Identifying_tracks_in_histology
Tracks are not necessarily easy to observe in simple nissl- or DAPI-stained slices. Therefore, labeling with DiI, a fluorescent dye, is recommended.
Dip the probe in DiI, let it dry (wait about 2s), and repeat (5 - 10 times seems to work very well). If it is inconvenient to dip the whole probe, the dye can also be applied by using a micropipette, holding a drop on the end of it and running the drop over the stationary probe several times. Usually you will be able to see the probe take a pink color when the light catches it at the right angle, after the DiI has been successfully applied.
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| Example probe holder, allowing easy probe dipping in DiI |
Normal DiI or other colors (e.g. DiO, DiD) can be used for normal histology. If tissue clearing will be used, a modified version of DiI can be used instead.
To track probes, slices can be imaged with a confocal, 2-photon or light-sheet microscope (after tissue-clearing).
To get highly accurate tracking results, a system with a microscope mounted on top of an automated slicer, like this one, is very useful.
Most fluorescence spectra are available here: https://www.fpbase.org/.
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| Example slice with a few DiI-stained probe tracks, imaged on a 2-photon at 920 nm |
To align histological slices to a reference atlas, several one open source and publicly available software designed for this purpose exist:
- SHARP-Track. See the bioRxiv paper for more details.
- AP_histology, inspired by SHARP-Track.
- Brain globe. See the paper for more details.