5 Free Image Analysis Software Tools for Microscopy

5 Free Image Analysis Software Tools for Microscopy

If your lab was like mine was, you struggle to afford new antibodies, let alone expensive image analysis software programs like Neurolucida to analyze your data.

Out of sheer necessity to analyze a massive image set before I turned grey, I scrounged around for free open source software programs to help analyze my confocal microscopy image stacks.

It took me years (actually!) to find some of them, so I thought I would pay-it-forward by sharing them with you! Kudos to everyone who takes the time to create these free open source programs.

Here are my Top 5 favorite time/life savers:

1. ImageJ/FIJI The Image Multi-Tool

ImageJ should be the first program you become familiar with when looking for image analysis software. Download it, search through the plugins to see what’s available and test them out. Being proficient at using ImageJ is essential for most image processing and analysis.

It can do simple things like crop, label, and alter the brightness and contrast of fluorescence images. It can also easily handle 3D stacks of confocal microscopy images, and perform complex quantitative analysis.

For more information visit their website: http://imagej.nih.gov/ij/

FIJI (FIJI Is Just ImageJ) is a bundle of the top plugins available for ImageJ. Downloading FIJI instead of ImageJ just starts you out with more base plugins so you don’t have to hunt around and add them to your ImageJ program. There were a few plugins that were only available in FIJI. The select grouping of plugins is geared for neuroscience.

For more information visit their website: http://fiji.sc/Fiji

2. Cell Profiler/Cell AnalystImage Batch Processing and Data Analysis


Cell Profiler can extract quantitative measurements from thousands of images through a custom pipeline that can first process and then analyze your images.

It can quantify antibody fluorescence intensity, colocalization, cell density, or even track cells through time-lapse movies. It’s as simple as setting up your pipeline and then going for a coffee.

I’ve never used Cell Analyst but I’ve heard good things about its ability to handle large datasets. Give it a try and let me know what you think.

For more information visit their website: http://www.cellprofiler.org/

3. NeuronstudioAutomatic Neuron Tracing Super-Tool

If you need to trace the dendritic arbor of a neuron then this is by far the best free open source program that I came across. Don’t be thrown by the lack of updates since 2009 or the fact that you need Windows 2000/XP/Vista to run it. It has a comprehensive help manual that will walk you through everything you need to know.

It has manual, semi-manual, and automatic tracing modes for complete 3D reconstruction. All-around the program is a breeze to use, and does a fantastic job of automatically tracing through a stack of confocal images based on the fluorescence intensity.

You can even find errors in your 3D reconstruction by flipping the trace file and image stack into a different dimension to look for anomalies in the trace, and then easily fix them by dragging the point to the correct location.

It will also perform a quantitative analysis on dendritic spine morphology. It can distinguish between different types, or you can set your own classification parameters.


It can perform a basic Sholl Analysis on the dendritic arbor and output branch order data, including spine counts.


The program also lets you output your trace as a .swc file, which allows you to reconstruct or analyze the traced neuron in other programs, such as L-measure (below).

For more information visit their website: http://research.mssm.edu/cnic/tools-ns.html

4. Volume Integration and Alignment System (VIAS) – Image Stack Alignment Software


VIAS enables you to tile multiple confocal microscopy image stacks into a single 3D image dataset. This is a software program made by the same group that created Neuronstudio. It has a similar intuitive user interface and a step-by-step online guide.

It allows for real-time interaction with the stacks, therefore you can easily drag the stacks around until they are perfectly aligned. An auto-alignment feature helps to tweak the alignment for a seamless result.

If your image stacks are of different depths, they can be easily aligned in the z-dimension. Once finished you can export as a .tif to use the new stack in Neuronstudio, or ImageJ.

For more information visit their website: http://research.mssm.edu/cnic/tools-vias.html

 5. L-measure – Quantitative Morphological Measurements to the Max


L-measure was my tool of choice to extract more complex quantitative measurements from my neuronal reconstructions. It has a huge range of morphological measurements that you cannot find built into other programs.

It will take your morphological analysis to a whole new level by analyzing the volume of dendrites, the asymmetry of branches or even the angle between branches.


The online help guide is fantastic and goes into great depth to explain the complex measurements. The user interface is super simple and allows for multiple traces files (.swc format) to be analyzed at once and will output one file for your subsequent statistical analysis.

For more information visit their online help guide: http://cng.gmu.edu:8080/Lm/help/index.htm
To Download L-measure visit their website: http://cng.gmu.edu:8080/Lm/

This is just a list of the programs that I loved during my graduate work. There are plenty of other great free open source image analysis programs available.

Have questions about any of these programs? Ask them in the comments section below!

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3 responses to “5 Free Image Analysis Software Tools for Microscopy”

  1. Sattar Abdullah says:

    I am very need of software to image analysis because I work in cytogenetics.


  2. Floyd Wilson says:

    I have a amscope camera attached to an Olympus microscope. Is there a way to work with live microscope images using imagej with this setup?


    • Leslie Rietveld says:

      Hi Floyd,

      As we are not responsible for creating any of this software, I can only speak from my experience with these programs. I have used Micro-manager (https://micro-manager.org/) in the past on an Olympus microscope (not sure what the camera was) to acquire images. It was super simple to use. I would recommend you checkout the list of acquisition plugins on the ImageJ website (https://imagej.nih.gov/ij/plugins/#acq). You may be able to find something that meets your needs.

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