Jan 07, 2026
Quantification of neurons in cleared large ganglionated tissues
- 1University of Melbourne
- SPARCTech. support email: [email protected]
Protocol Citation: John-Paul Fuller-Jackson, Peregrine B Osborne, Janet R Keast 2026. Quantification of neurons in cleared large ganglionated tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzjxn8lx1/v1
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
We use this protocol and it's working
Created: May 22, 2024
Last Modified: January 07, 2026
Protocol Integer ID: 100250
Keywords: imaris, segmentation, quantification, peripheral nervous system, autonomic ganglia, quantification of neuron, segmentation of neuron, large samples of ganglionate peripheral nerve tissue, cleared large ganglionated tissue, large ganglionated tissues this protocol, ganglionate peripheral nerve tissue, neuron distribution, 3d representation of the neuron distribution, segmentation, neuron, cleared large sample, quantification, accurate count, using imari
Funders Acknowledgements:
NIH SPARC
Grant ID: 3OT2OD023872
Abstract
This protocol is a semi-automated method using Imaris to quantify neurons in cleared large samples of ganglionate peripheral nerve tissue. The process involves the segmentation of neurons, which can then be filtered and manually curated. This not only provides accurate counts, but also a 3D representation of the neuron distribution.
Materials
Equipment
Ultramicroscope Blaze
NAME
Light sheet microscope
TYPE
Ultramicroscope
BRAND
140-006-739.04
SKU
LINK
Software
Imaris File Converter
NAME
Bitplane
DEVELOPER
Software
Imaris Stitcher
NAME
Bitplane
DEVELOPER
Large Volume Image Acquisition on Light Sheet Microscope
Mount sample on sample holder of light sheet microscope to optimize illumination of neuron-containing region.
Load sample into light sheet microscope, fully immersed in imaging medium.
At low magnification (1-2x), inspect the sample and identify the ganglionated region. If poorly illuminated, repeat Steps 1 and 2 until the region of interest is suitable illuminated by the light sheet.
At higher magnification (at least 8x), completely image the neuron-containing region using tile scanning if required. A z-step of 2 µm is suitable for neuron counting. Single-sided illumination is best due to differences in left and right illumination in asymmetrical samples. Ensure sufficient laser power and exposure for adequate illumination. Save files as OME-TIFF.
Image Preprocessing
Convert the image stack(s) from OME-TIFF to Imaris files (.IMS) using Imaris Converter.
Software
Imaris File Converter
NAME
Bitplane
DEVELOPER
For tile scan acquisitions, stitch the separate .IMS files together using Imaris Stitcher.
Software
Imaris Stitcher
NAME
Bitplane
DEVELOPER
Neuron Visualization in Imaris
Open the stitched .IMS file in Imaris.
In the Display Properties (Ctrl+D), rename each channel according to the immunolabel. Adjust each channel's Display Settings to best visualize the neurons in the image. A gamma correction of 1.2-1.6 is typical, to correct for differences in illumination across the sample.
Semi-automatic segmentation of neurons with Surfaces
In Object Panel, click on Surfaces.
In the first part of the Surface Creation Wizard, tick Segment only a Region of Interest. Leave all other options unticked. Click next (blue arrow icon at the bottom of panel).
In the second part of the Surface Creation Wizard, manually adjust the region of interest to encompass the neurons to be segmented (and thus counted). Click next.
In the third part of the Surface Creation Wizard, under Source Channel select the channel containing the neurons of interest. Tick Smooth and set Surfaces Detail to 5 µm. Under Thresholding select Background Subtraction (Local Contrast). Set the Diameter of the largest Sphere which fits into the Object to 10 µm. Based on subsequent neuron detection success, these values can be modified.
In the fourth part of the Surface Creation Wizard, a preview of the Surfaces is shown on the image. Under Threshold, the Surface can be adjusted based on background subtraction. Adjust the thresholding until the previewed Surface just obscures the signal it is detecting. Tick Seed Points and set Seed Point Diameter to 20 µm (this value can be modified to optimize selection of neurons and splitting neurons that are closely positioned).
For samples in which illumination of neurons varies drastically across the sample, it may be better to break down the Surface creation into separate Regions of Interest, to adjust each accordingly.
In the fifth part of the Surface Creation Wizard which is only added when Seed Points are enabled, the suggested Seed Points on the image can be filtered according to many metrics - in this example Quality was chosen and the selection thresholded to the point when all neurons were identified by Seed Points.
In the sixth and final step of the Surface Creation Wizard, the final Surfaces are shown, and can be filtered again based on many criteria. Number of Voxels filters Surfaces based on size, which is useful for quickly removing very large and very small things that are not neurons but were included (for example, axon tracts immunolabelled by the same marker). Another useful metric to filter by is Sphericity, which helps remove Surfaces based on non-spherical objects (again, axon tracts).
Click on the Green arrow icon to finish the creation of the Surface.
With the Surface segmentation of the neurons complete, manual editing can still be performed for anything that was not filtered out during the Surface Creation Wizard. With the Surface selected, click on the colourful cube icon to bring up display options for the Surface. Under Material a range of surface textures can be selected, with the semi-transparent most useful for identifying which Surfaces correctly represent neurons.
With the Surface selected, change to the Edit tab (pencil icon) to then select individual Surfaces and modify/delete them. Selected Surfaces will be highlighted with a more intense yellow hue.
To view the total number of neurons in any given Surface, click on the Measurement tab (icon of a line graph). Under Overall, the Total Number of Surfaces will be displayed. The Save icon below allows for this to be exported as a .CSV.