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 the minimum photon count 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.
Highlights
Multiphoton microscopy combined with genetically encoded calcium indicators (GECIs) is a powerful functional imaging technique widely applied to in vivo neurophysiological recordings (Lin and Schnitzer, 2016; Yang and Yuste, 2017)
2-photon microscopy (2PM) has enabled activity recording from thousands of neurons with single-cell resolution (Sofroniew et al, 2016; Stirman et al, 2016; Weisenburger et al, 2019)
Given DF=F and t1=e of a calcium indicator are fixed, the quality of calcium imaging can only be improved by increasing the baseline neuron brightness F0, which can be achieved by adjusting a number of imaging parameters, for example excitation repetition rate, pulse duration, pulse energy, focal spot size and laser dwell time on each neuron
Summary
Multiphoton microscopy combined with genetically encoded calcium indicators (GECIs) is a powerful functional imaging technique widely applied to in vivo neurophysiological recordings (Lin and Schnitzer, 2016; Yang and Yuste, 2017). 2-photon microscopy (2PM) has enabled activity recording from thousands of neurons with single-cell resolution (Sofroniew et al, 2016; Stirman et al, 2016; Weisenburger et al, 2019). 2-photon excitation (2PE) can effectively reduce out-of-focus fluorescence, the background intensity eventually becomes comparable to the signal in non-sparsely labeled samples, as the excitation power grows exponentially with imaging depth. 3-photon microscopy (3PM) has emerged as a useful tool for imaging in deep brain regions that are typically inaccessible to 2PM.
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