Effects of isoflurane anesthesia on physiological parameters in murine subcutaneous tumor allografts measured via diffuse reflectance spectroscopy.

Diffuse reflectance spectroscopy (DRS) has been used in murine studies to quantify tumor perfusion and therapeutic response. These studies frequently use inhaled isoflurane anesthesia, which depresses the respiration rate and results in the desaturation of arterial oxygen saturation, potentially affecting tissue physiological parameters. However, there have been no controlled studies quantifying the effect of isoflurane anesthesia on DRS-derived physiological parameters of murine tissue. The goal of this study was to perform DRS on Balb/c mouse (n = 10) tissue under various anesthesia conditions to quantify effects on tissue physiological parameters, including total hemoglobin concentration, tissue oxygen saturation, oxyhemoglobin and reduced scattering coefficient. Two independent variables were manipulated including metabolic gas type (pure oxygen vs. medical air) and isoflurane concentration (1.5 to 4.0%). The 1.5% isoflurane and 1 L/min oxygen condition most closely mimicked a no-anesthesia condition with oxyhemoglobin concentration within 89% ± 19% of control. The time-dependent effects of isoflurane anesthesia were tested, revealing that anesthetic induction with 4.0% isoflurane can affect DRS-derived physiological parameters up to 20 minutes post-induction. Finally, spectroscopy with and without isoflurane anesthesia was compared for colon tumor Balb/c-CT26 allografts (n = 5) as a representative model of subcutaneous murine tumor allografts. Overall, isoflurane anesthesia yielded experimentally-induced depressed oxyhemoglobin, and this depression was both concentration and time dependent. Investigators should understand the dynamic effects of isoflurane on tissue physiological parameters measured by DRS. These results may guide investigators in eliminating, limiting, or managing anesthesia-induced physiological changes in DRS studies in mouse models.

Effects of digital phase-conjugate light intensity on time-reversal imaging through animal tissue.

For transillumination imaging of animal tissues, we have attempted to suppress the scattering effect in a turbid medium using the time-reversal principle of phase-conjugate light. We constructed a digital phase-conjugate system to enable intensity modulation and phase modulation. Using this system, we clarified the effectiveness of the intensity information for restoration of the original light distribution through a turbid medium. By varying the scattering coefficient of the medium, we clarified the limit of time-reversal ability with intensity information of the phase-conjugate light. Experiment results demonstrated the applicability of the proposed technique to animal tissue.

Correction of an adding-doubling inversion algorithm for the measurement of the optical parameters of turbid media.

We present broadband measurements of the optical properties of tissue-mimicking solid phantoms using a single integrating sphere to measure the hemispherical reflectance and transmittance under a direct illumination at the normal incident angle. These measurements are traceable to reflectance and transmittance scales. An inversion routine using the output of the adding-doubling algorithm restricted to the reflectance and transmittance under a direct illumination was developed to produce the optical parameters of the sample along with an uncertainty budget at each wavelength. The results for two types of phantoms are compared to measurements by time-resolved approaches. The results between our method and these independent measurements agree within the estimated measurement uncertainties.

Light transport in homogeneous tissue with m-dependent anisotropic scattering II: Fourier transform solution and consistent relations.

This paper is the second of two focusing on the analytical solutions for light transport in infinite homogeneous tissue with an azimuth-dependent (m-dependent) anisotropic scattering kernel by two approaches, Case's singular eigenfuncions (CSEs) expansion and Fourier transform, and proving the consistence of the two solutions theoretically. In this paper, the analytical solution for the m-dependent truncated scattering kernel was derived via the Fourier transform and inversion, and expanded with the m-dependent generalized singular eigenfuncions (GSEs). Two kinds of GSEs that are defined by Ganapol in the case m = 0 are extended to arbitrary azimuthal orders and proven to be consistent with CSEs both in expression forms and in intrinsic behaviors. By applying the Fourier transform inversion on the solution for the three-term recurrences, the Green's function of radiance distributions is obtained successfully, and it conforms perfectly to the CSEs solution in the limit, which has already been discussed in our first accompanying paper. Meanwhile, as a byproduct, a series of identities about the m-dependent Chandrasekhar orthogonal polynomials were presented and will be greatly helpful for further studies.

Light transport in homogeneous tissue with m-dependent anisotropic scattering I: Case's singular eigenfunctions solution and Chandrasekhar polynomials.

This paper is the first of two deriving the analytical solutions for light transport in infinite homogeneous tissue with an azimuth-dependent (m-dependent) anisotropic scattering kernel by two approaches, Case's singular eigenfuncions expansion and Fourier transform, as well as proving the consistence of the two solutions. In this paper, Case's method was applied and extended to the general m-dependent anisotropic scattering case. The explicit Green's function of radiance distributions, which was regarded as the comparative standard for the equivalent solution via Fourier transform and inversion in our second accompanying paper, was expanded into a complete set of the discrete and continuous eigenfunctions. Considering that the two kinds of m-dependent Chandrasekhar orthogonal polynomials that play vital roles in these analytical solutions are very sensitive to the typical optical parameters of biological tissue as well as the degrees or orders, four numerical evaluation methods were benchmarked to find the stable, reliable and feasible numerical evaluation methods in high degrees and high orders.

In vivo study of optical speckle decorrelation time across depths in the mouse brain.

The strong optical scattering of biological tissue confounds our ability to focus light deeply into the brain beyond depths of a few hundred microns. This challenge can be potentially overcome by exploiting wavefront shaping techniques which allow light to be focused through or inside scattering media. However, these techniques require the scattering medium to be static, as changes in the arrangement of the scatterers between the wavefront recording and playback steps reduce the fidelity of the focus that is formed. Furthermore, as the thickness of the scattering medium increases, the influence of the dynamic nature becomes more severe due to the growing number of scattering events experienced by each photon. In this paper, by examining the scattering dynamics in the mouse brain in vivo via multispeckle diffusing wave spectroscopy (MSDWS) using a custom fiber probe that simulates a point-like source within the brain, we investigate the relationship between this decorrelation time and the depth of the point-like light source inside the living mouse brain at depths up to 3.2 mm.

Virtually increased acceptance angle for efficient estimation of spatially resolved reflectance in the subdiffusive regime: a Monte Carlo study.

Light propagation in biological tissues is frequently modeled by the Monte Carlo (MC) method, which requires processing of many photon packets to obtain adequate quality of the observed backscattered signal. The computation times further increase for detection schemes with small acceptance angles and hence small fraction of the collected backscattered photon packets. In this paper, we investigate the use of a virtually increased acceptance angle for efficient MC simulation of spatially resolved reflectance and estimation of optical properties by an inverse model. We devise a robust criterion for approximation of the maximum virtual acceptance angle and evaluate the proposed methodology for a wide range of tissue-like optical properties and various source configurations.

Erratum: In vivo study of optical speckle decorrelation time across depths in the mouse brain: erratum.

[This corrects the article on p. 4855 in vol. 8.].

In-silico and in-vitro investigation of a photonic monitor for intestinal perfusion and oxygenation.

The quantification of visceral organ oxygenation after trauma-related systemic hypovolemia and shock is critical to enable effective resuscitation. In this work, a photoplethysmography-based (PPG) sensor was specifically designed for probing the perfusion and oxygenation condition of intestinal tissue with the ultimate goal to monitor patients post trauma to guide resuscitation. Through Monte Carlo modeling, suitable optofluidic phantoms were determined, the wavelength and separation distance for the sensor was optimized, and sensor performance for the quantification of tissue perfusion and oxygenation was tested on the in-vitro phantom. In particular, the Monte Carlo simulated both a standard block three-layer model and a more realistic model including villi. Measurements were collected on the designed three layer optofluidic phantom and the results taken with the small form factor PPG device showed a marked improvement when using shorter visible wavelengths over the more conventional longer visible wavelengths. Overall, in this work a Monte Carlo model was developed, an optofluidic phantom was built, and a small form factor PPG sensor was developed and characterized using the phantom for perfusion and oxygenation over the visible wavelength range. The results show promise that this small form factor PPG sensor could be used as a future guide to shock-related resuscitation.

Line scanning, stage scanning confocal microscope (LSSSCM).

For rapid pathological assessment of large surgical tissue excisions with cellular resolution, we present a line scanning, stage scanning confocal microscope (LSSSCM). LSSSCM uses no scanning mirrors. Laser light is focused with a single cylindrical lens to a line of diffraction-limited width directly into the (Z) sample focal plane, which is parallel to and near the flattened specimen surface. Semi-confocal optical sections are derived from the linear array distribution (Y) and a single mechanical drive that moves the sample parallel to the focal plane and perpendicular to the focused line (X). LSSSCM demonstrates cellular resolution in the conditions of high nuclear density within micronodular basal cell carcinoma.

Lookup table-based sampling of the phase function for Monte Carlo simulations of light propagation in turbid media.

Analytical expressions for sampling the scattering angle from a phase function in Monte Carlo simulations of light propagation are available only for a limited number of phase functions. Consequently, numerical sampling methods based on tabulated values are often required instead. By using Monte Carlo simulated reflectance, we compare two existing and propose an improved numerical sampling method and show that both the number of the tabulated values and the numerical sampling method significantly influence the accuracy of the simulated reflectance. The provided results and guidelines should serve as a good starting point for conducting computationally efficient Monte Carlo simulations with numerical phase function sampling.

Momentum transfer Monte Carlo for the simulation of laser speckle imaging and its application in the skin.

Due to its simplicity and low cost, laser speckle imaging (LSI) has achieved widespread use in biomedical applications. However, interpretation of the blood-flow maps remains ambiguous, as LSI enables only limited visualization of vasculature below scattering layers such as the epidermis and skull. Here, we describe a computational model that enables flexible in-silico study of the impact of these factors on LSI measurements. The model uses Monte Carlo methods to simulate light and momentum transport in a heterogeneous tissue geometry. The virtual detectors of the model track several important characteristics of light. This model enables study of LSI aspects that may be difficult or unwieldy to address in an experimental setting, and enables detailed study of the fundamental origins of speckle contrast modulation in tissue-specific geometries. We applied the model to an in-depth exploration of the spectral dependence of speckle contrast signal in the skin, the effects of epidermal melanin content on LSI, and the depth-dependent origins of our signal. We found that LSI of transmitted light allows for a more homogeneous integration of the signal from the entire bulk of the tissue, whereas epi-illumination measurements of contrast are limited to a fraction of the light penetration depth. We quantified the spectral depth dependence of our contrast signal in the skin, and did not observe a statistically significant effect of epidermal melanin on speckle contrast. Finally, we corroborated these simulated results with experimental LSI measurements of flow beneath a thin absorbing layer. The results of this study suggest the use of LSI in the clinic to monitor perfusion in patients with different skin types, or inhomogeneous epidermal melanin distributions.

Characterization of fiber-optic light delivery and light-induced temperature changes in a rodent brain for precise optogenetic neuromodulation.

Understanding light intensity and temperature increase is of considerable importance in designing or performing in vivo optogenetic experiments. Our study describes the optimal light power at target depth in the rodent brain that would maximize activation of light-gated ion channels while minimizing temperature increase. Monte Carlo (MC) simulations of light delivery were used to provide a guideline for suitable light power at a target depth. In addition, MC simulations with the Pennes bio-heat model using data obtained from measurements with a temperature-measuring cannula having 12.3 mV/°C of thermoelectric sensitivity enabled us to predict tissue heating of 0.116 °C/mW on average at target depth of 563 µm and specifically, a maximum mean plateau temperature increase of 0.25 °C/mW at 100 µm depth for 473 nm light. Our study will help to improve the design and performance of optogenetic experiments while avoiding potential over- and under-illumination.

Subtraction-based approach for enhancing the depth sensitivity of time-resolved NIRS.

The aim of this study was to evaluate enhancing of the depth sensitivity of time-resolved near-infrared spectroscopy with a subtraction-based approach. Due to the complexity of light propagation in a heterogeneous media, and to prove the validity of the proposed method in a heterogeneous turbid media we conducted a broad analysis taking into account a number of parameters related to the method as well as various parameters of this media. The results of these experiments confirm that the depth sensitivity of the subtraction-based approach is better than classical approaches using continuous-wave or time-resolved methods. Furthermore, the results showed that the subtraction-based approach has a unique, selective sensitivity to a layer at a specific depth. In vivo application of the proposed method resulted in a greater magnitude of the hemodynamic changes during functional activation than with the standard approach.

Artificial neural networks for retrieving absorption and reduced scattering spectra from frequency-domain diffuse reflectance spectroscopy at short source-detector separation.

Diffuse reflectance spectroscopy (DRS) based on the frequency-domain (FD) technique has been employed to investigate the optical properties of deep tissues such as breast and brain using source to detector separation up to 40 mm. Due to the modeling and system limitations, efficient and precise determination of turbid sample optical properties from the FD diffuse reflectance acquired at a source-detector separation (SDS) of around 1 mm has not been demonstrated. In this study, we revealed that at SDS of 1 mm, acquiring FD diffuse reflectance at multiple frequencies is necessary for alleviating the influence of inevitable measurement uncertainty on the optical property recovery accuracy. Furthermore, we developed artificial neural networks (ANNs) trained by Monte Carlo simulation generated databases that were capable of efficiently determining FD reflectance at multiple frequencies. The ANNs could work in conjunction with a least-square optimization algorithm to rapidly (within 1 second), accurately (within 10%) quantify the sample optical properties from FD reflectance measured at SDS of 1 mm. In addition, we demonstrated that incorporating the steady-state apparatus into the FD DRS system with 1 mm SDS would enable obtaining broadband absorption and reduced scattering spectra of turbid samples in the wavelength range from 650 to 1000 nm.

Multiple pinhole collimator based X-ray luminescence computed tomography.

X-ray luminescence computed tomography (XLCT) is an emerging hybrid imaging modality, which is able to improve the spatial resolution of optical imaging to hundreds of micrometers for deep targets by using superfine X-ray pencil beams. However, due to the low X-ray photon utilization efficiency in a single pinhole collimator based XLCT, it takes a long time to acquire measurement data. Herein, we propose a multiple pinhole collimator based XLCT, in which multiple X-ray beams are generated to scan a sample at multiple positions simultaneously. Compared with the single pinhole based XLCT, the multiple X-ray beam scanning method requires much less measurement time. Numerical simulations and phantom experiments have been performed to demonstrate the feasibility of the multiple X-ray beam scanning method. In one numerical simulation, we used four X-ray beams to scan a cylindrical object with 6 deeply embedded targets. With measurements from 6 angular projections, all 6 targets have been reconstructed successfully. In the phantom experiment, we generated two X-ray pencil beams with a collimator manufactured in-house. Two capillary targets with 0.6 mm edge-to-edge distance embedded in a cylindrical phantom have been reconstructed successfully. With the two beam scanning, we reduced the data acquisition time by 50%. From the reconstructed XLCT images, we found that the Dice similarity of targets is 85.11% and the distance error between two targets is less than 3%. We have measured the radiation dose during XLCT scan and found that the radiation dose, 1.475 mSv, is in the range of a typical CT scan. We have measured the changes of the collimated X-ray beam size and intensity at different distances from the collimator. We have also studied the effects of beam size and intensity in the reconstruction of XLCT.

Nanoparticle-free tissue-mimicking phantoms with intrinsic scattering.

We present an alternative to the conventional approach, phantoms without scattering nanoparticles, where scattering is achieved by the material itself: spherical cavities trapped in a silicone matrix. We describe the properties and fabrication of novel optical phantoms based on a silicone elastomer polydimethylsiloxane (PDMS) and glycerol mixture. Optical properties (absorption coefficient µa , reduced scattering coefficient µs' , and anisotropy factor g) of the fabricated phantoms were retrieved from spectrophotometric measurements (in the 400-1100 nm wavelength range) using the inverse adding-doubling method. The internal structure of the phantoms was studied under a scanning electron microscope, and the chemical composition was assessed by Raman spectroscopy. Composition of the phantom material is reported along with the full characterization of the produced phantoms and ways to control their parameters.

Light distribution modulated diffuse reflectance spectroscopy.

Typically, a diffuse reflectance spectroscopy (DRS) system employing a continuous wave light source would need to acquire diffuse reflectances measured at multiple source-detector separations for determining the absorption and reduced scattering coefficients of turbid samples. This results in a multi-fiber probe structure and an indefinite probing depth. Here we present a novel DRS method that can utilize a few diffuse reflectances measured at one source-detector separation for recovering the optical properties of samples. The core of innovation is a liquid crystal (LC) cell whose scattering property can be modulated by the bias voltage. By placing the LC cell between the light source and the sample, the spatial distribution of light in the sample can be varied as the scattering property of the LC cell modulated by the bias voltage, and this would induce intensity variation of the collected diffuse reflectance. From a series of Monte Carlo simulations and phantom measurements, we found that this new light distribution modulated DRS (LDM DRS) system was capable of accurately recover the absorption and scattering coefficients of turbid samples and its probing depth only varied by less than 3% over the full bias voltage variation range. Our results suggest that this LDM DRS platform could be developed to various low-cost, efficient, and compact systems for in-vivo superficial tissue investigation.

In vivo assessment of optical properties of basal cell carcinoma and differentiation of BCC subtypes by high-definition optical coherence tomography.

High-definition optical coherence tomography (HD-OCT) features of basal cell carcinoma (BCC) have recently been defined. We assessed in vivo optical properties (IV-OP) of BCC, by HD-OCT. Moreover their critical values for BCC subtype differentiation were determined. The technique of semi-log plot whereby an exponential function becomes linear has been implemented on HD-OCT signals. The relative attenuation factor (µraf ) at different skin layers could be assessed.. IV-OP of superficial BCC with high diagnostic accuracy (DA) and high negative predictive values (NPV) were (i) decreased µraf in lower part of epidermis and (ii) increased epidermal thickness (E-T). IV-OP of nodular BCC with good to high DA and NPV were (i) less negative µraf in papillary dermis compared to normal adjacent skin and (ii) significantly decreased E-T and papillary dermal thickness (PD-T). In infiltrative BCC (i) high µraf in reticular dermis compared to normal adjacent skin and (ii) presence of peaks and falls in reticular dermis had good DA and high NPV. HD-OCT seems to enable the combination of in vivo morphological analysis of cellular and 3-D micro-architectural structures with IV-OP analysis of BCC. This permits BCC sub-differentiation with higher accuracy than in vivo HD-OCT analysis of morphology alone.

Toward reliable retrieval of functional information of papillary dermis using spatially resolved diffuse reflectance spectroscopy.

Spatially resolved diffuse reflectance spectroscopy (SRDRS) has been employed to quantify tissue optical properties and its interrogation volume is majorly controlled by the source-to-detector separations (SDSs). To noninvasively quantify properties of dermis, a SRDRS setup that includes SDS shorter than 1 mm is required. It will be demonstrated in this study that Monte Carlo simulations employing the Henyey-Greenstein phase function cannot always precisely predict experimentally measured diffuse reflectance at such short SDSs, and we speculated this could be caused by the non-negligible backward light scattering at short SDSs that cannot be properly modeled by the Henyey-Greenstein phase function. To accurately recover the optical properties and functional information of dermis using SRDRS, we proposed the use of the modified two-layer (MTL) geometry. Monte Carlo simulations and phantom experiment results revealed that the MTL probing geometry was capable of faithfully recovering the optical properties of upper dermis. The capability of the MTL geometry in probing the upper dermis properties was further verified through a swine study, and it was found that the measurement results were reasonably linked to histological findings. Finally, the MTL probe was utilized to study psoriatic lesions. Our results showed that the MTL probe was sensitive to the physiological condition of tissue volumes within the papillary dermis and could be used in studying the physiology of psoriasis.