The optic axis of birefringent samples indicates the direction of optical anisotropy, which should be described in three-dimensional (3-D) space. We present a method to quantify the complete 3-D optic axis orientation calculated from in-plane optic axis measurements from a polarization-sensitive optical coherence tomography system. The in-plane axis orientations with different illumination angles allow the calculation of the necessary polar angle. The method then provides the information to produce the actual birefringence. The method and results from a biological sample are presented.

Biomedical applications of terahertz (THz) radiation are appealing because THz radiation is nonionizing and has the demonstrated ability to detect intrinsic contrasts between cancerous and normal tissue. A linear polarization-sensitive detection technique for tumor margin delineation has already been demonstrated; however, utilization of a circular polarization-sensitive detection technique has yet to be explored at THz frequencies. A reflective, continuous-wave THz imaging system capable of illuminating a target sample at 584 GHz with either linearly or circularly polarized radiation, and capable of collecting both cross- and copolarized signals remitted from the target, is implemented. To demonstrate the system’s utility, a fresh ex vivo human skin tissue specimen containing nonmelanoma skin cancer was imaged. Both polarization-sensitive detection techniques showed contrast between tumor and normal skin tissue, although some differences in images were observed between the two techniques. Our results indicate that further investigation is required to explain the contrast mechanism, as well as to quantify the specificity and sensitivity of the circular polarization-sensitive detection technique.

This PDF file contains the editorial “Special Section Guest Editorial:Polarized Light for Biomedical Applications” for JBO Vol. 21 Issue 07

This PDF file contains the editorial “Special Section Guest Editorial:Antonello De Martino (1954–2014): in memoriam” for JBO Vol. 21 Issue 07

Accurate classification of malignant cells from benign ones can significantly enhance cancer diagnosis and prognosis by detection of circulating tumor cells (CTCs). We have investigated two approaches of quantitative morphology and polarization diffraction imaging on two prostate cell types to evaluate their feasibility as single-cell assay methods toward CTC detection after cell enrichment. The two cell types have been measured by a confocal imaging method to obtain their three-dimensional morphology parameters and by a polarization diffraction imaging flow cytometry (p-DIFC) method to obtain image texture parameters. The support vector machine algorithm was applied to examine the accuracy of cell classification with the morphology and diffraction image parameters. Despite larger mean values of cell and nuclear sizes of the cancerous prostate cells than the normal ones, it has been shown that the morphologic parameters cannot serve as effective classifiers. In contrast, accurate classification of the two prostate cell types can be achieved with high classification accuracies on measured data acquired separately in three measurements. These results provide strong evidence that the p-DIFC method has the potential to yield morphology-related “fingerprints” for accurate and label-free classification of the two prostate cell types.

Dental erosion starts with a chemical attack on dental tissue causing tooth demineralization, altering the tooth structure and making it more sensitive to mechanical erosion. Medical diagnosis of dental erosion is commonly achieved through a visual inspection by the dentist during dental checkups and is therefore highly dependent on the operator’s experience. The detection of this disease at preliminary stages is important since, once the damage is done, cares become more complicated. We investigate the difference in light-scattering properties between healthy and eroded teeth. A change in light-scattering properties is observed and a transition from volume to surface backscattering is detected by means of polarized laser speckle imaging as teeth undergo acid etching, suggesting an increase in enamel surface roughness.

We comprehensively analyze the performance of a type of optical rotation (OR) polarimeter, which has been designed from the outset to fit the special requirements of two major applications: general chiral detection during the separation of optical isomers by high-pressure liquid chromatography systems in the pharmaceutical industry, and monitoring of glucose in the interstitial fluid of diabetics by a fully implanted long-term optical sensor. Both very demanding applications call for an OR polarimeter that can be miniaturized while maintaining high resolution and accuracy in the microdegree range in the face of considerable noise from various sources. These two characteristics—miniature size and immunity to noise—set this polarimeter apart from the traditional OR polarimeters currently in use, which are both bulky and very susceptible to noise. The following detailed analysis demonstrates the advantages of this polarimeter and its potential as an analytic and diagnostic tool.

An endoscopic image processing technique for enhancing the appearance of microstructures on translucent mucosae is described. This technique employs two pairs of co- and cross-polarization images under two different linearly polarized lights, from which the averaged subtracted polarization image (AVSPI) is calculated. Experiments were then conducted using an acrylic phantom and excised porcine stomach tissue using a manual experimental setup with ring-type lighting, two rotating polarizers, and a color camera; better results were achieved with the proposed method than with conventional color intensity image processing. An objective evaluation method that uses texture analysis was developed and used to evaluate the enhanced microstructure images. This paper introduces two types of online, rigid-type, polarimetric endoscopic implementations using a polarized ring-shaped LED and a polarimetric camera. The first type uses a beam-splitter-type color polarimetric camera, and the second uses a single-chip monochrome polarimetric camera. Microstructures on the mucosa surface were enhanced robustly with these online endoscopes regardless of the difference in the extinction ratio of each device. These results show that polarimetric endoscopy using AVSPI is both effective and practical for hardware implementation.

This paper reports a technique based on spectrally differential measurement for determining the full Mueller matrix of a biological sample through an optical fiber. In this technique, two close wavelengths were used simultaneously, one for characterizing the fiber and the other for characterizing the assembly of fiber and sample. The characteristics of the fiber measured at one wavelength were used to decouple its contribution from the measurement on the assembly of fiber and sample and then to extract sample Mueller matrix at the second wavelength. The proof of concept was experimentally validated by measuring polarimetric parameters of various calibrated optical components through the optical fiber. Then, polarimetric images of histological cuts of human colon tissues were measured, and retardance, diattenuation, and orientation of the main axes of fibrillar regions were displayed. Finally, these images were successfully compared with images obtained by a free space Mueller microscope. As the reported method does not use any moving component, it offers attractive integration possibilities with an endoscopic probe.

Polarization gating is a popular and widely used technique in biomedical optics to sense superficial tissues (colinear detection), deeper volumes (crosslinear detection), and also selectively probe subsuperficial volumes (using elliptically polarized light). As opposed to the conventional linearly polarized illumination, we propose a new protocol of polarization gating that combines coelliptical and counter-elliptical measurements to selectively enhance the contrast of the images. This new method of eliminating multiple-scattered components from the images shows that it is possible to retrieve a greater signal and a better contrast for subsurface structures. In vivo experiments were performed on skin abnormalities of volunteers to confirm the results of the subtraction method and access subsurface information.

Mueller polarimetry is applied to study the samples of nails: natural (or reference) and irradiated to 2 Gy ionizing radiation dose. We measure the whole Mueller matrices of the samples as a function of the scattering angle at a wavelength of 632.8 nm. We apply depolarization analysis to measured Mueller matrices by calculating the depolarization metrics [depolarization index, Q(M)-metric, first and second Lorenz indices, Cloude and Lorenz entropy] to quantify separability of the different samples of nails under study based on differences in their Mueller matrix. The results show that nail samples strongly depolarize the output light in backscattering, and irradiation in all cases results in increasing of depolarization. Most sensitive among depolarization metrics are the Lorenz entropy and Q(M)-metric.

Mueller matrix polarimetry and polarization-sensitive optical coherence tomography (PS-OCT) are two emerging techniques utilized in the assessment of tissue anisotropy. While PS-OCT can provide cross-sectional images of local tissue birefringence through its polarimetric sensitivity, Mueller matrix polarimetry can be used to measure bulk polarimetric properties such as depolarization, diattenuation, and retardance. To this day true quantification of PS-OCT data can be elusive, partly due to the reliance on inverse models for the characterization of tissue birefringence and the influence of instrumentation noise. Similarly for Mueller matrix polarimetry, calculation of retardance or depolarization may be influenced by tissue heterogeneities that could be monitored with PS-OCT. Here, we propose an instrument that combines Mueller matrix polarimetry and PS-OCT. Through the co-registration of the two systems, we aim at achieving a better understanding of both modalities.

The optical model of formation of polarization structure of laser radiation scattered by polycrystalline networks of human skin in Fourier plane was elaborated. The results of investigation of the values of statistical (statistical moments of the 1st to 4th order) parameters of polarization-inhomogeneous images of skin surface in Fourier plane were presented. The diagnostic criteria of pathological process in human skin and its severity degree differentiation were determined.

Immersion optical clearing makes it possible to use transmission polarized-light microscopy for characterization of thick (200 to 2000  μm) layers of biological tissues. We discuss polarization properties of thick samples in the context of the problem of characterization of collagen fiber alignment in connective tissues such as sclera and dermis. Optical chirality caused by azimuthal variations of the macroscopic (effective) optic axis of the medium across the sample thickness should be considered in polarization mapping of thick samples of these tissues. We experimentally evaluate to what extent the optical chirality affects the measurement results in typical situations and show under what conditions it can be easily taken into account and does not hinder, but rather helps, in characterization of collagen fiber alignment.

Today the increasing cancer incidence rate is becoming one of the biggest threats to human health. Among all types of cancers, liver cancer ranks in the top five in both frequency and mortality rate all over the world. During the development of liver cancer, fibrosis often evolves as part of a healing process in response to liver damage, resulting in cirrhosis of liver tissues. In a previous study, we applied the Mueller matrix microscope to pathological liver tissue samples and found that both the Mueller matrix polar decomposition (MMPD) and Mueller matrix transformation (MMT) parameters are closely related to the fibrous microstructures. In this paper, we take this one step further to quantitatively facilitate the fibrosis detections and scorings of pathological liver tissue samples in different stages from cirrhosis to cancer using the Mueller matrix microscope. The experimental results of MMPD and MMT parameters for the fibrotic liver tissue samples in different stages are measured and analyzed. We also conduct Monte Carlo simulations based on the sphere birefringence model to examine in detail the influence of structural changes in different fibrosis stages on the imaging parameters. Both the experimental and simulated results indicate that the polarized light microscope and transformed Mueller matrix parameters can provide additional quantitative information helpful for fibrosis detections and scorings of liver cirrhosis and cancers. Therefore, the polarized light microscope and transformed Mueller matrix parameters have a good application prospect in liver cancer diagnosis.

Early detection through screening plays a major role in reducing the impact of cervical cancer on patients. When detected before the invasive stage, precancerous lesions can be eliminated with very limited surgery. Polarimetric imaging is a potential alternative to the standard screening methods currently used. In a previous proof-of-concept study, significant contrasts have been found in polarimetric images acquired for healthy and precancerous regions of excised cervical tissue. To quantify the ability of the technique to differentiate between healthy and precancerous tissue, polarimetric images of seventeen cervical conization specimens (cone-shaped or cylindrical wedges from the uterine cervix) are compared with results from histopathological diagnoses, which is considered to be the “gold standard.” The sensitivity and specificity of the technique are calculated for images acquired at wavelengths of 450, 550, and 600 nm, aiming to differentiate between high-grade cervical intraepithelial neoplasia (CIN 2-3) and healthy squamous epithelium. To do so, a sliding threshold for the scalar retardance parameter was used for the sample zones, as labeled after histological diagnosis. An optimized value of ∼83% is achieved for both sensitivity and specificity for images acquired at 450 nm and for a threshold scalar retardance value of 10.6 deg. This study paves the way for an application of polarimetry in the clinic.

This tutorial-review introduces the fundamentals of polarized light interaction with biological tissues and presents some of the recent key polarization optical methods that have made possible the quantitative studies essential for biomedical diagnostics. Tissue structures and the corresponding models showing linear and circular birefringence, dichroism, and chirality are analyzed. As the basis for a quantitative description of the interaction of polarized light with tissues, the theory of polarization transfer in a random medium is used. This theory employs the modified transfer equation for Stokes parameters to predict the polarization properties of single- and multiple-scattered optical fields. The near-order of scatterers in tissues is accounted for to provide an adequate description of tissue polarization properties. Biomedical diagnostic techniques based on polarized light detection, including polarization imaging and spectroscopy, amplitude and intensity light scattering matrix measurements, and polarization-sensitive optical coherence tomography are described. Examples of biomedical applications of these techniques for early diagnostics of cataracts, detection of precancer, and prediction of skin disease are presented. The substantial reduction of light scattering multiplicity at tissue optical clearing that leads to a lesser influence of scattering on the measured intrinsic polarization properties of the tissue and allows for more precise quantification of these properties is demonstrated.

This report describes how optical images acquired using linearly polarized light can specify the anisotropy of scattering (g) and the ratio of reduced scattering [μ'_s=μ_s(1−g)] to absorption (μ_a), i.e., N′=μ'_s/μ_a. A camera acquired copolarized (HH) and crosspolarized (HV) reflectance images of a tissue (skin), which yielded images based on the intensity ( I=HH+HV) and difference (Q=HH−HV) of reflectance images. Monte Carlo simulations generated an analysis grid (or lookup table), which mapped Q and I into a grid of g versus N′, i.e., g(Q,I) and N′(Q,I). The anisotropy g is interesting because it is sensitive to the submicrometer structure of biological tissues. Hence, polarized light imaging can monitor shifts in the submicrometer (50 to 1000 nm) structure of tissues. The Q values for forearm skin on two subjects (one Caucasian, one pigmented) were in the range of 0.046±0.007 (24), which is the mean±SD for 24 measurements on 8 skin sites×3 visible wavelengths, 470, 524, and 625 nm, which indicated g values of 0.67±0.07 (24).

This research presents investigation results of the diagnostic efficiency of an azimuthally stable Mueller-matrix method of analysis of laser autofluorescence of polycrystalline films of dried uterine cavity peritoneal fluid. A model of the generalized optical anisotropy of films of dried peritoneal fluid is proposed in order to define the processes of laser autofluorescence. The influence of complex mechanisms of both phase (linear and circular birefringence) and amplitude (linear and circular dichroism) anisotropies is taken into consideration. The interconnections between the azimuthally stable Mueller-matrix elements characterizing laser autofluorescence and different mechanisms of optical anisotropy are determined. The statistical analysis of coordinate distributions of such Mueller-matrix rotation invariants is proposed. Thereupon the quantitative criteria (statistic moments of the first to the fourth order) of differentiation of polycrystalline films of dried peritoneal fluid, group 1 (healthy donors) and group 2 (uterus endometriosis patients), are determined.

In the framework of further development of a unified computational tool for the needs of biomedical optics, we introduce an electric field Monte Carlo (MC) model for simulation of backscattering of coherent linearly polarized light from a turbid tissue-like scattering medium with a rough surface. We consider the laser speckle patterns formation and the role of surface roughness in the depolarization of linearly polarized light backscattered from the medium. The mutual phase shifts due to the photons’ pathlength difference within the medium and due to reflection/refraction on the rough surface of the medium are taken into account. The validation of the model includes the creation of the phantoms of various roughness and optical properties, measurements of co- and cross-polarized components of the backscattered/reflected light, its analysis and extensive computer modeling accelerated by parallel computing on the NVIDIA graphics processing units using compute unified device architecture (CUDA). The analysis of the spatial intensity distribution is based on second-order statistics that shows a strong correlation with the surface roughness, both with the results of modeling and experiment. The results of modeling show a good agreement with the results of experimental measurements on phantoms mimicking human skin. The developed MC approach can be used for the direct simulation of light scattered by the turbid scattering medium with various roughness of the surface.

Young patients with dense breasts have a relatively low-positive biopsy rate for breast cancer (∼1 in 7). South Korean women have higher breast density than Westerners. We investigated the benefit of using a functional and metabolic imaging technique, diffuse optical spectroscopic imaging (DOSI), to help the standard of care imaging tools to distinguish benign from malignant lesions in premenopausal Korean women. DOSI uses near-infrared light to measure breast tissue composition by quantifying tissue concentrations of water (ctH₂O), bulk lipid (ctLipid), deoxygenated (ctHHb), and oxygenated (ctHbO₂) hemoglobin. DOSI spectral signatures specific to abnormal tissue and absent in healthy tissue were also used to form a malignancy index. This study included 19 premenopausal subjects (average age 41±9), corresponding to 11 benign and 10 malignant lesions. Elevated lesion to normal ratio of ctH₂O, ctHHb, ctHbO₂, total hemoglobin (THb=ctHHb+ctHbO₂), and tissue optical index (ctHHb×ctH₂O/ctLipid) were observed in the malignant lesions compared to the benign lesions (p<0.02). THb and malignancy index were the two best single predictors of malignancy, with >90% sensitivity and specificity. Malignant lesions showed significantly higher metabolism and perfusion than benign lesions. DOSI spectral features showed high discriminatory power for distinguishing malignant and benign lesions in dense breasts of the Korean population.

Various types of collagens, e.g., type I and III, represent the main load-bearing components in biological tissues. Their composition changes during processes such as wound healing and fibrosis. When excited by ultraviolet light, collagens exhibit autofluorescence distinguishable by their unique fluorescent lifetimes across a range of emission wavelengths. Here, we designed a miniaturized spectral-lifetime detection system as a noninvasive probe for monitoring tissue collagen compositions. A sine-modulated LED illumination was applied to enable frequency domain fluorescence lifetime measurements under three wavelength bands, separated via a series of longpass dichroics at 387, 409, and 435 nm. We employed a lithography-based three-dimensional (3-D) printer with <50  μm resolution to create a custom designed optomechanics in a handheld form factor. We examined the characteristics of the optomechanics with finite element modeling to simulate the effect of thermal (from LED) and mechanical (from handling) strain on the optical system. The geometry was further optimized with ray tracing to form the final 3-D printed structure. Using this device, the phase shift and demodulation of collagen types were measured, where the separate spectral bands enhanced the differentiation of their lifetimes. This system represents a low cost, handheld probe for clinical tissue monitoring applications.

The timing of the first embryonic cell divisions may predict the ability of an embryo to establish pregnancy. Similarly, metabolic profiles may be markers of embryonic viability. However, in bovine, data about the metabolomics profile of these embryos are still not available. In the present work, we describe Raman-based metabolomic profiles of culture media of bovine embryos with different developmental kinetics (fast x slow) throughout the in vitro culture. The principal component analysis enabled us to classify embryos with different developmental kinetics since they presented specific spectroscopic profiles for each evaluated time point. We noticed that bands at 1076  cm−¹ (lipids), 1300  cm−¹ (Amide III), and 2719  cm−¹ (DNA nitrogen bases) gave the most relevant spectral features, enabling the separation between fast and slow groups. Bands at 1001  cm−¹ (phenylalanine) and 2892  cm−¹ (methylene group of the polymethylene chain) presented specific patterns related to embryonic stage and can be considered as biomarkers of embryonic development by Raman spectroscopy. The culture media analysis by Raman spectroscopy proved to be a simple and sensitive technique that can be applied with high efficiency to characterize the profiles of in vitro produced bovine embryos with different development kinetics and different stages of development.

To evaluate how different amounts of induced spherical aberration (SA) affect the light distortion (LD) phenomena, tests were performed using an experimental device to measure the distortion (haloes, glare, and so on) of a point source. To simulate the effect of SA, eight different phase plates between +0.300 and −0.300  μm of SA for a 5-mm aperture were used in a random and double-masked experimental design. Measurements were performed at a distance of 2 m in a darkened room in 10 eyes of five subjects with a mean age of 26.4±6.1 years and a mean refractive error of −0.50±0.70  D. Data were obtained with natural pupil and after pupil dilatation. The measurements with this experimental system showed a significant increase in all distortion parameters with cycloplegia for the phase plates with the higher positive SA (+0.300 and +0.150  μm). The disturbance index increased from 14.86±6.12% to 57.98±36.20% (p<0.05) with the +0.300  μm plate. The same effect was observed through at a much lower rate when the eye could accommodate. Plates inducing negative SA did not change the LD compared to the control condition without induction of SA or even decreased the effect of distortion. Pupillary dilation and cyclopegia led to a significant increase in the size of the LD when increasing values of SA were induced. Accommodation and pupillary constriction are capable of compensating the degradation of the optical quality induced.

Intravascular optical coherence tomography (IV-OCT) is a high-resolution imaging method used to visualize the internal structures of walls of coronary arteries in vivo. However, accurate characterization of atherosclerotic plaques with gray-scale IV-OCT images is often limited by various intrinsic artifacts. In this study, we present an algorithm for characterizing lipid-rich plaques with a spectroscopic OCT technique based on a Gaussian center of mass (GCOM) metric. The GCOM metric, which reflects the absorbance properties of lipids, was validated using a lipid phantom. In addition, the proposed characterization method was successfully demonstrated in vivo using an atherosclerotic rabbit model and was found to have a sensitivity and specificity of 94.3% and 76.7% for lipid classification, respectively.

Fourier transform infrared (FTIR) spectroscopy technique can detect the abnormality of a cervical cell that occurs before the morphological change could be observed under the light microscope as employed in conventional techniques. This paper presents developed features extraction for an automated screening system for cervical precancerous cell based on the FTIR spectroscopy as a second opinion to pathologists. The automated system generally consists of the developed features extraction and classification stages. Signal processing techniques are used in the features extraction stage. Then, discriminant analysis and principal component analysis are employed to select dominant features for the classification process. The datasets of the cervical precancerous cells obtained from the feature selection process are classified using a hybrid multilayered perceptron network. The proposed system achieved 92% accuracy.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract, and gastric adenocarcinomas are a common cancer worldwide. To differentiate GISTs from adenocarcinomas is important because the surgical processes for both are different; the former excises the tumor with negative margins, while the latter requires radical gastrectomy with lymph node dissection. Endoscopy with biopsy is used to distinguish GISTs from adenocarcinomas; however, it may cause tumor bleeding in GISTs. We reported here the confocal Raman microspectroscopy as an effective tool to differentiate GISTs, adenocarcinomas, and normal mucosae. Of 119 patients enrolled in this study, 102 patients underwent gastrectomy (40 GISTs and 62 adenocarcinomas), and 17 patients with benign lesions were obtained as normal mucosae. Raman signals were integrated for 100 s for each spot on the specimen, and 5 to 10 spots, depending on the sample size, were chosen for each specimen. There were significant differences among those tissues as evidenced by different Raman signal responding to phospholipids and protein structures. The spectral data were further processed and analyzed by using principal component analysis. A two-dimensional plot demonstrated that GISTs, adenocarcinomas, and normal gastric mucosae could be effectively differentiated from each other.

Laser-enhanced irrigation of complex root canals appears to be a very promising technique to improve the outcome of root canal treatment. This applies, in particular, if the technique can be effective at very low laser energies in irrigating not only the main canal but also the small lateral canals. This is important in order to avoid potential undesirable effects at higher laser energies such as temperature increase, dentin ablation, or extrusion of irrigating solution beyond the apical foramen. An improved understanding of the role of laser parameters, such as laser wavelength and pulse duration, in irrigation of lateral canals is therefore desired in order to optimize treatment efficacy. The influence of laser wavelength and pulse duration on cavitation phenomena was studied using shadow photography and a method of measuring fluid flow in lateral canals based on tracking of movements of small air bubbles naturally forming in liquid as a result of laser agitation. A simulated model of a root canal including a narrow lateral canal designed to represent typical root canal morphology was used for the water flow measurements. The following three laser wavelengths with relatively high absorption in water were studied: Er:YAG (2.94 μm), Er,Cr:YSGG (2.73 μm), and Nd:YAP (1.34 μm). Among the three wavelengths studied, the Er:YAG laser wavelength was found to be the most effective in formation of cavitation bubbles and in generating fluid motions within narrow lateral canals. A comparison between the shadow photography and fluid motion data indicates that it is the bubble’s radius and not the bubble’s volume that predominantly influences the fluid motion within lateral canals. Based on the results of our study, it appears that effective minimally invasive laser-assisted irrigation can be performed with low Er:YAG laser pulse energies below 10 mJ.

Accurate segmentation of the retinal microvasculature is a critical step in the quantitative analysis of the retinal circulation, which can be an important marker in evaluating the severity of retinal diseases. As manual segmentation remains the gold standard for segmentation of optical coherence tomography angiography (OCT-A) images, we present a method for automating the segmentation of OCT-A images using deep neural networks (DNNs). Eighty OCT-A images of the foveal region in 12 eyes from 6 healthy volunteers were acquired using a prototype OCT-A system and subsequently manually segmented. The automated segmentation of the blood vessels in the OCT-A images was then performed by classifying each pixel into vessel or nonvessel class using deep convolutional neural networks. When the automated results were compared against the manual segmentation results, a maximum mean accuracy of 0.83 was obtained. When the automated results were compared with inter and intrarater accuracies, the automated results were shown to be comparable to the human raters suggesting that segmentation using DNNs is comparable to a second manual rater. As manually segmenting the retinal microvasculature is a tedious task, having a reliable automated output such as automated segmentation by DNNs, is an important step in creating an automated output.

We are developing a label-free nanoscale photoacoustic tomography (nPAT) for imaging a single living cell. nPAT uses a laser-induced acoustic pulse to generate a nanometer-scale image. The primary motivation behind this imaging technique is the imaging of biological cells in the context of diagnosis without fluorescent tagging. During this procedure, thermal damage due to the laser pulse is a potential risk that may damage the cells. A physical model is built to estimate the temperature rise and thermal relaxation during the imaging procedure. Through simulations using finite element methods, two lasers (532 nm at 5 ps pulse duration and 830 nm at 0.2 ps pulse duration) were simulated for imaging red blood cells (RBCs). We demonstrate that a single 5-ps pulse laser with a 400-Hz repetition rate will generate a steady state temperature rise of less than a Kelvin on the surface of the RBCs. All the simulation results show that there is no significant temperature rise in an RBC in either single pulse or multiple pulse illumination with a 532-nm laser with 219 W fluence. Therefore, our simulation results demonstrate the thermal safety of an nPAT system. The photoacoustic signal generated by this laser is on the order of 2.5 kPa, so it should still be large enough to generate high-resolution images with nPAT. Frequency analysis of this signal shows a peak at 1.47 GHz, with frequencies as high as 3.5 GHz still being present in the spectrum. We believe that nPAT will open an avenue for disease diagnosis and cell biology studies at the nanometer-level.

The analysis of biological systems by spectroscopic techniques involves the evaluation of hundreds to thousands of variables. Hence, different statistical approaches are used to elucidate regions that discriminate classes of samples and to propose new vibrational markers for explaining various phenomena like disease monitoring, mechanisms of action of drugs, food, and so on. However, the technical statistics are not always widely discussed in applied sciences. In this context, this work presents a detailed discussion including the various steps necessary for proper statistical analysis. It includes univariate parametric and nonparametric tests, as well as multivariate unsupervised and supervised approaches. The main objective of this study is to promote proper understanding of the application of various statistical tools in these spectroscopic methods used for the analysis of biological samples. The discussion of these methods is performed on a set of in vivo confocal Raman spectra of human skin analysis that aims to identify skin aging markers. In the Appendix, a complete routine of data analysis is executed in a free software that can be used by the scientific community involved in these studies.

The goal of this work was to objectively characterize the external morphology, topography, and optics of the cornea after orthokeratology (ortho-k). A number of 24 patients between the ages of 17 and 30 years (median=24  years) were fitted with Corneal Refractive Therapy® contact lenses to correct myopia between −2.00 and −5.00 diopters (D) (median=−3.41  D). A classification algorithm was applied to conduct an automatic segmentation based on the mean local curvature. As a result, three zones (optical zone, transition zone, and peripheral zone) were delimited. Topographical analysis was provided through global and zonal fit to a general ellipsoid. Ray trace on partially customized eye models provided wave aberrations and retinal image quality. Monozone topographic description of the ortho-k cornea loses accuracy when compared with zonal description. Primary (C₄⁰) and secondary (C₆⁰) spherical aberration (SA) coefficients for a 5-mm pupil increased 3.68 and 19 times, respectively, after the treatments. The OZ area showed a strong correlation with C₄⁰ (r=−0.49, p<0.05) and a very strong correlation with C₆⁰ (r=0.78, p<0.01). The OZ, as well as the TZ, areas did not correlate with baseline refraction. The increase in the eye’s positive SA after ortho-k is the major factor responsible for the decreased retinal optical quality of the unaccommodated eye.

We evaluated the damage caused to optically trapped red blood cells (RBCs) after 1 or 2 min of exposure to near-infrared (NIR) laser beams at 785 or 1064 nm. Damage was quantified by measuring cell elasticity using an automatic, real-time, homemade, optical tweezer system. The measurements, performed on a significant number (hundreds) of cells, revealed an overall deformability decrease up to ∼104% after 2 min of light exposure, under 10 mW optical trapping for the 785-nm wavelength. Wavelength dependence of the optical damage is attributed to the light absorption by hemoglobin. The results provided evidence that RBCs have their biomechanical properties affected by NIR radiation. Our findings establish limits for laser applications with RBCs.

Fluorescence lifetime in heterogeneous multiple light scattering systems is analyzed by an algorithm without solving the diffusion or radiative transfer equations. The algorithm assumes that the optical properties of medium are constant in the excitation and emission wavelength regions. If the assumption is correct and the fluorophore is a single species, the fluorescence lifetime can be determined by a set of measurements of temporal point-spread function of the excitation light and fluorescence at two different concentrations of the fluorophore. This method is not dependent on the heterogeneity of the optical properties of the medium as well as the geometry of the excitation–detection on an arbitrary shape of the sample. The algorithm was validated by an indocyanine green fluorescence in phantom measurements and demonstrated by an in vivo measurement.

A blockage of the middle cerebral artery (MCA) on the cortical branch will seriously affect the blood supply of the cerebral cortex. Real-time monitoring of MCA hemodynamic parameters is critical for therapy and rehabilitation. Optical coherence tomography (OCT) is a powerful imaging modality that can produce not only structural images but also functional information on the tissue. We use OCT to detect hemodynamic changes after MCA branch occlusion. We injected a selected dose of endothelin-1 (ET-1) at a depth of 1 mm near the MCA and let the blood vessels follow a process first of occlusion and then of slow reperfusion as realistically as possible to simulate local cerebral ischemia. During this period, we used optical microangiography and Doppler OCT to obtain multiple hemodynamic MCA parameters. The change trend of these parameters from before to after ET-1 injection clearly reflects the dynamic regularity of the MCA. These results show the mechanism of the cerebral ischemia-reperfusion process after a transient middle cerebral artery occlusion and confirm that OCT can be used to monitor hemodynamic parameters.

Unlike laser Doppler flowmetry, there has yet to be presented a clear description of the physical variables that laser speckle contrast imaging (LSCI) is sensitive to. Herein, we present a theoretical basis for demonstrating that LSCI is sensitive to total flux and, in particular, the summation of diffusive flux and advective flux. We view LSCI from the perspective of mass transport and briefly derive the diffusion with drift equation in terms of an LSCI experiment. This equation reveals the relative sensitivity of LSCI to both diffusive flux and advective flux and, thereby, to both concentration and the ordered velocity of the scattering particles. We demonstrate this dependence through a short series of flow experiments that yield relationships between the calculated speckle contrast and the concentration of the scatterers (manifesting as changes in scattering coefficient), between speckle contrast and the velocity of the scattering fluid, and ultimately between speckle contrast and advective flux. Finally, we argue that the diffusion with drift equation can be used to support both Lorentzian and Gaussian correlation models that relate observed contrast to the movement of the scattering particles and that a weighted linear combination of these two models is likely the most appropriate model for relating speckle contrast to particle motion.

Several variables may affect the local contrast values in laser speckle contrast imaging (LSCI), irrespective of relative motion. It has been suggested that the optical properties of the moving fluid and surrounding tissues can affect LSCI values. However, a detailed study of this has yet to be presented. In this work, we examined the combined effects of the reduced scattering and absorption coefficients on LSCI. This study employs fluid phantoms with different optical properties that were developed to mimic whole blood with varying hematocrit levels. These flow phantoms were imaged with an LSCI system developed for this study. The only variable parameter was the optical properties of the flowing fluid. A negative linear relationship was seen between the changes in contrast and changes in reduced scattering coefficient, absorption coefficient, and total attenuation coefficient. The change in contrast observed due to an increase in the scattering coefficient was greater than what was observed with an increase in the absorption coefficient. The results indicate that optical properties affect contrast values and that they should be considered in the interpretation of LSCI data.

Objective measurement of straylight in the human eye with a Shack–Hartmann (SH) wavefront aberrometer is limited in imaging angle. We propose a measurement principle and a point spread function (PSF) reconstruction algorithm to overcome this limitation. In our optical setup, a variable stop replaces the stop conventionally used to suppress reflections and scatter in SH aberrometers. We record images with 21 diameters of the stop. From each SH image, the average intensity of the pupil is computed and normalized. The intensities represent integral values of the PSF. We reconstruct the PSF, which is the derivative of the intensities with respect to the visual angle. A modified Stiles Holladay approximation is fitted to the reconstructed PSF, resulting in a straylight parameter. A proof-of-principle study was carried out on eight healthy young volunteers. Scatter filters were positioned in front of the volunteers’ eyes to simulate straylight. The straylight parameter was compared to the C-Quant measurements and the filter values. The PSF parameter shows strong correlation with the density of the filters and a linear relation to the C-Quant straylight parameter. Our measurement and reconstruction techniques allow for objective straylight analysis of visual angles up to 4 deg.

Noninvasive cell analyses are increasingly important in the medical field. A coherent anti-Stokes Raman scattering (CARS) microscope is the noninvasive imaging equipment and enables to obtain images indicating molecular distribution. However, due to low-signal intensity, it is still challenging to obtain images of the fingerprint region, in which many spectrum peaks correspond to compositions of a cell. Here, to identify cell differentiation by using multiplex CARS, we investigated hyperspectral imaging of the fingerprint region of living cells. To perform multiplex CARS, we used a prototype of a compact light source generating both pump light and broadband Stokes light. Assuming application to regenerative medicine, we chose a cartilage cell, whose differentiation is difficult to be identified by change of the cell morphology. Because one of the major components of cartilage is collagen, we focused on distribution of proline, which accounts for approximately 20% of collagen. The spectrum quality was improved by optical adjustments of the power branching ratio and divergence of Stokes light. Periphery of a cartilage cell was highlighted in a CARS image of proline, and this result suggests correspondence with collagen generated as an extracellular matrix. The possibility of noninvasive analyses by using CARS hyperspectral imaging was indicated.

A method for depth enhancement is presented using a bidirectional imaging modality for spectral domain optical coherence tomography (SD-OCT). Two precisely aligned sample arms along with two reference arms were utilized in the optical configuration to scan the samples. Using exemplary images of the optical resolution target, Scotch tape, a silicon sheet with two needles, and a leaf, we demonstrated how the developed bidirectional SD-OCT imaging method increases the ability to characterize depth-enhanced images. The results of the developed system were validated by comparing the images with the standard OCT configuration (single-sample arm setup). Given the advantages of higher resolution and the ability to visualize deep morphological structures, this method can be utilized to increase the depth dependent fall-off in samples with limited thickness. Thus, the proposed bidirectional imaging modality is apt for cross-sectional imaging of entire samples, which has the potential capability to improve the diagnostic ability.

Molecular imaging techniques are becoming increasingly important in biomedical research and potentially in clinical practice. We present a continuous-terahertz (THz)-wave molecular imaging system for biomedical applications, in which an infrared (IR) laser is integrated into a 0.2-THz reflection-mode continuous-THz-wave imaging system to induce surface plasmon polaritons on the nanoparticles and further improve the intensity of the reflected signal from the water around the nanoparticles. A strong and rapid increment of the reflected THz signal in the nanoparticle solution upon the IR laser irradiation is demonstrated, using either gold or silver nanoparticles. This low-cost, simple, and stable continuous-THz-wave molecular imaging system is suitable for miniaturization and practical imaging applications; in particular, it shows great promise for cancer diagnosis and nanoparticle drug-delivery monitoring.

Photoacoustic computed tomography (PACT) has emerged as a unique and promising technology for multiscale biomedical imaging. To fully realize its potential for various preclinical and clinical applications, development of systems with high imaging speed, reasonable cost, and manageable data flow are needed. Sparse-sampling PACT with advanced reconstruction algorithms, such as compressed-sensing reconstruction, has shown potential as a solution to this challenge. However, most such algorithms require iterative reconstruction and thus intense computation, which may lead to excessively long image reconstruction times. Here, we developed a principal component analysis (PCA)-based PACT (PCA-PACT) that can rapidly reconstruct high-quality, three-dimensional (3-D) PACT images with sparsely sampled data without requiring an iterative process. In vivo images of the vasculature of a human hand were obtained, thus validating the PCA-PACT method. The results showed that, compared with the back-projection (BP) method, PCA-PACT required ∼50% fewer measurements and ∼40% less time for image reconstruction, and the imaging quality was almost the same as that for BP with full sampling. In addition, compared with compressed sensing-based PACT, PCA-PACT had approximately sevenfold faster imaging speed with higher imaging accuracy. This work suggests a promising approach for low-cost, 3-D, rapid PACT for various biomedical applications.

Speckle artifacts can strongly hamper quantitative analysis of optical coherence tomography (OCT), which is necessary to provide assessment of ocular disorders associated with vision loss. Here, we introduce a method for speckle reduction, which leverages from low-rank + sparsity decomposition (LRpSD) of the logarithm of intensity OCT images. In particular, we combine nonconvex regularization-based low-rank approximation of an original OCT image with a sparsity term that incorporates the speckle. State-of-the-art methods for LRpSD require a priori knowledge of a rank and approximate it with nuclear norm, which is not an accurate rank indicator. As opposed to that, the proposed method provides more accurate approximation of a rank through the use of nonconvex regularization that induces sparse approximation of singular values. Furthermore, a rank value is not required to be known a priori. This, in turn, yields an automatic and computationally more efficient method for speckle reduction, which yields the OCT image with improved contrast-to-noise ratio, contrast and edge fidelity. The source code will be available at www.mipav.net/English/research/research.html.

Morphology and distribution of melanocytes are critical imaging information for the diagnosis of melanocytic lesions. However, how to image intratumoral melanocytes noninvasively in pigmented skin tumors is seldom investigated. Third-harmonic generation (THG) is shown to be enhanced by melanin, whereas high accuracy has been demonstrated using THG microscopy for in vivo differential diagnosis of nonmelanocytic pigmented skin tumors. It is thus desirable to investigate if label-free THG microscopy was capable to in vivo identify intratumoral melanocytes. In this study, histopathological correlations of label-free THG images with the immunohistochemical images stained with human melanoma black (HMB)-45 and cluster of differentiation 1a (CD1a) were made. The correlation results indicated that the intratumoral THG-bright dendritic-cell-like signals were endogenously derived from melanocytes rather than Langerhans cells (LCs). The consistency between THG-bright dendritic-cell-like signals and HMB-45 melanocyte staining showed a kappa coefficient of 0.807, 84.6% sensitivity, and 95% specificity. In contrast, a kappa coefficient of −0.37, 21.7% sensitivity, and 30% specificity were noted between the THG-bright dendritic-cell-like signals and CD1a staining for LCs. Our study indicates the capability of noninvasive label-free THG microscopy to differentiate intratumoral melanocytes from LCs, which is not feasible in previous in vivo label-free clinical-imaging modalities.

Quantification of choroidal neovascularization (CNV) as visualized by optical coherence tomography angiography (OCTA) may have importance clinically when diagnosing or tracking disease. Here, we present an automated algorithm to quantify the vessel skeleton of CNV as vessel length. Initial segmentation of the CNV on en face angiograms was achieved using saliency-based detection and thresholding. A level set method was then used to refine vessel edges. Finally, a skeleton algorithm was applied to identify vessel centerlines. The algorithm was tested on nine OCTA scans from participants with CNV and comparisons of the algorithm’s output to manual delineation showed good agreement.

Superresolution localization microscopy initially produces a dataset of fluorophore coordinates instead of a conventional digital image. Therefore, superresolution localization microscopy requires additional data analysis to present a final superresolution image. However, methods of employing the structural information within the localization dataset to improve the data analysis performance remain poorly developed. Here, we quantify the structural information in a localization dataset using structural anisotropy, and propose to use it as a figure of merit for localization event filtering. With simulated as well as experimental data of a biological specimen, we demonstrate that exploring structural anisotropy has allowed us to obtain superresolution images with a much cleaner background.

The attenuation of near-infrared (NIR) light intensity as it propagates in a turbid medium like biological tissue is described by modified the Beer–Lambert law (MBLL). The MBLL is generally used to quantify the changes in tissue chromophore concentrations for NIR spectroscopic data analysis. Even though MBLL is effective in terms of providing qualitative comparison, it suffers from its applicability across tissue types and tissue dimensions. In this work, we introduce Lambert-W function-based modeling for light propagation in biological tissues, which is a generalized version of the Beer–Lambert model. The proposed modeling provides parametrization of tissue properties, which includes two attenuation coefficients μ₀ and η. We validated our model against the Monte Carlo simulation, which is the gold standard for modeling NIR light propagation in biological tissue. We included numerous human and animal tissues to validate the proposed empirical model, including an inhomogeneous adult human head model. The proposed model, which has a closed form (analytical), is first of its kind in providing accurate modeling of NIR light propagation in biological tissues.

Polarization-sensitive optical coherence tomography (PS-OCT) is a light-based, high-resolution, real-time, noninvasive, and nondestructive imaging modality yielding quasimicroscopic cross-sectional images of cartilage. As yet, comprehensive parameterization and quantification of birefringence and tissue properties have not been performed on human cartilage. PS-OCT and algorithm-based image analysis were used to objectively grade human cartilage degeneration in terms of surface irregularity, tissue homogeneity, signal attenuation, as well as birefringence coefficient and band width, height, depth, and number. Degeneration-dependent changes were noted for the former three parameters exclusively, thereby questioning the diagnostic value of PS-OCT in the assessment of human cartilage degeneration.

Gas microbubbles (MBs) are investigated as intravascular optical coherence tomography (OCT) contrast agents. Agar + intralipid scattering tissue phantoms with two embedded microtubes were fabricated to model vascular blood flow. One was filled with human blood, and the other with a mixture of human blood + MB. Swept-source structural and speckle variance (sv) OCT images, as well as speckle decorrelation times, were evaluated under both no-flow and varying flow conditions. Faster decorrelation times and higher structural and svOCT image contrasts were detected in the presence of MB in all experiments. The effects were largest in the svOCT imaging mode, and uniformly diminished with increasing flow velocity. These findings suggest the feasibility of utilizing MB for tissue hemodynamic investigations and for microvasculature contrast enhancement in OCT angiography.

We introduce a metric in graph search and demonstrate its application for segmenting retinal optical coherence tomography (OCT) images of macular pathology. Our proposed “adjusted mean arc length” (AMAL) metric is an adaptation of the lowest mean arc length search technique for automated OCT segmentation. We compare this method to Dijkstra’s shortest path algorithm, which we utilized previously in our popular graph theory and dynamic programming segmentation technique. As an illustrative example, we show that AMAL-based length-adaptive segmentation outperforms the shortest path in delineating the retina/vitreous boundary of patients with full-thickness macular holes when compared with expert manual grading.

We demonstrate recovery of optical properties using arrays of interstitial cylindrical diffusing fibers as sources and detectors. A single 1-cm diffuser delivered laser illumination at 665 nm, while seven 1- and 2-cm diffusers at 1-cm grid spacing acted as detectors. Extraction of optical properties from these measurements was based upon a diffusion model of emission and detection distributions for these diffuser fibers, informed by previous measurements of heterogeneous axial detection. Verification of the technique was performed in 15 liquid tissue-simulating phantoms consisting of deionized water, India ink as absorber, and Intralipid 20% as scatterer. For the range of optical properties tested, mean errors were 4.4% for effective attenuation coefficient, 12.6% for absorption coefficient, and 7.6% for reduced scattering coefficient. Error in recovery tended to increase with decreasing transport albedo. For therapeutic techniques involving the delivery of light to locations deep within the body, such as interstitial photodynamic and photothermal therapies, the methods described here would allow the treatment diffuser fibers also to be used as sources and detectors for recovery of optical properties. This would eliminate the need for separately inserted fibers for spectroscopy, reducing clinical complexity and improving the accuracy of treatment planning.

The fluorescent dye indocyanine green (ICG) is clinically approved and has been applied for ophthalmic and intraoperative angiography, measurement of cardiac output and liver function, or as contrast agent in cancer surgery. Though ICG is known for its photochemical effects, it has played a minor role so far in photodynamic therapy or techniques for targeted protein-inactivation. Here, we investigated ICG as an antibody-conjugate for the selective inactivation of the protein Ki-67 in the nucleus of cells. Conjugates of the Ki-67 antibody TuBB-9 with different amounts of ICG were synthesized and delivered into HeLa and OVCAR-5 cells through conjugation to the nuclear localization sequence. Endosomal escape of the macromolecular antibodies into the cytoplasm was optically triggered by photochemical internalization with the photosensitizer BPD. The second light irradiation at 690 nm inactivated Ki-67 and subsequently caused cell death. Here, we show that ICG as an antibody-conjugate can be an effective photosensitizing agent. Best effects were achieved with 1.8 ICG molecules per antibody. Conjugated to antibodies, the ICG absorption peaks vary proportionally with concentration. The absorption of ICG above 650 nm within the optical window of tissue opens the possibility of selective Ki-67 inactivation deep inside of tissues.

Activation of sonosensitizers via focused ultrasound (FUS), i.e., sonodynamic therapy has been proposed as an extension to light-activated photodynamic therapy for the treatment of brain as well as other tumors. The use of FUS, as opposed to light, allows treatment to tumor sites buried deep within tissues as well as through the intact skull. We have examined ultrasonic activation of sonosensitizers together with the anticancer agent bleomycin (BLM), i.e., sonochemical internalization (SCI). SCI is a technique that utilizes FUS for the enhanced delivery of endo-lysosomal trapped macromolecules into the cell cytoplasm in a similar manner to light-based photochemical internalization. The released agent can, therefore, exert its full biological activity, in contrast to being degraded by lysosomal hydrolases. Our results indicate that, compared to drug or FUS treatment alone, FUS activation of the sonosensitizer AlPcS_2a together with BLM significantly inhibits the ability of treated glioma cells to grow as three-dimensional tumor spheroids in vitro.