Modeling nonlinear optical microscopy in scattering media, part I. Propagation from lens to focal volume: tutorial.

The development and application of nonlinear optical (NLO) microscopy methods in biomedical research have experienced rapid growth over the past three decades. Despite the compelling power of these methods, optical scattering limits their practical use in biological tissues. This tutorial offers a model-based approach illustrating how analytical methods from classical electromagnetism can be employed to comprehensively model NLO microscopy in scattering media. In Part I, we quantitatively model focused beam propagation in non-scattering and scattering media from the lens to focal volume. In Part II, we model signal generation, radiation, and far-field detection. Moreover, we detail modeling approaches for major optical microscopy modalities including classical fluorescence, multi-photon fluorescence, second harmonic generation, and coherent anti-Stokes Raman microscopy.

Modeling nonlinear optical microscopy in scattering media, part II. Radiation from focal volume to far-field: tutorial.

The development and application of nonlinear optical (NLO) microscopy methods in biomedical research has experienced rapid growth over the past three decades. Despite the compelling power of these methods, optical scattering limits their practical use in biological tissues. This tutorial offers a model-based approach illustrating how analytical methods from classical electromagnetism can be employed to comprehensively model NLO microscopy in scattering media. In Part I, we quantitatively model focused beam propagation in non-scattering and scattering media from the lens to focal volume. In Part II, we model signal generation, radiation, and far-field detection. Moreover, we detail modeling approaches for major optical microscopy modalities including classical fluorescence, multi-photon fluorescence, second harmonic generation, and coherent anti-Stokes Raman microscopy.

Corneal transparency and scleral opacity arises from the nanoarchitecture of the constituent collagen fibrils.

While human scleral and corneal tissues possess similar structural morphology of long parallel cylindrical collagen fibrils, their optical characteristics are markedly different. Using pseudospectral time-domain (PSTD) simulations of Maxwell's equations, we model light propagation through realistic representations of scleral and corneal nanoarchitecture and analyze the transmittance and spatial correlation in the near field. Our simulation results provide differing predictions for scleral opacity and corneal transparency across the vacuum ultraviolet to the mid-infrared spectral region in agreement with experimental data. The simulations reveal that the differences in optical transparency between these tissues arise through differences in light scattering emanating from the specific nanoscale arrangement and polydispersity of the constituent collagen fibrils.

MCCL: an open-source software application for Monte Carlo simulations of radiative transport.

The Monte Carlo Command Line application (MCCL) is an open-source software package that provides Monte Carlo simulations of radiative transport through heterogeneous turbid media. MCCL is available on GitHub through our virtualphotonics.org website, is actively supported, and carries extensive documentation. Here, we describe the main technical capabilities, the overall software architecture, and the operational details of MCCL.

Effect of scattering on coherent anti-Stokes Raman scattering (CARS) signals.

We develop a computational framework to examine the factors responsible for scattering-induced distortions of coherent anti-Stokes Raman scattering (CARS) signals in turbid samples. We apply the Huygens-Fresnel wave-based electric field superposition (HF-WEFS) method combined with the radiating dipole approximation to compute the effects of scattering-induced distortions of focal excitation fields on the far-field CARS signal. We analyze the effect of spherical scatterers, placed in the vicinity of the focal volume, on the CARS signal emitted by different objects (2µm diameter solid sphere, 2µm diameter myelin cylinder and 2µm diameter myelin tube). We find that distortions in the CARS signals arise not only from attenuation of the focal field but also from scattering-induced changes in the spatial phase that modifies the angular distribution of the CARS emission. Our simulations further show that CARS signal attenuation can be minimized by using a high numerical aperture condenser. Moreover, unlike the CARS intensity image, CARS images formed by taking the ratio of CARS signals obtained using x- and y-polarized input fields is relatively insensitive to the effects of spherical scatterers. Our computational framework provide a mechanistic approach to characterizing scattering-induced distortions in coherent imaging of turbid media and may inspire bottom-up approaches for adaptive optical methods for image correction.

Reflection-mode multiple-illumination photoacoustic sensing to estimate optical properties.

OBJECTIVES: We analyze a reflection-mode multiple-illumination photoacoustic method which allows us to estimate optical scattering properties of turbid media based on fitting light-transport models and explore its limits in optical property estimation and depth-dependent fluence compensation. BACKGROUND: Recent simulation results show significant promise for a technique called multiple-illumination photoacoustic tomography (MI-PAT) to quantitatively reconstruct both absorption and scattering heterogeneities in turbid medium. Prior to experiments, it is essential to develop and analyze a measurement technique and probe capabilities of quantitative measurements that focus on sensing rather than imaging. METHODS: This technique involved translation of a 532 nm pulsed-laser light spot while focusing an ultrasound receiver on a sub-surface optical absorber immersed in a scattering medium at 3, 4 and 5 mm below the surface. Measured photoacoustic amplitudes for media with different reduced scattering coefficients are fitted with a light propagation model to estimate optical properties. RESULTS: When the absorber was located at 5 mm below the membrane in media with a reduced scattering coefficient of 4.4 and 5.5 cm(-1), the true values were predicted with an error of 5.7% and 12.7%, respectively. We observe accuracy and the ability of estimating optical scattering properties decreased with the increased reduced scattering coefficient. Nevertheless, the estimated parameters were sufficient for demonstrating depth-dependent fluence compensation for improved quantitation in photoacoustic imaging.

Rapid computation of the amplitude and phase of tightly focused optical fields distorted by scattering particles.

We develop an efficient method for accurately calculating the electric field of tightly focused laser beams in the presence of specific configurations of microscopic scatterers. This Huygens-Fresnel wave-based electric field superposition (HF-WEFS) method computes the amplitude and phase of the scattered electric field in excellent agreement with finite difference time-domain (FDTD) solutions of Maxwell's equations. Our HF-WEFS implementation is 2-4 orders of magnitude faster than the FDTD method and enables systematic investigations of the effects of scatterer size and configuration on the focal field. We demonstrate the power of the new HF-WEFS approach by mapping several metrics of focal field distortion as a function of scatterer position. This analysis shows that the maximum focal field distortion occurs for single scatterers placed below the focal plane with an offset from the optical axis. The HF-WEFS method represents an important first step toward the development of a computational model of laser-scanning microscopy of thick cellular/tissue specimens.

Photoacoustic and high-frequency power Doppler ultrasound biomicroscopy: a comparative study.

Both photoacoustic imaging and power Doppler ultrasound are capable of producing images of the vasculature of living subjects, however, the contrast mechanisms of the two modalities are very different. We present a quantitative and objective comparison of the two methods using phantom data, highlighting relative merits and shortcomings. An imaging system for combined photoacoustic and high-frequency power Doppler ultrasound microscopy is presented. This system uses a swept-scan 25-MHz ultrasound transducer with confocal dark-field laser illumination optics. A pulse-sequencer enables ultrasonic and laser pulses to be interlaced so that photoacoustic and power Doppler ultrasound images can be coregistered. Experiments are performed on flow phantoms with various combinations of vessel size, flow velocity, and optical wavelength. For the task of blood volume detection, power Doppler is seen to be advantageous for large vessels and high flow speeds. For small vessels with low flow speeds, photoacoustic imaging is seen to be more effective than power Doppler at the detection of blood as quantified by receiver operating characteristic analysis. A combination of the two modes could provide improved estimates of fractional blood volume in comparison with either mode used alone.

Optical resolution photoacoustic microscopy using novel high-repetition-rate passively Q-switched microchip and fiber lasers.

Optical-resolution photoacoustic microscopy (OR-PAM) is a novel imaging technology for visualizing optically absorbing superficial structures in vivo with lateral spatial resolution determined by optical focusing rather than acoustic detection. Since scanning of the illumination spot is required, OR-PAM imaging speed is limited by both scanning speed and laser pulse repetition rate. Unfortunately, lasers with high repetition rates and suitable pulse durations and energies are not widely available and can be cost-prohibitive and bulky. We are developing compact, passively Q-switched fiber and microchip laser sources for this application. The properties of these lasers are discussed, and pulse repetition rates up to 100 kHz are demonstrated. OR-PAM imaging was conducted using a previously developed photoacoustic probe, which enabled flexible scanning of the focused output of the lasers. Phantom studies demonstrate the ability to image with lateral spatial resolution of 7±2 µm with the microchip laser system and 15±5 µm with the fiber laser system. We believe that the high pulse repetition rates and the potentially compact and fiber-coupled nature of these lasers will prove important for clinical imaging applications where real-time imaging performance is essential.

Combined photoacoustic and oblique-incidence diffuse reflectance system for quantitative photoacoustic imaging in turbid media.

While photoacoustic imaging is capable of producing high-resolution biomedical images with optical absorption contrast, optical property quantification has thus far remained challenging. One reason for this is that laser-induced photoacoustic signal amplitudes are proportional to not only the local optical absorption coefficient, but also the local laser fluence in the tissue. Unfortunately, local laser fluence is often unknown, but could possibly be estimated if local bulk tissue optical properties were known. One method to estimate tissue optical properties is a technique known as oblique incidence diffuse reflectance (OIR). We report on an integrated OIR and photoacoustic imaging system and demonstrate, using phantom experiments, improved ability to quantitatively estimate optical properties of a turbid medium.

Combined photoacoustic and ultrasound biomicroscopy.

We report on the development of an imaging system capable of combined ultrasound and photoacoustic imaging based on a fast-scanning single-element 25-MHz ultrasound transducer and a unique light-delivery system. The system is capable of 20 ultrasound frames per second and slower photoacoustic frame rates limited by laser pulse-repetition rates. Laser and ultrasound pulses are interlaced for co-registration of photoacoustic and ultrasound images. In vivo imaging of a human finger permits ultrasonic visualization of vessel structures and speckle changes indicative of blood flow, while overlaid photoacoustic images highlight some small vessels that are not clear from the ultrasound scan. Photoacoustic images provide optical absorption contrast co-registered in the structural and blood-flow context of ultrasound with high-spatial resolution and may prove important for clinical diagnostics and basic science of the microvasculature.

Photoacoustic technique for assessing optical scattering properties of turbid media.

Measurement of tissue optical properties impacts both optical diagnostic and theraputic applications. Although existing photoacoustic imaging techniques provide optical absorption contrast, we present a photoacoustic technique that demonstrates sensitivity to the optical scattering coefficient of a turbid medium. By incrementing the distance between a surface illumination spot and a subsurface absorber and measuring the photoacoustic amplitude of the absorber, we can effectively estimate the Green's function of light transport in a turbid medium. Our results for different concentrations of Intralipid indicate that this technique has the ability to distinguish small changes of the reduced scattering coefficient. It has the potential to be used for in vivo studies to obtain reduced scattering coefficients of biological tissues. These findings will potentially improve the calculation of subcutaneous fluence in photoacoustic-based techniques and laser dosimetry studies in live tissues.

Effects of inhomogeneous myofibril morphology on optical diffraction in single muscle fibers.

Laser diffraction is commonly used in physiological research that explores single muscle fibers. Although variations in sarcomere morphological properties have often been observed, their effects on laser diffraction have not been studied in detail. In this study, we applied three-dimensional coupled wave theory to a physical sarcomere model to investigate the effects of inhomogeneous morphological profiles in muscle fibers. The simulation results were compared with several those of published experimental studies. Our results indicate that by incorporating various myofibril inhomogeneities such as skew and domain effect in the theoretical model, a variety of observations in single fiber diffraction under different experimental conditions can be reproduced in the simulation.

Polarization-sensitive reflectance imaging in skeletal muscle.

We acquired polarization-sensitive reflectance images in freshly excised skeletal muscle samples. The obtained raw images varied depending on the incident and detection polarization states. The Stokes vectors were measured for incident light of four different polarization states, and the whole Mueller matrix images were also calculated. We found that the images obtained in skeletal muscles exhibited different features from those obtained in a typical polystyrene sphere solution. The back-reflected light in muscle maintained a higher degree of polarization along the axis perpendicular to muscle fiber orientation. Our analysis indicates that the unique muscle sarcomere structure plays an important role in modulating the propagation of polarized light in whole muscle.

Imaging 2D optical diffuse reflectance in skeletal muscle.

We discovered a unique pattern of optical reflectance from fresh prerigor skeletal muscles, which can not be described using existing theories. A numerical fitting function was developed to quantify the equiintensity contours of acquired reflectance images. Using this model, we studied the changes of reflectance profile during stretching and rigor process. We found that the prominent anisotropic features diminished after rigor completion. These results suggested that muscle sarcomere structures played important roles in modulating light propagation in whole muscle. When incorporating the sarcomere diffraction in a Monte Carlo model, we showed that the resulting reflectance profiles quantitatively resembled the experimental observation.