol, microscopy, fluorescent labeling across entire stack, fluorescent labeling, colocalization quantification, analysis of colocalization, colocalization, b3d, automate statistical analysis, automated analysis
Funders Acknowledgements:
ANR
Grant ID: ANR-19-CE37-0019 Relax
FRM
Grant ID: FDT202404018302
Abstract
This protocol introduces Bioloc3D, a fully open-source tool designed to quantify fluorescent labeling across entire stacks and automate statistical analysis. It offers a solution to the lack of tools capable of colocalization quantification between multiple identified elements in 3D. Expected results includes graphical representations of the results through images and estimation plots.
Image Attribution
Thibault Dhellemmes
Materials
Raw image stacks with fluorescent signals to analyze in any format adapted to ImageJ.
Linux/Mac/Windows computer with ImageJ, Excel and Bioloc3D-Plt installed. ImageJ installation file is available online at https://imagej.net.
Troubleshooting
Before start
To operate in the best condition, the minimal requirement for colocalization analysis is a stack of 2 channels with 2 images (i); in 8-bit color depth (ii); preferably in TIFF format (iii)
B3D workflow and protocol
- Schematic representation of the analysis workflow using B3D
- Diagram of the simplified procedure
- User-interface (UI) of B3D-Img toolset in ImageJ and B3D-Plt standalone application
Illustrations of B3D workflow and protocol
! CAUTION Figures mentioned under brackets (e.g. (Supplementary Fig. 1C)) refer to the method publication in which the experimental design of B3D toolset is detailed (cf. "Manuscript citation" section)
Installation
3m
Install B3D-Img in ImageJ macro library, by drag/drop the .ijm file in the toolsets folder of the application (Fiji/ImageJ → macros → toolsets)
Install latest version of 3D Objects Counter plugin available online on GitHub, by drag/drop the .jar file in the plugins folder of the application (Fiji/ImageJ → plugins)
Launch ImageJ software
Software
ImageJ (Fiji)
NAME
Windows 10
OS
National Institutes of Health (USA)
DEVELOPER
SOURCE LINK
Through ImageJ install MorphoLibJ, 3D ImageJ Suite and Read and Write Excel additional plugins available in plugin list (Help → Update… → Manage Update Sites → tick plugins → Apply and Close)
Close ImageJ software
Download the B3D-Plt application depending of the user operating system at https://github.com/Bioloc3D/B3D-Plt
Software
Bioloc3D
NAME
Windows / MacOS
OS
Thibault Dhellemmes / Rémi Kinet
DEVELOPER
REPOSITORY
SOURCE LINK
Image analysis : B3D-Imaging (B3D-Img)
Launch B3D toolset in ImageJ software
30s
Start ImageJ software
Click on the "More Tools" menu (≫) and select B3D-Img
Defining the correct parameters
2m 30s
Click on B3D parameters icon (cf. Supplementary Fig. 1A)
Select the stack to test the parameters and the folder in which test data will be saved. Parameters must be tested in an iterative matter, select one channel from the stack (cf. Supplementary Fig. 1C, Step 1 of the diagram).
Click "OK"
Define the threshold adapted to the signal of the selected channel (cf. Supplementary Fig. 1C, Step 2 of the diagram).
Click "OK"
Use the threshold defined in sub-step 11.4 and define minimal and maximal size threshold to test (Supplementary Fig. 1C, Step 3 of the diagram). If needed, define minimal area for soma detection (cf. Supplementary Fig. 1C, Step 4 of the diagram)
Click "OK": Test analysis is launched
Analysis of one stack and raw data export
! CAUTION As discussed in the "advantages and limitations" section, time of analysis depends on several aspects, including computation power of the machine, size, potential number of objects to count per channel and signal-to-noise ratio of your images. To estimate the time of each step of the procedure, we used a stack presenting the following characteristics: size= 1024 x 1024; z= 25; objects counted per channel= 1000. In addition, sample is presenting a moderate signal-to-background (SBR) but a low signal-to-noise ratio (SNR).
Click on B3D analysis icon (cf. Fig. 2A)
Select the folder containing stack(s) to analyze and numbered your batch of analysis (Fig2C, section 1 on the diagram). Normalization of the name of exported data files is compulsory for the automatic computation. If more than one file is present in this folder, "headless" option must be defined on "Yes"
! CAUTION Select folder must contains only tiff file(s) before starting the analysis in ImageJ.
Click "OK"
Enter parameters defined in step 11, in the GUI (Fig. 2C, section 3 on the diagram) and select options for quantification based on expected information
Option A :
Simple quantification (3D-Simple) of primary channels, without quantification of additional features (control of colocalizations sites and soma)
(i) Select "Yes" for "Simple segmentation of C1 & C2:"
(ii) Select "No" for "Check colocalization with an additional channel"
(iii) Select "No" for "Complete counting with an additional channel"
30s
Option B :
Complete quantification (3D-OC) of primary channels, without quantification of additional features
(i) Select "No" for "Simple segmentation of C1 & C2:"
(ii) Select "No" for "Check colocalization with an additional channel"
(iii) Select "No" for "Complete counting with an additional channel"
3m
Option C :
Simple quantification (3D-Simple) of primary channels, including quantification of additional features
(i) Select "Yes" for "Simple segmentation of C1 & C2:"
(ii) Select "Yes" for "Check colocalization with an additional channel"
(iii) Enter parameters for colocalization control (threshold and size)
(iv) Select "Yes" for "Complete counting with an additional channel"
(v) Enter parameters for soma counting (minimum area and threshold)
40s
Option D :
Complete quantification (3D-OC) of primary channels, including quantification of additional features
(i) Select "No" for "Simple segmentation of C1 & C2:"
(ii) Select "Yes" for "Check colocalization with an additional channel"
(iii) Enter parameters for colocalization control (threshold and size)
(iv) Select "Yes" for "Complete counting with an additional channel"
(v) Enter parameters for soma counting (minimum area and threshold)
3m 30s
Click "OK"
(Optional) Click “Help” to be redirected to GitHub repository
(Optional) Proofreading of 3D quantification on one stack
! CAUTION If only one stack is present in the folder, illustrations of the results can be displayed by selecting “No” for headless mode (Fig2C, section 2 on the diagram).
5m
For a more precise proofreading apply option B or D (Sub-steps 12.6 or 12.8)
Overview the different output stacks with the excel of the data aside to check if it counts what you wanted
Computation, plotting and statistical analysis : B3D-Plotting (B3D-Plt)
Open the B3D-Plt application
Click on "Load file" and select the folder containing the Excels to analyze
Click on "Save file" and select the folder in which graphs and analysis will be saved
Click on the drop-down menu and select the number of conditions you have to analyze
In the bars that appear, enter the name of the condition and the color you want to associate with it (seaborn or hexadecimal #FF0000 style, but can stay empty)
Seaborn color link : https://seaborn.pydata.org/tutorial/color_palettes.html
Click on "Launch analysis"
! CAUTION Timing indicated for this step has been tested on a file containing 60 excel files
15m
The name of the document currently analyzed is print to monitor progress
Analysis end is notified through a pop-up window
Find the figures and Excel in the folder selected in step 16