Continuous monitoring of interstitial tissue oxygen using subcutaneous oxygen microsensors: In vivo characterization in healthy volunteers.

Measurements of regional tissue oxygen serve as a proxy to monitor local perfusion and have the potential to guide therapeutic decisions in multiple clinical disciplines. Transcutaneous oximetry (tcpO2) is a commercially available noninvasive technique that uses an electrode to warm underlying skin tissue and measure the resulting oxygen tension at the skin surface. A novel approach is to directly measure interstitial tissue oxygen using subcutaneous oxygen microsensors composed of a biocompatible hydrogel carrier platform with embedded oxygen sensing molecules. After initial injection of the hydrogel into subcutaneous tissue, noninvasive optical measurements of phosphorescence-based emissions at the skin surface are used to sense oxygen in the subcutaneous interstitial space. The object of the present study was to characterize the in vivo performance of subcutaneous microsensors and compare with transcutaneous oximetry (tcpO2). Vascular occlusion tests were performed on the arms of 7 healthy volunteers, with repeated tests occurring 1 to 10 weeks after sensor injection, yielding 95 total tests for analysis. Comparative analysis characterized the response of both devices to decreases in tissue oxygen during occlusion and to increases in tissue oxygen following release of the occlusion. Results indicated: (I) time traces returned by microsensors and tcpO2 were highly correlated, with the median (interquartile range) correlation coefficient of r = 0.93 (0.10); (II) both microsensors and tcpO2 sensed a statistically significant decrease in normalized oxygen during occlusion (p < 0.001 for each device); (III) microsensors detected faster rates change (p < 0.001) and detected overshoot during recovery more frequently (38% vs. 4% of tests); (IV) inter-measurement analysis showed no correlation of baseline values between microsensors and tcpO2 (r = 0.03), but comparison of integrated oxygen dynamics showed similar variation in the normalized response to occlusion between devices (p = 0.06), (V) intra-measurement analysis revealed that microsensors detect greater physiological fluctuations than tcpO2 (p < 0.001) and may provide enhanced sensitivity to processes such as vasomotion. Additionally, the functional response of microsensors was not significantly different across time groupings (per month) post-injection (p = 0.61). Although the compared devices have differences in the mechanisms used to sense oxygen, these findings demonstrate that subcutaneous oxygen microsensors measure changes in interstitial tissue oxygen in human subjects in vivo.

Separation of Solid Stress From Interstitial Fluid Pressure in Pancreas Cancer Correlates With Collagen Area Fraction.

Elevated total tissue pressure (TTP) in pancreatic adenocarcinoma is often associated with stress applied by cellular proliferation and hydrated hyaluronic acid osmotic swelling; however, the causal roles of collagen in total tissue pressure have yet to be clearly measured. This study illustrates one direct correlation between total tissue pressure and increased deposition of collagen within the tissue matrix. This observation comes from a new modification to a conventional piezoelectric pressure catheter, used to independently separate and quantify total tissue pressure, solid stress (SS), and interstitial fluid pressure (IFP) within the same tumor location, thereby clarifying the relationship between these parameters. Additionally, total tissue pressure shows a direct correlation with verteporfin uptake, demonstrating the impediment of systemically delivered molecules with increased tissue hypertension.

Sub-diffuse optical biomarkers characterize localized microstructure and function of cortex and malignant tumor.

This study uses a sub-diffusive light transport model to analyze fiber-optic measurements of reflectance spectra to recover endogenous tissue biomarkers and to correct raw fluorescence emissions for distortions from background optical properties. Measurements in tissue-simulating phantoms validated accurate recovery of the reduced scattering coefficient [(0.3-3.4 mm-1), error 10%], blood volume fraction [(1-3 vol%), error 7%], and a dimensionless metric of anisotropic scattering, γ, that is sensitive to submillimeter tissue ultrastructure [(1.29-2.06), error 11%]. In vivo sub-diffusive optical data acquired during clinical neurosurgeries characterize differences in microstructure (γ), perfusion (blood volume), and metabolism (PpIX fluorescence) between normal cortex and malignant tumor.

Hyperspectral dark-field microscopy of human breast lumpectomy samples for tumor margin detection in breast-conserving surgery.

Significance: Hyperspectral dark-field microscopy (HSDFM) and data cube analysis algorithms demonstrate successful detection and classification of various tissue types, including carcinoma regions in human post-lumpectomy breast tissues excised during breast-conserving surgeries. Aim: We expand the application of HSDFM to the classification of tissue types and tumor subtypes in pre-histopathology human breast lumpectomy samples. Approach: Breast tissues excised during breast-conserving surgeries were imaged by the HSDFM and analyzed. The performance of the HSDFM is evaluated by comparing the backscattering intensity spectra of polystyrene microbead solutions with the Monte Carlo simulation of the experimental data. For classification algorithms, two analysis approaches, a supervised technique based on the spectral angle mapper (SAM) algorithm and an unsupervised technique based on the K-means algorithm are applied to classify various tissue types including carcinoma subtypes. In the supervised technique, the SAM algorithm with manually extracted endmembers guided by H&E annotations is used as reference spectra, allowing for segmentation maps with classified tissue types including carcinoma subtypes. Results: The manually extracted endmembers of known tissue types and their corresponding threshold spectral correlation angles for classification make a good reference library that validates endmembers computed by the unsupervised K-means algorithm. The unsupervised K-means algorithm, with no a priori information, produces abundance maps with dominant endmembers of various tissue types, including carcinoma subtypes of invasive ductal carcinoma and invasive mucinous carcinoma. The two carcinomas' unique endmembers produced by the two methods agree with each other within <2% residual error margin. Conclusions: Our report demonstrates a robust procedure for the validation of an unsupervised algorithm with the essential set of parameters based on the ground truth, histopathological information. We have demonstrated that a trained library of the histopathology-guided endmembers and associated threshold spectral correlation angles computed against well-defined reference data cubes serve such parameters. Two classification algorithms, supervised and unsupervised algorithms, are employed to identify regions with carcinoma subtypes of invasive ductal carcinoma and invasive mucinous carcinoma present in the tissues. The two carcinomas' unique endmembers used by the two methods agree to <2% residual error margin. This library of high quality and collected under an environment with no ambient background may be instrumental to develop or validate more advanced unsupervised data cube analysis algorithms, such as effective neural networks for efficient subtype classification.

Pulsed-light imaging for fluorescence guided surgery under normal room lighting.

Fluorescence guided surgery (FGS) is an emerging technology that has demonstrated improved surgical outcomes. However, dim lighting conditions required by current FGS systems are disruptive to standard surgical workflow. We present a novel FGS system capable of imaging fluorescence under normal room light by using pulsed excitation and gated acquisition. Images from tissue-simulating phantoms confirm visual detection down to 0.25 µM of protoporphyrin IX under 125 µW/cm2 of ambient light, more than an order of magnitude lower than that measured with the Zeiss Pentero in the dark. Resection of orthotopic brain tumors in mice also suggests that the pulsed-light system provides superior sensitivity in vivo.

Extraction of intrinsic fluorescence from single fiber fluorescence measurements on a turbid medium.

This study utilizes Monte Carlo simulations of single fiber fluorescence to develop an empirical model that corrects for the influence of scattering and absorption on fluorescence intensity (F(SF)). The model expresses F(SF) in terms of the reduced scattering coefficient (µs') and absorption coefficient (µ(a)), each determined independently at excitation and emission wavelengths (λ(x) and λ(m)), and the fiber diameter (d(f)). This model returns accurate descriptions (mean residual <6%) of F(SF) across a biologically relevant range of µs' and µ(a) values and is insensitive to the form of the scattering phase function.

Method to quantitate absorption coefficients from single fiber reflectance spectra without knowledge of the scattering properties.

This study presents a methodology to accurately extract the absorption coefficient from single fiber reflectance spectra measured in turbid media without a priori knowledge of either the reduced scattering coefficient or the phase function. This novel approach accounts for the interrelated effects these properties have on the photon path length, yielding estimates of an absorption coefficient on average within <7.5% of true values over a wide range of biologically relevant optical properties.

Method to quantitatively estimate wavelength-dependent scattering properties from multidiameter single fiber reflectance spectra measured in a turbid medium.

This study utilizes experimentally validated Monte Carlo simulations to identify a mathematical formulation of the reflectance intensity collected by a single fiber probe expressed in terms of the reduced scattering coefficient (µs'), fiber diameter d(fiber), and a property of the first two moments of the scattering phase function (γ). This model is then utilized to accurately obtain wavelength-dependent estimates of µs'(λ) and γ(λ) from multiple single fiber spectral measurements of a turbid medium obtained with different diameters. This method returns accurate descriptions (mean residual <3%) of both µs' and γ across the biologically relevant range.

Special Section Guest Editorial: Wearable, Implantable, Mobile, and Remote Biomedical Optics and Photonics.

Guest editors Jessica Ramella-Roman, Amir H. Gandjbakhche, Stephen C. Kanick, Babak Shadgan, and Bruce J. Tromberg introduce and summarize the articles included in the 6-part JBO Special Section on Wearable, Implantable, Mobile, and Remote Biomedical Optics Photonics.

Empirical model description of photon path length for differential path length spectroscopy: combined effect of scattering and absorption.

Differential path length spectroscopy (DPS) is a method of reflectance spectroscopy that utilizes a specialized fiber geometry to make the photon path length (tau) insensitive to variations in tissue optical properties over a wide range of absorption (mu(a)) and total scattering (mu(s)) coefficients, which are common within the ultraviolet/visible (UV/VIS) wavelength region. This study extends the description of tau to larger mu(a) and smaller mu(s) values, optical properties that are representative of the near-infrared region (NIR), a region where the DPS path length may be dependent on both coefficients. This study presents a novel empirical relationship between tau and the combined effect of both mu(a) (range: 0.1-12 mm(-1)) and mu(s) (range: 1.5-42 mm(-1)), anisotropy of 0.8, and is applicable to DPS probes containing a wide range of fiber diameters (range: 100-1000 microm). The results indicate that the simple empirical formula, including only one fitted parameter, is capable of accurately predicting tau over a wide range (r=0.985; range: 80-940 microm) and predictions are not biased versus mu(a) or mu(s). This novel relationship is applicable to analysis of DPS measurements of tissue in both the UV/VIS and NIR wavelength regions and may provide information about the wavelength-specific tissue volume optically sampled during measurement.

Wide-field color imaging of scatter-based tissue contrast using both high spatial frequency illumination and cross-polarization gating.

This study characterizes the scatter-specific tissue contrast that can be obtained by high spatial frequency (HSF) domain imaging and cross-polarization (CP) imaging, using a standard color imaging system, and how combining them may be beneficial. Both HSF and CP approaches are known to modulate the sensitivity of epi-illumination reflectance images between diffuse multiply scattered and superficially backscattered photons, providing enhanced contrast from microstructure and composition than what is achieved by standard wide-field imaging. Measurements in tissue-simulating optical phantoms show that CP imaging returns localized assessments of both scattering and absorption effects, while HSF has uniquely specific sensitivity to scatter-only contrast, with a strong suppression of visible contrast from blood. The combination of CP and HSF imaging provided an expanded sensitivity to scatter compared with CP imaging, while rejecting specular reflections detected by HSF imaging. ex vivo imaging of an atlas of dissected rodent organs/tissues demonstrated the scatter-based contrast achieved with HSF, CP and HSF-CP imaging, with the white light spectral signal returned by each approach translated to a color image for intuitive encoding of scatter-based contrast within images of tissue. The results suggest that visible CP-HSF imaging could have the potential to aid diagnostic imaging of lesions in skin or mucosal tissues and organs, where just CP is currently the standard practice imaging modality.

Red-light excitation of protoporphyrin IX fluorescence for subsurface tumor detection.

OBJECTIVE The objective of this study was to detect 5-aminolevulinic acid (ALA)-induced tumor fluorescence from glioma below the surface of the surgical field by using red-light illumination. METHODS To overcome the shallow tissue penetration of blue light, which maximally excites the ALA-induced fluorophore protoporphyrin IX (PpIX) but is also strongly absorbed by hemoglobin and oxyhemoglobin, a system was developed to illuminate the surgical field with red light (620-640 nm) matching a secondary, smaller absorption peak of PpIX and detecting the fluorescence emission through a 650-nm longpass filter. This wide-field spectroscopic imaging system was used in conjunction with conventional blue-light fluorescence for comparison in 29 patients undergoing craniotomy for resection of high-grade glioma, low-grade glioma, meningioma, or metastasis. RESULTS Although, as expected, red-light excitation is less sensitive to PpIX in exposed tumor, it did reveal tumor at a depth up to 5 mm below the resection bed in 22 of 24 patients who also exhibited PpIX fluorescence under blue-light excitation during the course of surgery. CONCLUSIONS Red-light excitation of tumor-associated PpIX fluorescence below the surface of the surgical field can be achieved intraoperatively and enables detection of subsurface tumor that is not visualized under conventional blue-light excitation. Clinical trial registration no.: NCT02191488 (clinicaltrials.gov).

Light scattering measured with spatial frequency domain imaging can predict stromal versus epithelial proportions in surgically resected breast tissue.

This study aims to determine if light scatter parameters measured with spatial frequency domain imaging (SFDI) can accurately predict stromal, epithelial, and adipose fractions in freshly resected, unstained human breast specimens. An explicit model was developed to predict stromal, epithelial, and adipose fractions as a function of light scattering parameters, which was validated against a quantitative analysis of digitized histology slides for N = 31 specimens using leave-one-out cross-fold validation. Specimen mean stromal, epithelial, and adipose volume fractions predicted from light scattering parameters strongly correlated with those calculated from digitized histology slides (r = 0.90, 0.77, and 0.91, respectively, p-value <1 × 10 - 6). Additionally, the ratio of predicted epithelium to stroma classified malignant specimens with a sensitivity and specificity of 90% and 81%, respectively, and also classified all pixels in malignant lesions with 63% and 79%, at a threshold of 1. All specimens and pixels were classified as malignant, benign, or fat with 84% and 75% accuracy, respectively. These findings demonstrate how light scattering parameters acquired with SFDI can be used to accurately predict and spatially map stromal, epithelial, and adipose proportions in fresh unstained, human breast tissue, and suggest that these estimations could provide diagnostic value.

Noninvasive and nondestructive optical spectroscopic measurement of motexafin gadolinium in mouse tissues: comparison to high-performance liquid chromatography.

Efficient design of anti-cancer treatments involving radiation- and photo-sensitizing therapeutics requires knowledge of tissue-specific drug concentrations. This study investigates the use of the optical pharmacokinetic system (OPS) to measure concentrations of the anti-cancer agent motexafin gadolinium (MGd) in mouse tissues noninvasively and nondestructively using elastic-scattering spectroscopy. The magnitude of MGd absorbance was quantitated by integration of the MGd peak absorbance area, and MGd concentrations were estimated by comparison with standard curves that were validated by high performance liquid chromatography (HPLC). In tissue-simulating phantoms in vitro, MGd peak absorbance area correlated with MGd concentration. Female C.B-17 SCID mice, bearing subcutaneous MDA-MB-231 human breast cancer xenografts, were dosed with 23 mg/kg MGd i.v. At specific times between 5 min and 24h after dosing, noninvasive OPS measurements were made on skin overlaying the subcutaneous tumor and skin on the opposite flank in vivo, and following exsanguination, nondestructive measurements were made on tumor, skin, and internal tissues in situ. OPS measurements on tissues in vivo detected MGd present in both tissue and blood perfusing the tissue. Both the OPS and the HPLC detected selective localization of MGd in malignant tissues compared with surrounding non-malignant tissues, and neither technique detected MGd in brain tissue. Comparison of MGd concentrations measured by HPLC and OPS is complicated by mismatch between measured tissue volumes, heterogeneous spatial distribution of MGd in tissues, and blood-localized MGd at early time points. Tumor-specific MGd concentrations measured by HPLC correlated with those measured by OPS in vivo and in situ. Best fit lines to the concentration estimates (forced through zero) had slopes of 0.900 and 1.185, respectively; however, the variability was significant (r(2)=0.477 and 0.269). The clinical utility of the OPS to quantitate MGd concentrations remains to be validated.

Mathematical model to interpret localized reflectance spectra measured in the presence of a strong fluorescence marker.

Quantification of multiple fluorescence markers during neurosurgery has the potential to provide complementary contrast mechanisms between normal and malignant tissues, and one potential combination involves fluorescein sodium (FS) and aminolevulinic acid-induced protoporphyrin IX (PpIX). We focus on the interpretation of reflectance spectra containing contributions from elastically scattered (reflected) photons as well as fluorescence emissions from a strong fluorophore (i.e., FS). A model-based approach to extract µa and µ's in the presence of FS emission is validated in optical phantoms constructed with Intralipid (1% to 2% lipid) and whole blood (1% to 3% volume fraction), over a wide range of FS concentrations (0 to 1000 µg/ml 1000 µg/ml ). The results show that modeling reflectance as a combination of elastically scattered light and attenuation-corrected FS-based emission yielded more accurate tissue parameter estimates when compared with a nonmodified reflectance model, with reduced maximum errors for blood volume (22% versus 90%), microvascular saturation (21% versus 100%), and µs' (13% versus 207%). Additionally, quantitative PpIX fluorescence sampled in the same phantom as FS showed significant differences depending on the reflectance model used to estimate optical properties (i.e., maximum error 29% versus 86%). These data represent a first step toward using quantitative optical spectroscopy to guide surgeries through simultaneous assessment of FS and PpIX.

Revisiting photodynamic therapy dosimetry: reductionist & surrogate approaches to facilitate clinical success.

Photodynamic therapy (PDT) can be a highly complex treatment, with many parameters influencing treatment efficacy. The extent to which dosimetry is used to monitor and standardize treatment delivery varies widely, ranging from measurement of a single surrogate marker to comprehensive approaches that aim to measure or estimate as many relevant parameters as possible. Today, most clinical PDT treatments are still administered with little more than application of a prescribed drug dose and timed light delivery, and thus the role of patient-specific dosimetry has not reached widespread clinical adoption. This disconnect is at least partly due to the inherent conflict between the need to measure and understand multiple parameters in vivo in order to optimize treatment, and the need for expedience in the clinic and in the regulatory and commercialization process. Thus, a methodical approach to selecting primary dosimetry metrics is required at each stage of translation of a treatment procedure, moving from complex measurements to understand PDT mechanisms in pre-clinical and early phase I trials, towards the identification and application of essential dose-limiting and/or surrogate measurements in phase II/III trials. If successful, identifying the essential and/or reliable surrogate dosimetry measurements should help facilitate increased adoption of clinical PDT. In this paper, examples of essential dosimetry points and surrogate dosimetry tools that may be implemented in phase II/III trials are discussed. For example, the treatment efficacy as limited by light penetration in interstitial PDT may be predicted by the amount of contrast uptake in CT, and so this could be utilized as a surrogate dosimetry measurement to prescribe light doses based upon pre-treatment contrast. Success of clinical ALA-based skin lesion treatment is predicted almost uniquely by the explicit or implicit measurements of photosensitizer and photobleaching, yet the individualization of treatment based upon each patients measured bleaching needs to be attempted. In the case of ALA, lack of PpIX is more likely an indicator that alternative PpIX production methods must be implemented. Parsimonious dosimetry, using surrogate measurements that are clinically acceptable, might strategically help to advance PDT in a medical world that is increasingly cost and time sensitive. Careful attention to methodologies that can identify and advance the most critical dosimetric measurements, either direct or surrogate, are needed to ensure successful incorporation of PDT into niche clinical procedures.

Imaging Techniques for Clinical Burn Assessment with a Focus on Multispectral Imaging.

Significance: Burn assessments, including extent and severity, are some of the most critical diagnoses in burn care, and many recently developed imaging techniques may have the potential to improve the accuracy of these evaluations. Recent Advances: Optical devices, telemedicine, and high-frequency ultrasound are among the highlights in recent burn imaging advancements. We present another promising technology, multispectral imaging (MSI), which also has the potential to impact current medical practice in burn care, among a variety of other specialties. Critical Issues: At this time, it is still a matter of debate as to why there is no consensus on the use of technology to assist burn assessments in the United States. Fortunately, the availability of techniques does not appear to be a limitation. However, the selection of appropriate imaging technology to augment the provision of burn care can be difficult for clinicians to navigate. There are many technologies available, but a comprehensive review summarizing the tissue characteristics measured by each technology in light of aiding clinicians in selecting the proper device is missing. This would be especially valuable for the nonburn specialists who encounter burn injuries. Future Directions: The questions of when burn assessment devices are useful to the burn team, how the various imaging devices work, and where the various burn imaging technologies fit into the spectrum of burn care will continue to be addressed. Technologies that can image a large surface area quickly, such as thermography or laser speckle imaging, may be suitable for initial burn assessment and triage. In the setting of presurgical planning, ultrasound or optical microscopy techniques, including optical coherence tomography, may prove useful. MSI, which actually has origins in burn care, may ultimately meet a high number of requirements for burn assessment in routine clinical use.

Wide-field quantitative imaging of tissue microstructure using sub-diffuse spatial frequency domain imaging.

Localized measurements of scattering in biological tissue provide sensitivity to microstructural morphology but have limited utility to wide-field applications, such as surgical guidance. This study introduces sub-diffusive spatial frequency domain imaging (sd-SFDI), which uses high spatial frequency illumination to achieve wide-field sampling of localized reflectances. Model-based inversion recovers macroscopic variations in the reduced scattering coefficient [Formula: see text] and the phase function backscatter parameter (γ). Measurements in optical phantoms show quantitative imaging of user-tuned phase-function-based contrast with accurate decoupling of parameters that define both the density and the size-scale distribution of scatterers. Measurements of fresh ex vivo breast tissue samples revealed, for the first time, unique clustering of sub-diffusive scattering properties for different tissue types. The results support that sd-SFDI provides maps of microscopic structural biomarkers that cannot be obtained with diffuse wide-field imaging and characterizes spatial variations not resolved by point-based optical sampling.

Molecular dyes used for surgical specimen margin orientation allow for intraoperative optical assessment during breast conserving surgery.

A variety of optical techniques utilizing near-infrared (NIR) light are being proposed for intraoperative breast tumor margin assessment. However, immediately following a lumpectomy excision, the margins are inked, which preserves the orientation of the specimen but prevents optical interrogation of the tissue margins. Here, a workflow is proposed that allows for both NIR optical assessment following full specimen marking using molecular dyes which have negligible absorption and scattering in the NIR. The effect of standard surgical inks in contrast to molecular dyes for an NIR signal is shown. Further, the proposed workflow is demonstrated with full specimen intraoperative imaging on all margins directly after the lumpectomy has been excised and completely marked. This work is an important step in the path to clinical feasibility of intraoperative breast tumor margin assessment using NIR optical methods without having to compromise on the current clinical practice of inking resected specimens for margin orientation.

Macroscopic optical imaging technique for wide-field estimation of fluorescence depth in optically turbid media for application in brain tumor surgical guidance.

A diffuse imaging method is presented that enables wide-field estimation of the depth of fluorescent molecular markers in turbid media by quantifying the deformation of the detected fluorescence spectra due to the wavelength-dependent light attenuation by overlying tissue. This is achieved by measuring the ratio of the fluorescence at two wavelengths in combination with normalization techniques based on diffuse reflectance measurements to evaluate tissue attenuation variations for different depths. It is demonstrated that fluorescence topography can be achieved up to a 5 mm depth using a near-infrared dye with millimeter depth accuracy in turbid media having optical properties representative of normal brain tissue. Wide-field depth estimates are made using optical technology integrated onto a commercial surgical microscope, making this approach feasible for real-world applications.