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Scattering is one of the main issues that limit the imaging depth in deep tissue optical imaging. To characterize the role of scattering, we have developed a forward model based on the beam propagation method and established the link between the macroscopic optical properties of the media and the statistical parameters of the phase masks applied to the wavefront. Using this model, we have analyzed the degradation of the point-spread function of the illumination beam in the transition regime from ballistic to diffusive light transport. Our method provides a wave-optic simulation toolkit to analyze the effects of scattering on image quality degradation in scanning microscopy. Our open-source implementation is available at https://github.com/BUNPC/Beam-Propagation-Method.

Non-degenerate two-photon excitation (ND-TPE) has been explored in two-photon excitation microscopy. However, a systematic study of the efficiency of ND-TPE to guide the selection of fluorophore excitation wavelengths is missing. We measured the relative non-degenerate two-photon absorption cross-section (ND-TPACS) of several commonly used fluorophores (two fluorescent proteins and three small-molecule dyes) and generated 2-dimensional ND-TPACS spectra. We observed that the shape of a ND-TPACS spectrum follows that of the corresponding degenerate two-photon absorption cross-section (D-TPACS) spectrum, but is higher in magnitude. We found that the observed enhancements are higher than theoretical predictions.

In non-degenerate two-photon microscopy (ND-TPM), the required energy for fluorescence excitation occurs via absorption of two photons of different energies derived from two synchronized pulsed laser beams. ND-TPM is a promising imaging technology offering flexibility in the choice of the photon energy for each beam. However, a formalism to quantify the efficiency of two-photon absorption (TPA) under non-degenerate excitation, relative to the resonant degenerate excitation, is missing. Here, we derive this formalism and experimentally validate our prediction for a common fluorophore, fluorescein. An accurate quantification of non-degenerate TPA is important to optimize the choice of photon energies for each fluorophore.

Non-degenerate 2-photon excitation (ND-2PE) of a fluorophore with two laser beams of different photon energies offers an independent degree of freedom in tuning of the photon flux for each beam. This feature takes advantage of the infrared wavelengths used in degenerate 3-photon excitation (D-3PE) microscopy to achieve increased penetration depths, while preserving a relatively high 2-photon excitation cross section in comparison to that of D-3PE. Here, using spatially and temporally aligned Ti:Sapphire laser and optical parametric oscillator beams operating at near infrared (NIR) and short-wavelength infrared (SWIR) optical frequencies, we employ ND-2PE and provide a practical demonstration that a constant fluorophore emission intensity is achievable deeper into a scattering medium using ND-2PE as compared to the commonly used degenerate 2-photon excitation (D-2PE).

There is an increasing need for quantitative and computationally affordable models for analyzing tissue metabolism and hemodynamics in microvascular networks. In this work, we develop a hybrid model to solve for the time-varying oxygen advection-diffusion equation in the vessels and tissue. To obtain a three-dimensional temporal evolution of tissue oxygen concentration for realistic complex vessel networks, we used a graph-based advection model combined with a finite-element based diffusion model and an implicit time-advancing scheme. We validated this algorithm for both static and dynamic conditions. We also applied it to a complex vascular network obtained from a rodent somatosensory cortex. Qualitative agreement was found with in-vivo experiments.

Laser speckle contrast imaging is becoming an established method for full-field imaging of cerebral blood flow dynamics in animal models. The sensitivity and noise in the measurement of blood flow changes depend on the camera exposure time. The relation among sensitivity, noise, and camera exposure time was investigated experimentally by imaging the speckle contrast changes in the brain after electrical forepaw stimulation in rats. The sensitivity to relative changes in speckle contrast was found to increase at longer exposure times and to reach a plateau for exposure times greater than approximately 2 ms. However, the speckle contrast noise also increases with exposure time and thus the contrast-to-noise ratio was found to peak at an exposure time of approximately 5 ms. Our results suggests that approximately 5 ms is an optimal exposure time for imaging of stimulus-induced changes in cerebral blood flow in rodents.