The aim of electrophysiological recordings using silicon probes is to record neuronal activity across brain regions and animals. It is therefore fundamental to record precisely the localisation of the probe (or multiple probes) in each brain and be able to compare probes across animals.
Here we describe a tool for the analysis of silicon probe tracks (e.g. Neuropixels) in brains imaged post-hoc in a standard coordinate space. This tool is packaged as part of the cellfinder suite of tools for whole-brain microscopy image analysis.
In order to label the probe penetration track in the brain, the probe is delicately coated with DiI (Molecular Probes, Cat# V22885) using a pipette tip (Fig. 1, left). The probe is then attached to a micromanipulator and the probe tip lowered to touch the surface of the brain (e.g. in head-fixed mice). This position is recorded as position “zero”. The probe is then introduced in the brain at a speed of ~10μm per second to the desired penetration depth (in our case 1750μm; Fig. 1, centre).
When the experiment is done and the probe is retracted, the animal is anaesthetised and perfused with PFA 4% following standard perfusion protocols. The brain is carefully extracted and left in PFA 4% overnight.
The brain is then thoroughly washed with 100mM PBS and imaged (e.g. by Serial 2-Photon Tomography; Fig. 1, right). We imaged 2 channels (one where DiI signal is detected and one with background fluorescence only) at a resolution of x = 5μm, y = 5μm, z = 20μm.
To track the probe in standard space, the brain must first be registered to an atlas using amap.
Before registration, cellfinder (which includes amap) needs to be installed. Please follow the instructions here. Once cellfinder is installed, we can proceed to register the imaged brain.
You will need:
The path where the brain image stack (DiI signal channel) is located
The path where the brain stack (background fluorescence channel) is located.
The path where you want the registration result to be saved
The resolution at which the brain was imaged
To register your brain to an atlas, please follow the instructions for amap here.
An example registration command is as follows:
amap /path/to/signal/channel1.tiff /path/to/output/directory -x 5 -y 5 -z 20 /path/to/background/channel2.tiff
A new output directory has been created, which contains the registered brain. We are now ready to manually trace the probe track.
To open the graphical user interface, type the following command:
manual_seggraphical user interface opens and shows a set of tools. A detailed description of these tools can be found in the legend of Fig 2. For a step-by-step description of how to manually trace a probe track with cellfinder, please see the video at the top of this page. There is also some more information at the main page for this tool, here.
a. The layers tool box. At the bottom can be found the layers shown in the central screen. In this case, only one layer is present, called
Image in standard space, highlighted in blue. At the top are a set of tools that affect the layer highlighted. In this case, the layer highlighted called
Image in standard space is the brain image stack, therefore the tools available allow to adjust the visualisation of the image stack.
b. The project tool box. Here, the image stack can be loaded (
Load project). An atlas can be overlaid to the brain stack (
Load atlas). A probe track tracing can be added (
Add track). Finally, track markers (also called points) can be fitted (
Trace tracks). The fitting properties can be adjusted by fit degrees, spline smoothing and spline points (i.e. the number of points used to sample the fit). A marker at the exact position of the surface can be added by ticking the
Add surface point box.
c. The brainrender tool box. The manually traced probe tracks can be previewed in brainrender. In addition to probe tracks, one brain region can be visualised by choosing it in the drop-down list
Region to render. The transparency of the region to render can be adjusted with
Atlas region alpha. The probe track can be saved in the registration directory, under
name_of_track.csv file shows the localisation of the probe track following standard brain atlas region annotations.
By Mateo Vélez-Fort