Fluorescent dyes exhibiting peak action cross-sections within the water absorption window are generally unsuitable for in vivo two-photon fluorescence imaging using photons of the same wavelength. We show that undertaking the two-photon excitation process with two photons of different wavelengths, namely non-degenerate two-photon excitation (ND-2PE), enables imaging in the water absorption window using two spatially and temporally overlapped excitation sources at 1300 nm and 1600 nm. We explore the relative brightness spectra of indocyanine green (ICG) and assess its suitability for imaging at wavelengths susceptible to water absorption. Further, we demonstrate damage-free in vivo imaging of the rodent cortex vascular structure up to 1.2 mm using ND-2PE.
KEYWORDS: Pulse signals, Education and training, In vivo imaging, Signal generators, Signal to noise ratio, Ultrafast phenomena, Two photon excitation microscopy, Imaging systems, Electrooptic modulation, Neurophotonics
SignificanceTwo-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.AimWe 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.ApproachAn 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.ResultsWe 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 1050 μm.ConclusionsWe 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.
Photonic Crystal Fibers have proved to be an efficient medium for propagation of electromagnetic radiation especially in the Terahertz regime. Located between the infra-red and microwave region, the Terahertz frequency range lies within 0.1 to 1 Terahertz. We report a design of a graduating ring rectangular-core Photonic Crystal Fiber, having background material as Cyclic-Olefin Copolymer (COC), with extremely low confinement loss of 4.9399 × 10−7 dB/cm and material loss of 0.2 cm−1 at 0.22 mm pitch value. Due to the very low loss values, such a structure of the Photonic Crystal Fiber can be used for efficient low-loss data communication.
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