Pulse train gating to improve signal generation for in vivo two-photon fluorescence microscopy

Two-photon microscopy is used routinely for in vivo imaging of neural and vascular structures and functions in rodents with a high resolution. Image quality, however, often degrades in deeper portions of the cerebral cortex. Strategies to improve deep imaging are therefore needed. We introduce such a strategy using the gating of high repetition rate ultrafast pulse trains to increase the signal level.

We investigate how the signal generation, signal-to-noise ratio (SNR), and signal-to-background ratio (SBR) improve with pulse gating while imaging in vivo mouse cerebral vasculature.

An electro-optic modulator with a high-power (6 W) 80 MHz repetition rate ytterbium fiber amplifier is used to create gates of pulses at a 1 MHz repetition rate. We first measure signal generation from a Texas Red solution in a cuvette to characterize the system with no gating and at a 50%, 25%, and 12.5% duty cycle. We then compare the signal generation, SNR, and SBR when imaging Texas Red-labeled vasculature using these conditions.

We find up to a 6.73-fold increase in fluorescent signal from a cuvette when using a 12.5% duty cycle pulse gating excitation pattern as opposed to a constant 80 MHz pulse train at the same average power. We verify similar increases for in vivo imaging to that observed in cuvette testing. For deep imaging, we find that pulse gating results in a 2.95-fold increase in the SNR and a 1.37-fold increase in the SBR on average when imaging mouse cortical vasculature at depths ranging from 950 to $$.

We demonstrate that a pulse gating strategy can either be used to limit heating when imaging superficial brain regions or used to increase signal generation in deep regions. These findings should encourage others to adopt similar pulse gating excitation schemes for imaging neural structures through two-photon microscopy.