We employed two commercially available femtosecond lasers, a Ti:sapphire and a ytterbium-based oscillator, to directly compare from a user’s practical point-of-view in one common experimental setup the efficiencies of transient laser-induced cell membrane permeabilization, i.e., of so-called optoporation. The experimental setup consisted of a modified multiphoton laser-scanning microscope employing high-NA focusing optics. An automatic cell irradiation procedure was realized with custom-made software that identified cell positions and controlled relevant hardware components. The Ti:sapphire and ytterbium-based oscillators generated broadband sub-15-fs pulses around 800 nm and 250-fs pulses at 1044 nm, respectively. A higher optoporation rate and posttreatment viability were observed for the shorter fs pulses, confirming the importance of multiphoton effects for efficient optoporation.

A simultaneous multispectral fluorescence imaging system incorporating multiplexed volume holographic grating (VHG) is developed to acquire multispectral images of an object in one shot. With the multiplexed VHG, the imaging system can provide the distribution and spectral characteristics of multiple fluorophores in the scene. The implementation and performance of the simultaneous multispectral imaging system are presented. Further, the system’s capability in simultaneously obtaining multispectral fluorescence measurements is demonstrated with in vivo experiments on a mouse. The demonstrated imaging system has the potential to obtain multispectral images fluorescence simultaneously.

Two-photon excited fluorescence (TPEF) imaging of the cellular cofactors nicotinamide adenine dinucleotide and oxidized flavin adenine dinucleotide is widely used to measure cellular metabolism, both in normal and pathological cells and tissues. When dual-wavelength excitation is used, ratiometric TPEF imaging of the intrinsic cofactor fluorescence provides a metabolic index of cells—the “optical redox ratio” (ORR). With increased interest in understanding and controlling cellular metabolism in cancer, there is a need to evaluate the performance of ORR in malignant cells. We compare TPEF metabolic imaging with seahorse flux analysis of cellular oxygen consumption in two different breast cancer cell lines (MCF-7 and MDA-MB-231). We monitor metabolic index in living cells under both normal culture conditions and, for MCF-7, in response to cell respiration inhibitors and uncouplers. We observe a significant correlation between the TPEF-derived ORR and the flux analyzer measurements (R=0.7901, p<0.001). Our results confirm that the ORR is a valid dynamic index of cell metabolism under a range of oxygen consumption conditions relevant for cancer imaging.

The surface effect close to the boundary of a small light-scattering object in a highly scattering medium is experimentally demonstrated. This is the first attempt to measure the surface effect of a small spherical scattering object in 1% intralipid solution by use of developed diffuse photon-pairs density wave (DPPDW) in terms of the amplitude and phase detection. Theoretically, the surface effect of a small scattering object in turbid media is localized close to the boundary according to the perturbation theory, concerning an inhomogeneous distribution of the diffusion coefficient in the frequency-domain diffusion equation. Hence, an improvement of the spatial resolution of the image via an inverse algorithm, which relates to detection sensitivity of localization to the boundary of the image object in a multiple scattering medium, is anticipated. In this study, we demonstrate that DPPDW is able to sense the surface effect of a 2-mm spherical scattering object in 1% intralipid solution, with high sensitivity. Subsequently, an improvement of spatial resolution of imaging in turbid media by using DPPDW in comparison with conventional diffuse photon density wave (DPDW) using inverse algorithm is discussed.

Infrared control is a new technique that uses pulsed infrared lasers to thermally alter electrical activity. Originally developed for nerves, we have applied this technology to embryonic hearts using a quail model, previously demonstrating infrared stimulation and, here, infrared inhibition. Infrared inhibition enables repeatable and reversible block, stopping cardiac contractions for several seconds. Normal beating resumes after the laser is turned off. The block can be spatially specific, affecting propagation on the ventricle or initiation on the atrium. Optical mapping showed that the block affects action potentials and not just calcium or contraction. Increased resting intracellular calcium was observed after a 30-s exposure to the inhibition laser, which likely resulted in reduced mechanical function. Further optimization of the laser illumination should reduce potential damage. Stopping cardiac contractions by disrupting electrical activity with infrared inhibition has the potential to be a powerful tool for studying the developing heart.

This PDF file contains the editorial “Special Section Guest Editorial:Selected Topics in Biophotonics: Photoacoustic Tomography and Fiber-Based Lasers and Supercontinuum Sources” for JBO Vol. 21 Issue 06

Myoglobin is an essential oxygen-binding hemoprotein in skeletal and cardiac muscles that buffers intracellular oxygen (O₂) concentration in response to hypoxia or elevated muscle activities. We present a method that uses photoacoustic computed tomography to measure the distribution of myoglobin in tissue and the oxygen saturation of myoglobin (sO₂-Mb). From photoacoustic measurements of mice in different oxygenation states, we performed calibration-free quantification of the sO₂-Mb change in the backbone muscle invivo.

We generated a highly deuterated cholesterol analog (D38-cholesterol) and demonstrated its use for selective vibrational imaging of cholesterol storage in mammalian cells. D38-cholesterol produces detectable signals in stimulated Raman scattering (SRS) imaging, is rapidly taken up by cells, and is efficiently metabolized by acyl-CoA cholesterol acyltransferase to form cholesteryl esters. Using hyperspectral SRS imaging of D38-cholesterol, we visualized cholesterol storage in lipid droplets. We found that some lipid droplets accumulated preferentially unesterified D38-cholesterol, whereas others stored D38-cholesteryl esters. In steroidogenic cells, D38-cholesteryl esters and triacylglycerols were partitioned into distinct sets of lipid droplets. Thus, hyperspectral SRS imaging of D38-cholesterol demonstrates a heterogeneous incorporation of neutral lipid species, i.e., free cholesterol, cholesteryl esters, and triacylglycerols, between individual lipid droplets in a cell.

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 μ_a 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). 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 μ′_s (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.

We demonstrate a record bandwidth high energy supercontinuum source suitable for multispectral photoacoustic microscopy. The source has more than 150  nJ/10  nm bandwidth over a spectral range of 500 to 1600 nm. This performance is achieved using a carefully designed fiber taper with large-core input for improved power handling and small-core output that provides the desired spectral range of the supercontinuum source.

We present an ultrahigh-resolution spectral domain optical coherence tomography (OCT) system in 800 nm with a low-noise supercontinuum source (SC) optimized for myocardial imaging. The system was demonstrated to have an axial resolution of 2.72  μm with a large imaging depth of 1.78 mm and a 6-dB falloff range of 0.89 mm. The lateral resolution (5.52  μm) was compromised to enhance the image penetration required for myocardial imaging. The noise of the SC source was analyzed extensively and an imaging protocol was proposed for SC-based OCT imaging with appreciable contrast. Three-dimensional datasets were acquired ex vivo on the endocardium side of tissue specimens from different chambers of fresh human and swine hearts. With the increased resolution and contrast, features such as elastic fibers, Purkinje fibers, and collagen fiber bundles were observed. The correlation between the structural information revealed in the OCT images and tissue pathology was discussed as well.

Photoacoustic tomography (PAT) has become one of the fastest growing fields in biomedical optics. Unlike pure optical imaging, such as confocal microscopy and two-photon microscopy, PAT employs acoustic detection to image optical absorption contrast with high-resolution deep into scattering tissue. So far, PAT has been widely used for multiscale anatomical, functional, and molecular imaging of biological tissues. We focus on PAT’s basic principles, major implementations, imaging contrasts, and recent applications.

Spinal cord injury (SCI) triggers several lipid alterations in nervous tissue. It is characterized by extensive demyelination and the inflammatory response leads to accumulation of activated microglia/macrophages, which often transform into foam cells by accumulation of lipid droplets after engulfment of the damaged myelin sheaths. Using an experimental rat model, Raman microspectroscopy was applied to retrieve the modifications of the lipid distribution following SCI. Coherent anti-Stokes Raman scattering (CARS) and endogenous two-photon fluorescence (TPEF) microscopies were used for the detection of lipid-laden inflammatory cells. The Raman mapping of CH₂ deformation mode intensity at 1440  cm−¹ retrieved the lipid-depleted injury core. Preserved white matter and inflammatory regions with myelin fragmentation and foam cells were localized by specifically addressing the distribution of esterified lipids, i.e., by mapping the intensity of the carbonyl Raman band at 1743  cm−¹, and were in agreement with CARS/TPEF microscopy. Principal component analysis revealed that the inflammatory regions are notably rich in saturated fatty acids. Therefore, Raman spectroscopy enabled to specifically detect inflammation after SCI and myelin degradation products.

Imaging small blood vessels and measuring their functional information in finger joint are still challenges for clinical imaging modalities. In this study, we developed a multi-transducer functional photoacoustic tomography (PAT) system and successfully imaged human finger-joint vessels from ∼1  mm to <0.2  mm in diameter. In addition, the oxygen saturation (SO₂) values of these vessels were also measured. Our results demonstrate that PAT can provide both anatomical and functional information of individual finger-joint vessels with different sizes, which might help the study of finger-joint diseases, such as rheumatoid arthritis.

Fiber-based lasers and master oscillator power fiber amplifier configurations are described. These allow spectral versatility coupled with pulse width and pulse repetition rate selection in compact and efficient packages. This is enhanced through the use of nonlinear optical conversion in fibers and fiber-coupled nonlinear crystals, which can be integrated to provide all-fiber pump sources for diverse application. The advantages and disadvantages of sources based upon supercontinuum generation, stimulated Raman conversion, four-wave mixing, parametric generation and difference frequency generation, allowing spectral coverage from the UV to the mid-infrared, are considered.

We conducted an initial feasibility study using real-time magneto-motive optical Doppler tomography (MM-ODT) with enhanced contrast to investigate the detection of superparamagnetic iron oxide (SPIO) magnetic nanoparticles implanted into in vivo melanoma tissue. The MM-ODT signals were detected owing to the phase shift of the implanted magnetic nanoparticles, which occurred due to the action of an applied magnetic field. An amplifier circuit-based solenoid was utilized for generating high-intensity oscillating magnetic fields. The MM-ODT system was confirmed as an effective in vivo imaging method for detecting melanoma tissue, with the performance comparable to those of conventional optical coherence tomography and optical Doppler tomography methods. Moreover, the optimal values of the SPIO nanoparticles concentration and solenoid voltage for obtaining the uppermost Doppler velocity were derived as well. To improve the signal processing speed for real-time imaging, we adopted multithread programming techniques and optimized the signal path. The results suggest that this imaging modality can be used as a powerful tool to identify the intracellular and extracellular SPIO nanoparticles in melanoma tissues in vivo.

We experimentally verify a speckle-based technique for noncontact measurement of glucose concentration in the bloodstream. The final device is intended to be a single wristwatch-style device containing a laser, a camera, and an alternating current (ac) electromagnet generated by a solenoid. The experiments presented are performed in vitro as proof of the concept. When a glucose substance is inserted into a solenoid generating an ac magnetic field, it exhibits Faraday rotation, which affects the temporal changes of the secondary speckle pattern distributions. The temporal frequency resulting from the ac magnetic field was found to have a lock-in amplification role, which increased the observability of the relatively small magneto-optic effect. Experimental results to support the proposed concept are presented.

Optical coherence tomography angiography (OCTA) has increasingly become a clinically useful technique in ophthalmic imaging. We evaluate the repeatability and reproducibility of blood perfusion in the optic nerve head (ONH) measured using optical microangiography (OMAG)-based OCTA. Ten eyes from 10 healthy volunteers are recruited and scanned three times with a 68-kHz Cirrus HD-OCT 5000-based OMAG prototype system (Carl Zeiss Meditec Inc., Dublin, California) centered at the ONH involving two separate visits within six weeks. Vascular images are generated with OMAG processing by detecting the differences in OCT signals between consecutive B-scans acquired at the same retina location. ONH perfusion is quantified as flux, vessel area density, and normalized flux within the ONH for the prelaminar, lamina cribrosa, and the full ONH. Coefficient of variation (CV) and intraclass correlation coefficient (ICC) are used to evaluate intravisit and intervisit repeatability, and interobserver reproducibility. ONH perfusion measurements show high repeatability [CV≤3.7% (intravisit) and ≤5.2% (intervisit)] and interobserver reproducibility (ICC≤0.966) in all three layers by three metrics. OCTA provides a noninvasive method to visualize and quantify ONH perfusion in human eyes with excellent repeatability and reproducibility, which may add additional insight into ONH perfusion in clinical practice.

We have developed an adaptive calibration algorithm and protocol (ACA-Pro) that corrects from the instrumental response of various spatially resolved diffuse reflectance spectroscopy (DRSsr) systems to enable the quantification of absorption and scattering properties based on a Monte Carlo-based look-up-table approach. The protocol involves the use of a calibration reference base built with measurements of a range of different diffusive intralipid phantoms. Moreover, an advanced strategy was established to take into account the experimental variations with an additional measurement of a common solid material, allowing the use of a single calibration reference base for all experiments. The ACA-Pro is validated in contact and noncontact probe-based DRSsr systems. Furthermore, the first results of a setup replacing the probe with a CCD detector are shown to confirm the robustness of the approach.

Combining finite-difference time-domain (FDTD) methods and modeling of optical microscopy modalities, we previously developed an open-source software package called Angora, which is essentially a “microscope in a computer.” However, the samples being simulated were limited to nondispersive media. Since media dispersions are common in biological samples (such as cells with staining and metallic biomarkers), we have further developed a module in Angora to simulate samples having complicated dispersion properties, thereby allowing the synthesis of microscope images of most biological samples. We first describe a method to integrate media dispersion into FDTD, and we validate the corresponding Angora dispersion module by applying Mie theory, as well as by experimentally imaging gold microspheres. Then, we demonstrate how Angora can facilitate the development of optical imaging techniques with a case study.

The surface topographical, compositional, and structural modifications induced in human enamel by femtosecond laser ablation is studied. The laser treatments were performed using a Yb:KYW chirped-pulse-regenerative amplification laser system (560 fs and 1030 nm) and fluences up to 14  J/cm². The ablation surfaces were studied by scanning electron microscopy, grazing incidence x-ray diffraction, and micro-Raman spectroscopy. Regardless of the fluence, the ablation surfaces were covered by a layer of resolidified material, indicating that ablation is accompanied by melting of hydroxyapatite. This layer presented pores and exploded gas bubbles, created by the release of gaseous decomposition products of hydroxyapatite (CO₂ and H₂O) within the liquid phase. In the specimen treated with 1-kHz repetition frequency and 14  J/cm², thickness of the resolidified material is in the range of 300 to 900 nm. The micro-Raman analysis revealed that the resolidified material contains amorphous calcium phosphate, while grazing incidence x-ray diffraction analysis allowed detecting traces of a calcium phosphate other than hydroxyapatite, probably β-tricalcium phosphate Ca₃(PO₄)₂, at the surface of this specimen. The present results show that the ablation of enamel involves melting of enamel’s hydroxyapatite, but the thickness of the altered layer is very small and thermal damage of the remaining material is negligible.

Rod-dominated transient retinal phototropism (TRP) has been recently observed in freshly isolated mouse and frog retinas. Comparative confocal microscopy and optical coherence tomography revealed that the TRP was predominantly elicited from the rod outer segment (OS). However, the biophysical mechanism of rod OS dynamics is still unknown. Mouse and frog retinal slices, which displayed a cross-section of retinal photoreceptors and other functional layers, were used to test the effect of light stimulation on rod OSs. Time-lapse microscopy revealed stimulus-evoked conformational changes of rod OSs. In the center of the stimulated region, the length of the rod OS shrunk, while in the peripheral region, the rod OS swung toward the center region. Our experimental observation and theoretical analysis suggest that the TRP may reflect unbalanced rod disc-shape changes due to localized visible light stimulation.

Lasers have the potential for reducing the required debonding force and can prevent the mechanical damage given to the enamel surface as a result of conventional debonding procedure. However, excessive thermal effects limit the use of lasers for debonding purposes. The aim of this study was to investigate the optimal parameters of 1940-nm Tm:fiber laser for debonding ceramic brackets. Pulling force and intrapulpal temperature measurements were done during laser irradiation simultaneously. A laser beam was delivered in two different modes: scanning the fiber tip on the bracket surface with a Z shape movement or direct application of the fiber tip at one point in the center of the bracket. Results showed that debonding force could be decreased significantly compared to the control samples, in which brackets were debonded by only mechanical force. Intrapulpal temperature was kept equal or under the 5.5°C threshold value of probable thermal damage to pulp. Scanning was found to have no extra contribution to the process. It was concluded that using 1940-nm Tm:fiber laser would facilitate the debonding of ceramic brackets and can be proposed as a promising debonding tool with all the advantageous aspects of fiber lasers.

Although numerous studies have been performed to fabricate various optical tissue phantom (OTP) models, the fabrication of OTPs that simulate skin layers is laborious and time-consuming owing to the intricate characteristics of skin tissue. This study presents various OTP models that optically and structurally simulate the epidermis–dermis skin layer. The spin-coating method was employed to reproduce a uniform thin layer that mimics the epidermis layer, and the fabrication parameters were optimized for epoxy and silicone reference materials. Various OTP models simulating blood vessels and hyperpigmentation lesions were fabricated using the two reference materials to determine their feasibility. The suitability of each of the two reference materials for OTP fabrication was qualitatively evaluated by comparing the quality of the OTP models.

A fiber optic imaging approach is presented using structured illumination for quantification of almost pure epithelial backscattering. We employ multiple spatially modulated projection patterns and camera-based reflectance capture to image depth-dependent epithelial scattering. The potential diagnostic value of our approach is investigated on cervical ex vivo tissue specimens. Our study indicates a strong backscattering increase in the upper part of the cervical epithelium caused by dysplastic microstructural changes. Quantization of relative depth-dependent backscattering is confirmed as a potentially useful diagnostic feature for detection of precancerous lesions in cervical squamous epithelium.

Optical coherence tomography (OCT) is a noninvasive interferometric imaging modality providing anatomical information at depths of millimeters and a resolution of micrometers. Conventional OCT images limit our knowledge to anatomical structures alone, without any contrast enhancement. Therefore, here we have, for the first time, optimized an OCT-based contrast-enhanced imaging system for imaging single cells and blood vessels in vivo inside the living mouse retina at subnanomolar sensitivity. We used bioconjugated gold nanorods (GNRs) as exogenous OCT contrast agents. Specifically, we used anti-mouse CD45 coated GNRs to label mouse leukocytes and mPEG-coated GNRs to determine sensitivity of GNR detection in vivo inside mice retinae. We corroborated OCT observations with hyperspectral dark-field microscopy of formalin-fixed histological sections. Our results show that mouse leukocytes that otherwise do not produce OCT contrast can be labeled with GNRs leading to significant OCT intensity equivalent to a 0.5 nM GNR solution. Furthermore, GNRs injected intravenously can be detected inside retinal blood vessels at a sensitivity of ∼0.5  nM, and GNR-labeled cells injected intravenously can be detected inside retinal capillaries by enhanced OCT contrast. We envision the unprecedented resolution and sensitivity of functionalized GNRs coupled with OCT to be adopted for longitudinal studies of retinal disorders.

Islet transplantation (IT) is an established clinical therapy for select patients with type-1 diabetes. Clinically, the hepatic portal vein serves as the site for IT. Despite numerous advances in clinical IT, limitations remain, including early islet cell loss posttransplant, procedural complications, and the inability to effectively monitor islet grafts. Hence, alternative sites for IT are currently being explored, with the subcutaneous space as one potential option. When left unmodified, the subcutaneous space routinely fails to promote successful islet engraftment. However, when employing the previously developed subcutaneous “deviceless” technique, a favorable microenvironment for islet survival and function is established. In this technique, an angiocatheter was temporarily implanted subcutaneously, which facilitated angiogenesis to promote subsequent islet engraftment. This technique has been employed in preclinical animal models, providing a sufficient means to develop techniques to monitor functional aspects of the graft such as angiogenesis. Here, we utilize photoacoustic imaging to track angiogenesis during the priming of the subcutaneous site by the implanted catheter at 1 to 4 weeks postcatheter. Quantitative analysis on vessel densities shows gradual growth of vasculature in the implant position. These results demonstrate the ability to track angiogenesis, thus facilitating a means to optimize and assess the pretransplant microenvironment.

A Monte Carlo simulation was utilized to investigate a simple model for the transition between the ballistic and the diffusive regimes in diffusive media. The simulation focuses on the propagation of visible and near-infrared light in biological tissues. This research has mainly two findings: (1) the transition can be described, as was found experimentally, with good accuracy by only two terms (ballistic and diffusive). (2) The model can be utilized for cases where the absorption coefficient is not negligible compared to the scattering coefficient by adding a power-law prefactor to the diffusive term.

Grueneisen relaxation photoacoustic microscopy (GR-PAM) can achieve optically defined axial resolution, but it has been limited to ex vivo demonstrations so far. Here, we present the first in vivo image of a mouse brain acquired with GR-PAM. To induce the GR effect, an intensity-modulated continuous-wave laser was employed to heat absorbing objects. In phantom experiments, an axial resolution of 12.5  μm was achieved, which is sixfold better than the value achieved by conventional optical-resolution PAM. This axial-resolution improvement was further demonstrated by imaging a mouse brain in vivo, where significantly narrower axial profiles of blood vessels were observed. The in vivo demonstration of GR-PAM shows the potential of this modality for label-free and high-resolution anatomical and functional imaging of biological tissues.

This paper reports the application of the motion history image (MHI) method for biospeckle processing of a bacterial growth. The method avoids the complexity as well as the large computation in sequence-matching-based methods and detects whether the speckle structure has changed or not. Encouraging experimental results on the real-time evolution of the growing bacteria during 12 h demonstrate the effectiveness of the proposed method. The MHI presented an online result without loss of resolution and definition. In turn, the MHI also presented the ability to provide a close answer to the traditional offline method of generalized differences.

Super-resolution optical fluctuation imaging (SOFI) is a fast and low-cost live-cell optical nanoscopy for extracting subdiffraction information from the statistics of fluorescence intensity fluctuation. As SOFI is based on the fluctuation statistics, rather than the detection of single molecules, it poses unique requirements for imaging detectors, which still lack a systematic evaluation. Here, we analyze the influences of pixel sizes, frame rates, noise levels, and different gains in SOFI with simulations and experimental tests. Our analysis shows that the smaller pixel size and faster readout speed of scientific-grade complementary metal oxide semiconductor (sCMOS) enables SOFI to achieve high spatiotemporal resolution with a large field-of-view, which is especially beneficial for live-cell super-resolution imaging. Overall, as the performance of SOFI is relatively insensitive to the signal-to-noise ratio (SNR), the gain in pixel size and readout speed exceeds the loss in SNR, indicating sCMOS is superior to electron multiplying charge coupled device in context to SOFI in many cases. Super-resolution imaging of cellular microtubule structures with high-order SOFI is experimentally demonstrated at large field-of-view, taking advantage of the large pixel number and fast frame rate of sCMOS cameras.

Optical coherence tomography angiography (OCTA) is clinically useful for the qualitative assessment of the macular microvasculature. However, there is a need for comprehensive quantitative tools to help objectively analyze the OCT angiograms. Few studies have reported the use of a single quantitative index to describe vessel density in OCT angiograms. In this study, we introduce a five-index quantitative analysis of OCT angiograms in an attempt to detect and assess vascular abnormalities from multiple perspectives. The indices include vessel area density, vessel skeleton density, vessel diameter index, vessel perimeter index, and vessel complexity index. We show the usefulness of the proposed indices with five illustrative cases. Repeatability is tested on both a healthy case and a stable diseased case, giving interclass coefficients smaller than 0.031. The results demonstrate that our proposed quantitative analysis may be useful as a complement to conventional OCTA for the diagnosis of disease and monitoring of treatment.

Functional near-infrared spectroscopy (fNIRS) signals originate in hemoglobin changes in both the superficial layer of the head and the brain. Under the assumption that the changes in the blood flow in the scalp are spatially homogeneous in the region of interest, a variety of methods for reducing the superficial signals has been proposed. To clarify the spatial distributions of the superficial signals, the superficial signals from the forehead during a verbal-fluency task were investigated by using ten source–detector pairs separated by 5 mm, whereas fNIRS signals were also detected from two source–detector pairs separated by 30 mm. The fNIRS signals strongly correlated with the superficial signals at some channels on the forehead. Hierarchical cluster analysis was performed on the temporal cross-correlation coefficients for two channels of both the NIRS signals, and the analysis results demonstrate spatially heterogeneous distributions and network structures of the superficial signals from within the forehead. The results also show that the assumption stated above is invalid for homogeneous superficial signals from any region of interest of 15-mm diameter or larger on the forehead. They also suggest that the spatially heterogeneous distributions may be attributable to vascular networks, including supraorbital, supratrochlear, and superficial temporal vessels.

Dynamic fluorescence molecular tomography (DFMT) is a valuable method to evaluate the metabolic process of contrast agents in different organs in vivo, and direct reconstruction methods can improve the temporal resolution of DFMT. However, challenges still remain due to the large time consumption of the direct reconstruction methods. An acceleration strategy using graphics processing units (GPU) is presented. The procedure of conjugate gradient optimization in the direct reconstruction method is programmed using the compute unified device architecture and then accelerated on GPU. Numerical simulations and in vivo experiments are performed to validate the feasibility of the strategy. The results demonstrate that, compared with the traditional method, the proposed strategy can reduce the time consumption by ∼90% without a degradation of quality.

We report quantitative photoacoustic elastography (QPAE) capable of measuring Young’s modulus of biological tissue in vivo in humans. By combining conventional PAE with a stress sensor having known stress–strain behavior, QPAE can simultaneously measure strain and stress, from which Young’s modulus is calculated. We first demonstrate the feasibility of QPAE in agar phantoms with different concentrations. The measured Young’s modulus values fit well with both the empirical expectation based on the agar concentrations and those measured in an independent standard compression test. Next, QPAE was applied to quantify the Young’s modulus of skeletal muscle in vivo in humans, showing a linear relationship between muscle stiffness and loading. The results demonstrated the capability of QPAE to assess the absolute elasticity of biological tissue noninvasively in vivo in humans, indicating its potential for tissue biomechanics studies and clinical applications.

The application of near-infrared spectroscopy (NIRS) to assess microvascular function has shown promising results. An important limitation when using a single source-detector pair, however, is the lack of depth sensitivity. Diffuse optical tomography (DOT) overcomes this limitation using an array of sources and detectors that allow the reconstruction of volumetric hemodynamic changes. This study compares the key parameters of postocclusive reactive hyperemia measured in the forearm using standard NIRS and DOT. We show that while the mean parameter values are similar for the two techniques, DOT achieves much better reproducibility, as measured by the intraclass correlation coefficient (ICC). We show that DOT achieves high reproducibility for muscle oxygen consumption (ICC: 0.99), time to maximal HbO₂ (ICC: 0.94), maximal HbO₂ (ICC: 0.99), and time to maximal HbT (ICC: 0.99). Absolute reproducibility as measured by the standard error of measurement is consistently smaller and close to zero (ideal value) across all parameters measured by DOT compared to NIRS. We conclude that DOT provides a more robust characterization of the reactive hyperemic response and show how the availability of volumetric hemodynamic changes allows the identification of areas of temporal consistency, which could help characterize more precisely the microvasculature.

We developed a simultaneous visible-light (Vis) and near-infrared (NIR) dual-band optical coherence tomography (OCT) system using a single supercontinuum laser source. The goal was to benchmark our newly developed Vis-OCT against the well-developed NIR-OCT. The Vis-OCT subsystem operated at 91 nm full-width-at-half-maximum (FWHM) bandwidth centered at 566 nm; the NIR-OCT subsystem operated at 93 nm FWHM bandwidth centered at 841 nm. The axial resolutions were 1.8 and 4.4  μm in air for the Vis- and NIR-OCT subsystems, respectively. We compared the respective performances, including anatomical imaging, angiography, absolute retinal blood flow measurements, and spectroscopic analysis for retinal blood oxygen saturation (sO₂), between the two subsystems in rodents in vivo. While demonstrating minor discrepancies related to operation wavelengths, both subsystems showed comparable performances in the first three tests. However, we were only able to retrieve sO₂ using the Vis-OCT subsystem.

Optical spectroscopy is sensitive to morphological composition and has potential applications in intraoperative margin assessment. Here, we evaluate ex vivo breast tissue and corresponding quantified hematoxylin & eosin images to correlate optical scattering signatures to tissue composition stratified by patient characteristics. Adipose sites (213) were characterized by their cell area and density. All other benign and malignant sites (181) were quantified using a grid method to determine composition. The relationships between mean reduced scattering coefficient (〈μ'_s〉), and % adipose, % collagen, % glands, adipocyte cell area, and adipocyte density were investigated. These relationships were further stratified by age, menopausal status, body mass index (BMI), and breast density. We identified a positive correlation between 〈μ'_s〉 and % collagen and a negative correlation between 〈μ'_s〉 and age and BMI. Increased collagen corresponded to increased 〈μ'_s〉 variability. In postmenopausal women, 〈μ'_s〉 was similar regardless of fibroglandular content. Contributions from collagen and glands to 〈μ'_s〉 were independent and equivalent in benign sites; glands showed a stronger positive correlation than collagen to 〈μ'_s〉 in malignant sites. Our data suggest that scattering could differentiate highly scattering malignant from benign tissues in postmenopausal women. The relationship between scattering and tissue composition will support improved scattering models and technologies to enhance intraoperative optical margin assessment.

The response to polarization of second-harmonic generation (SHG) microscopy images of samples with different collagen distributions (quasialigned, partially organized, and nonorganized) has been analyzed. A linear decay relationship between the external arrangement and polarization sensitivity was found. SHG signal from nonorganized samples presented a large structural dispersion and a weak dependence with incident polarization. Polarization dependence is also associated with the internal organization of the collagen fibers, directly related to the ratio of hyperpolarizabilities ρ. This parameter can experimentally be computed from the modulation of the SHG signal. The results show that both external and internal collagen structures are closely related. This provides a tool to obtain information of internal properties from the polarimetric response of the external spatial distribution of collagen, which might be useful in clinical diagnosis of pathologies related to changes in collagen structure.

Photoaging is associated with increasing pigmentary heterogeneity and darkening of skin color. However, little is known about age-related changes in skin pigmentation on sun-protected areas. The aim of this explorative study was to measure skin color and dyspigmentation using image processing and to evaluate the reliability of these parameters. Twenty-four volunteers of three age-groups were included in this explorative study. Measurements were conducted at sun-exposed and sun-protected areas. Overall skin-color estimates were similar among age groups. The hyper- and hypopigmentation indices differed significantly by age groups and their correlations with age ranged between 0.61 and 0.74. Dorsal forearm skin differed from the other investigational areas (p<0.001). We observed an increase in dyspigmentation at all skin areas, including sun-protected skin areas, already in young adulthood. Associations between age and dyspigmentation estimates were higher compared to color parameters. All color and dyspigmentation estimates showed high reliability. Dyspigmentation parameters seem to be better biomarkers for UV damage than the overall color measurements.

We performed stimulated emission depletion (STED) imaging of isolated olfactory sensory neurons (OSNs) using a custom-built microscope. The STED microscope uses a single pulsed laser to excite two separate fluorophores, Atto 590 and Atto 647N. A gated timing circuit combined with temporal interleaving of the different color excitation/STED laser pulses filters the two channel detection and greatly minimizes crosstalk. We quantified the instrument resolution to be ∼81 and ∼44  nm, for the Atto 590 and Atto 647N channels. The spatial separation between the two channels was measured to be under 10 nm, well below the resolution limit. The custom-STED microscope is incorporated onto a commercial research microscope allowing brightfield, differential interference contrast, and epifluorescence imaging on the same field of view. We performed immunolabeling of OSNs in mice to image localization of ciliary membrane proteins involved in olfactory transduction. We imaged Ca²⁺-permeable cyclic nucleotide gated (CNG) channel (Atto 594) and adenylyl cyclase type III (ACIII) (Atto 647N) in distinct cilia. STED imaging resolved well-separated subdiffraction limited clusters for each protein. We quantified the size of each cluster to have a mean value of 88±48  nm and 124±43  nm, for CNG and ACIII, respectively. STED imaging showed separated clusters that were not resolvable in confocal images.

An investigation of microwave (MW) heating effects on biotissue for enhancing photoacoustic radar (PAR) signals was conducted. Localized tissue heating generated by MWs was used to improve PAR imaging depth and signal-to-noise ratio (SNR). Elevated temperatures were measured with thermocouples in ex vivo bovine muscle. The measured temperature rise on the heated spot surface by MWs was in agreement with theoretical predictions. The study showed localized MW heating can increase the photoacoustic imaging depth by 11%, and the SNR by 5% in ex vivo bovine muscle.

There is an urgent need for sensitive and specific tools to accurately image early stage, organ-confined human prostate cancers to facilitate active surveillance and reduce unnecessary treatment. Recently, we developed an acoustic lens that enhances the sensitivity of photoacoustic imaging. Here, we report the use of this device in conjunction with two molecular imaging agents that specifically target the prostate-specific membrane antigen (PSMA) expressed on the tumor cell surface of most prostate cancers. We demonstrate successful imaging of phantoms containing cancer cells labeled with either of two different PSMA-targeting agents, the ribonucleic acid aptamer A10-3.2 and a urea-based peptidomimetic inhibitor, each linked to the near-infrared dye IRDye800CW. By specifically targeting cells with these agents linked to a dye chosen for optimal signal, we are able to discriminate prostate cancer cells that express PSMA.

We present a method for low-cost fabrication of polydimethylsiloxane (PDMS) tissue simulating phantoms with tunable scattering spectra, spanning visible, and near-infrared regimes. These phantoms use optical polishing agents (aluminum oxide powders) at various grit sizes to approximate in vivo tissue scattering particles across multiple size distributions (range: 17 to 3  μm). This class of tunable scattering phantoms is used to mimic distinct changes in wavelength-dependent scattering properties observed in tissue pathologies such as partial thickness burns. Described by a power-law dependence on wavelength, the scattering magnitude of these phantoms scale linearly with particle concentration over a physiologic range [μ'_s=(0.5 to 2.0  mm−¹)] whereas the scattering spectra, specific to each particle size distribution, correlate to distinct exponential coefficients (range: 0.007 to 0.32). Aluminum oxide powders used in this investigation did not detectably contribute to the absorption properties of these phantoms. The optical properties of these phantoms are verified through inverse adding-doubling methods and the tolerances of this fabrication method are discussed.