Quantitative analysis of 1300-nm three-photon calcium imaging in the mouse brain.
basic_science · Level V
Where this comes from
- Record sourced from PubMed, PMID 31999253.
- Also identified by DOI 10.7554/eLife.53205 and PMC identifier 7028383.
- Licence recorded as CC BY.
- The licence permits redistribution, so the abstract is shown in full and the full text is available from the publisher.
Abstract
1300 nm three-photon calcium imaging has emerged as a useful technique to allow calcium imaging in deep brain regions. Application to large-scale neural activity imaging entails a careful balance between recording fidelity and perturbation to the sample. We calculated and experimentally verified the excitation pulse energy to achieve <i>the minimum photon count</i> required for the detection of calcium transients in GCaMP6s-expressing neurons for 920 nm two-photon and 1320 nm three-photon excitation. By considering the combined effects of in-focus signal attenuation and out-of-focus background generation, we quantified the cross-over depth beyond which three-photon microscopy outpeforms two-photon microscopy in recording fidelity. Brain tissue heating by continuous three-photon imaging was simulated with Monte Carlo method and experimentally validated with immunohistochemistry. Increased immunoreactivity was observed with 150 mW excitation power at 1 and 1.2 mm imaging depths. Our analysis presents a translatable model for the optimization of three-photon calcium imaging based on experimentally tractable parameters.
Medical subject headings
- Brain
- Calcium
- Microscopy, Fluorescence, Multiphoton