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Significance5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) fluorescence is currently used for image-guided glioma resection. Typically, this widefield imaging method highlights the bulk of high-grade gliomas, but it underperforms at the infiltrating edge where PpIX fluorescence is not visible to the eyes. Fluorescence lifetime imaging (FLIm) has the potential to detect PpIX fluorescence below the visible detection threshold. Moreover, simultaneous acquisition of time-resolved nicotinamide adenine (phosphate) dinucleotide [NAD(P)H] fluorescence may provide metabolic information from the tumor environment to further improve overall tumor detection.AimWe investigate the ability of pulse sampling, fiber-based FLIm to simultaneously image PpIX and NAD(P)H fluorescence of glioma infiltrative margins in patients.ApproachA mesoscopic fiber-based point-scanning FLIm device (355nm pulses) was used to simultaneously resolve the fluorescence decay of PpIX (629/53nm) and NAD(P)H (470/28nm). The FLIm device enabled data acquisition at room light and rapid (

Significance: Mechanical assessment of the cornea can provide important structural and functional information regarding its health. Current clinically available tools are limited in their efficacy at measuring corneal mechanical properties. Elastography allows for the direct estimation of mechanical properties of tissues in vivo but is generally performed using external excitation force. Aim: To show that heartbeat optical coherence elastography (Hb-OCE) can be used to assess the mechanical properties of the cornea in vivo. Approach: Hb-OCE was utilized to detect Hb-induced deformations in the rabbit cornea in vivo without the need for external excitation. Furthermore, we demonstrate how this technique can distinguish corneal stiffness between untreated (UT) and crosslinked (CXL) tissue. Results: Our results demonstrate that stiffness changes in the cornea can be detected using only the Hb-induced deformations in the cornea. Additionally, we demonstrate a statistically significant difference in strain between the UT and CXL corneas. Conclusions: Hb-OCE may be an effective tool for assessing the mechanical properties of the cornea in vivo without the need for external excitation. This tool may be effective for clinical assessment of corneal mechanical properties because it only requires optical coherence tomography imaging and data processing.

Significance: Common-path interferometers have the advantage of producing ultrastable interferometric fringes compared with conventional interferometers, such as Michelson or Mach–Zehnder that are sensitive to environmental instabilities. Isolating interferometric measurements from mechanical disturbances is important in biodynamic imaging because Doppler spectroscopy of intracellular dynamics requires extreme stability for phase-sensitive interferometric detection to capture fluctuation frequencies down to 10 mHz. Aim: The aim of this study was to demonstrate that Doppler spectra produced from a common-path interferometer using a grating and a spatial filter (SF) are comparable to, and more stable than, spectra from conventional biodynamic imaging. Approach: A common-path interferometer using a holographic diffraction grating and an SF was employed with a low-coherence source. Simulations evaluated the spatial resolution. DLD-1 (human colon adenocarcinoma) spheroids were used as living target tissue samples. Power spectra under external vibrations and drug-response spectrograms were compared between common-path and Fourier-domain holographic systems. Results: The common-path holography configuration shows enhanced interferometric stability against mechanical vibrations through common-mode rejection while maintaining sensitivity to Doppler frequency fluctuations caused by intracellular motions. Conclusions: A common-path interferometer using a grating and an SF can provide enhanced interferometric stability in tissue-dynamics spectroscopy for drug screening assays.

Significance: Near-infrared (NIR) fluorescence imaging using indocyanine green (ICG) has proven to be a feasible application for real-time intraoperative assessment of tissue perfusion, although quantification of NIR fluorescence signals is pivotal for standardized assessment of tissue perfusion.Aim: Four patients are described with possible compromised bowel perfusion after mesenteric resection. Based on these patients we want to emphasize the difficulties in the quantification of NIR fluorescence imaging for perfusion analysis.Approach: During image-guided fluorescence assessment, 5 mg of ICG (2.5 mg/ml) was intravenously administered by the anesthesiologist. NIR fluorescence imaging was done with the open camera system of Quest Medical Imaging. Fluorescence data taken from the regions of interest (bowel at risk, transition zone of bowel at risk and adjacent normally perfused bowel, and normally perfused reference bowel) were quantitatively analyzed after surgery for fluorescence intensity-and perfusion time-related parameters.Results: Bowel perfusion, as assessed clinically by independent surgeons based on NIR fluorescence imaging, resulted in different treatment strategies, three with excellent clinical outcome, but one with a perfusion related complication. Post-surgery quantitative analysis of fluorescence dynamics showed different patterns in the affected bowel segment compared to the unaffected reference segments for the four patients.Conclusions: Similar intraoperative fluorescence results could lead to different surgical treatment strategies, which demonstrated the difficulties in interpretation of uncorrected fluorescence signals. Real-time quantification and standardization of NIR fluorescence perfusion imaging could probably aid surgeons in the nearby future. (C) The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License.

Significance: Photothermal optical coherence tomography (PT-OCT) has the promise to offer structural images coregistered with chemical composition information, which can offer a significant impact in early detection of diseases such as atherosclerosis. Aim: We take the first step in understanding the relation between PT-OCT signals and the endogenous tissue composition by considering the interplay between the opto-thermo-physical properties of tissue as a function of its lipid composition and the ensuing effects on the PT-OCT signals. Approach: Multiparameter theoretical estimates for PT-OCT signal as a function of composition in a two-component lipid–water model are derived and discussed. Experimental data from various concentrations of lipid in the form of droplets and injections under bovine cardiac muscle align with theoretical predictions. Results: Theoretical and experimental results suggest that the variations of heat capacity and mass density with tissue composition significantly contribute to the amount of optical path length difference measured by OCT phase. Conclusion: PT-OCT has the potential to offer key insights into the chemical composition of the subsurface lipid pools in tissue; however, the interpretation of results needs to be carried out by keeping the nonlinear interplay between the tissue of opto-thermo-physical properties and PT-OCT signals in mind.

Significance: Melanocytic nevi represent the most common dermal melanocytic lesions in humans. Nevus is typically diagnosed clinically with the naked eye or with dermoscopy. However, it is essential to identify the type of nevus by invasive biopsy for histopathological examination. The use of noninvasive imaging tools can be used to evaluate the types of nevi to reduce unnecessary excisions of benign entities. Aim: To evaluate the feasibility of using en face and cross-sectional full-field optical coherence tomography (FF-OCT) in differentiation of melanocytic nevi that can facilitate the reduction of unnecessary excisions of benign entities. Approach: Dual-mode Mirau-type FF-OCT for cross-sectional imaging (B-scan) and en face imaging were used to distinguish the types of nevi. Results: Although the B-scan reveals the distribution of melanosomes, users can set a specific depth of the en face image to explore the morphology of surrounding skin cells instantly. According to the locations of nevus nests, the different types of nevi, including junction nevus and compound nevus, can be identified using this dual-mode FF-OCT system. Conclusions: Combining B-scan and en face imaging in vivo FF-OCT enables the examination and navigation of skin tissues in real time and in three dimensions.

Significance: The application of near-infrared spectroscopy (NIRS) to determine the concentrations of tissue chromophores has typically relied on three alternative technological approaches: continuous-wave, frequency-domain, and time-domain. It is often the case that uncertain and variable coupling of light into and out of the skin surface renders absolute measurements unreliable, and NIRS methods are mostly used to measure changes of chromophore concentrations and of physiological parameters such as blood volume and oxygenation. Aim: The aim has been to investigate whether an approach based on a wavelength-modulated source may enable measurements to be acquired, which are independent of surface coupling and may facilitate derivation of absolute values of tissue parameters. Approach: An analysis is performed using the modified Beer–Lambert law. Results: It is shown that the relative modulation in detected intensity resulting from a wavelength-modulated source could be used to estimate absolute concentrations of chromophores if unknown surface coupling losses and geometrical factors are insensitive to small changes in wavelength. Conclusions: Wavelength modulated NIRS could be an effective tool for quantitative in vivo analysis of tissues, although it may be technically challenging.

Significance: Optical coherence tomography (OCT) has been widely used in clinical studies. However, the image quality of OCT decreases with increasing imaging depth since the light is rapidly attenuated in biological tissues. Aim: We present a compensation approach to preserve weak high-frequency signals from deep structures and compress the dynamic range of the detected signal for superior analog-to-digital conversion and image display capability. Approach: A homemade frequency gain compensation amplifier is designed and fabricated to amplify the electrical signal from a balanced photodetector and compensate for the signal attenuation in swept-source OCT (SSOCT). Results: It is demonstrated in imaging various objects that this cost-efficient technique effectively enhances the contrast of the deep tissue image. Conclusions: A frequency gain compensation amplifier is designed and used to compress the dynamic range of the electrical signal detected by the photodetector of an SSOCT system, which enables weak signals from deep structures to be acquired by the ADC and displayed with enhanced local contrast.

Significance: Visible light optical coherence tomography (OCT) is emerging for spectroscopic and ultrahigh resolution imaging, but challenges remain. Depth-dependent dispersion limits retinal image quality and current correction approaches are cumbersome. Inconsistent group refractive indices during image reconstruction also limit reproducibility. Aim: To introduce and evaluate water wavenumber calibration (WWC), which corrects depth-dependent dispersion and provides an accurate depth axis in water. Approach: Enabled by a visible light OCT spectrometer configuration with a 3- to 4-dB sensitivity roll-off over 1 mm in air across a 90-nm bandwidth, we determine the spectral phase of a 1-mm water cell, an affine function of water wavenumber. Via WWC, we reconstruct visible light OCT human retinal images with 1.3-μm depth resolution in water. Results: Images clearly reveal Bruch’s membrane, inner plexiform layer lamination, and a thin nerve fiber layer in the temporal parafovea. WWC halves the processing time, while achieving the same image definition as an assumption-free gold standard approach, suggesting that water wavenumber is a suitable proxy for tissue wavenumber. WWC also provides a depth axis in water without explicitly assuming a group refractive index. Conclusions: WWC is a simple method that helps to realize the full potential of visible light OCT.

Significance: The retina is critical for vision, and several diseases may alter its biomechanical properties. However, assessing the biomechanical properties of the retina nondestructively is a challenge due to its fragile nature and location within the eye globe. Advancements in Brillouin spectroscopy have provided the means for nondestructive investigations of retina biomechanical properties. Aim: We assessed the biomechanical properties of mouse retinas using Brillouin microscopy noninvasively and showed the potential of Brillouin microscopy to differentiate the type and layers of retinas based on stiffness. Approach: We used Brillouin microscopy to quantify stiffness of fresh and paraformaldehyde (PFA)-fixed retinas. As further proof-of-concept, we demonstrated a change in the stiffness of a retina with N-methyl-D-aspartate (NMDA)-induced damage, compared to an undamaged sample. Results: We found that the retina layers with higher cell body density had higher Brillouin modulus compared to less cell-dense layers. We have also demonstrated that PFA-fixed retina samples were stiffer compared with fresh samples. Further, NMDA-induced neurotoxicity leads to retinal ganglion cell (RGC) death and reactive gliosis, increasing the stiffness of the RGC layer. Conclusion: Brillouin microscopy can be used to characterize the stiffness distribution of the layers of the retina and can be used to differentiate tissue at different conditions based on biomechanical properties.

Significance: Photoacoustic (PA) tomography has demonstrated versatile biomedical applications. However, an array-based PA computed tomography (PACT) system is complex and expensive, whereas a single-element detector-based scanning PA system is too slow to detect some fast biological dynamics in vivo. New PA imaging methods are sought after. Aim: To overcome these limitations, we developed photoacoustic topography through an ergodic relay (PATER), a novel high-speed imaging system with a single-element detector. Approach: PATER images widefield PA signals encoded by the acoustic ergodic relay with a single-laser shot. Results: We applied PATER in vivo to monitor changes in oxygen saturation in a mouse brain and also to demonstrate high-speed matching of vascular patterns for biometric authentication. Conclusions: PATER has achieved a high-speed temporal resolution over a large field of view. Our results suggest that PATER is a promising and economical alternative to PACT for fast imaging.

Significance: Hyperspectral microscopy has been intensively explored in biomedical applications. However, due to its huge three-dimensional hyperspectral data cube, it typically suffers from slow data acquisition, mass data transmission and storage, and computationally expensive postprocessing. Aim: To overcome the above limitations, a programmable hyperspectral microscopy technique was developed, which can perform hardware-based hyperspectral data postprocessing by the physical process of optical imaging in a snapshot. Approach: A programmable hyperspectral microscopy system was developed to collect coded microscopic images from samples under multiplexed illumination. Principal component analysis followed by linear discriminant analysis scheme was coded into multiplexed illumination and realized by the physical process of optical imaging. The contrast enhancement was evaluated on two representative types of microscopic samples, i.e., tissue section and cell samples. Results: Compared to the microscopic images collected under white light illumination, the contrasts of coded microscopic images were significantly improved by 41% and 59% for tissue section and cell samples, respectively. Conclusions: The proposed method can perform hyperspectral data acquisition and postprocessing simultaneously by its physical process, while preserving the most important spectral information to maximize the difference between the target and background, thus opening a new avenue for high-contrast microscopic imaging in a snapshot.

The potential to image subsurface fluorescent contrast agents at high spatial resolution has facilitated growing interest in short-wave infrared (SWIR) imaging for biomedical applications. The early but growing literature showing improvements in resolution in small animal models suggests this is indeed the case, yet to date, images from larger animal models that more closely recapitulate humans have not been reported. We report the first imaging of SWIR fluorescence in a large animal model. Specifically, we imaged the vascular kinetics of an indocyanine green (ICG) bolus injection during open craniotomy of a mini-pig using a custom SWIR imaging instrument and a clinical-grade surgical microscope that images ICG in the near-infrared-I (NIR-I) window. Fluorescence images in the SWIR were observed to have higher spatial and contrast resolutions throughout the dynamic sequence, particularly in the smallest vessels. Additionally, vessels beneath a surface pool of blood were readily visualized in the SWIR images yet were obscured in the NIR-I channel. These first-in-large-animal observations represent an important translational step and suggest that SWIR imaging may provide higher spatial and contrast resolution images that are robust to the influence of blood.

We present three-dimensional (3-D) high-resolution spectral-domain optical coherence microscopy (SD-OCM) by using a supercontinuum (SC) fiber laser source with 300-nm spectral bandwidth (full-width at half-maximum) in the 1700-nm spectral band. By using low-coherence interferometry with SC light and a confocal detection scheme, we realized lateral and axial resolutions of 3.4 and 3.8 μm in tissue (n=1.38), respectively. This is, to the best of our knowledge, the highest 3-D spatial resolution reported among those of Fourier-domain optical coherence imaging techniques in the 1700-nm spectral band. In our SD-OCM, to enhance the imaging depth, a full-range method was implemented, which suppressed the formation of a coherent ghost image and allowed us to set the zero-delay position inside the samples. We demonstrated the 3-D high-resolution imaging capability of 1700-nm SD-OCM through the measurement of an interference signal from a mirror surface and imaging of a single 200-nm polystyrene bead and a pig thyroid gland. Deep tissue imaging at a depth of up to 1.8 mm was also demonstrated. This is the first demonstration of 3-D high-resolution SD-OCM in the 1700-nm spectral band.

Microscopic and mesoscale optical imaging techniques allow for three-dimensional (3-D) imaging of biological tissue across millimeter-scale regions, and imaging phantom models are invaluable for system characterization and clinical training. Phantom models that replicate complex 3-D geometries with both structural and molecular contrast, with resolution and lateral dimensions equivalent to those of imaging techniques ([Formula: see text]), have proven elusive. We present a method for fabricating phantom models using a combination of two-photon polymerization (2PP) to print scaffolds, and microinjection of tailored tissue-mimicking materials to simulate healthy and diseased tissue. We provide a first demonstration of the capabilities of this method with intravascular optical coherence tomography, an imaging technique widely used in clinical practice. We describe the design, fabrication, and validation of three types of phantom models: a first with subresolution wires (5- to [Formula: see text] diameter) arranged circumferentially, a second with a vessel side-branch, and a third containing a lipid inclusion within a vessel. Silicone hybrid materials and lipids, microinjected within a resin framework created with 2PP, served as tissue-mimicking materials that provided realistic optical scattering and absorption. We demonstrate that optical phantom models made with 2PP and microinjected tissue-mimicking materials can simulate complex anatomy and pathology with exquisite detail.

Burn wounds and wound healing invoke several biological processes that may complicate the interpretation of spectral imaging data. Through analysis of spatial frequency domain spectroscopy data (450 to 1000nm) obtained from longitudinal investigations using a graded porcine burn wound healing model, we have identified features in the absorption spectrum that appear to suggest the presence of hemoglobin breakdown products, e.g., methemoglobin. Our results show that the calculated concentrations of methemoglobin directly correlate with burn severity, 24h after the injury. In addition, tissue parameters such as oxygenation (StO2) and water fraction may be underestimated by 20% and 78%, respectively, if methemoglobin is not included in the spectral analysis.

Optical coherence tomography (OCT) is an emerging technology for in vivo airway and lung imaging. However, OCT lacks sensitivity to the metabolic changes caused by inflammation, which drives chronic respiratory diseases such as asthma and chronic obstructive pulmonary disorder. Redox imaging (RI) is a label-free technique that uses the autofluorescence of the metabolic coenzymes NAD(P)H and flavin adenine dinucleotide (FAD) to probe cellular metabolism and could provide complimentary information to OCT for airway and lung imaging. We demonstrate OCT and RI of respiratory ciliated epithelial function in ex vivo mouse tracheae. We applied RI to measure cellular metabolism via the redox ratio [intensity of NAD(P)H divided by FAD] and particle tracking velocimetry OCT to quantify cilia-driven fluid flow. To model mitochondrial dysfunction, a key aspect of the inflammatory process, cyanide was used to inhibit oxidative metabolism and reduce ciliary motility. Cyanide exposure over 20 min significantly increased the redox ratio and reversed cilia-driven fluid flow. We propose that RI provides complementary information to OCT to assess inflammation in the airway and lungs.

Significance: Ensuring spectral quality is prerequisite to Raman spectroscopy applied to surgery. This is because the inclusion of poor-quality spectra in the training phase of Raman-based pathology detection models can compromise prediction robustness and generalizability to new data. Currently, there exists no quantitative spectral quality assessment technique that can be used to either reject low-quality data points in existing Raman datasets based on spectral morphology or, perhaps more importantly, to optimize the in vivo data acquisition process to ensure minimal spectral quality standards are met. Aim: To develop a quantitative method evaluating Raman signal quality based on the variance associated with stochastic noise in important tissue bands, including C─C stretch, CH2/CH3 deformation, and the amide bands. Approach: A single-point hand-held Raman spectroscopy probe system was used to acquire 315 spectra from 44 brain cancer patients. All measurements were classified as either high or low quality based on visual assessment (qualitative) and using a quantitative quality factor (QF) metric. Receiver-operator-characteristic (ROC) analyses were performed to evaluate the performance of the quantitative metric to assess spectral quality and improve cancer detection accuracy. Results: The method can separate high- and low-quality spectra with a sensitivity of 89% and a specificity of 90% which is shown to increase cancer detection sensitivity and specificity by up to 20% and 12%, respectively. Conclusions: The QF threshold is effective in stratifying spectra in terms of spectral quality and the observed false negatives and false positives can be linked to limitations of qualitative spectral quality assessment.

Light-emitting diode (LED) light sources have recently been introduced to photoacoustic imaging (PAI). The LEDs enable a smaller footprint for PAI systems when compared to laser sources, thereby improving system portability and allowing for improved access. An LED-based PAI system has been employed to identify inflammatory arthritis in human hand joints. B-mode ultrasound (US), Doppler, and PAIs were obtained from 12 joints with clinically active arthritis, five joints with subclinically active arthritis, and 12 normal joints. The quantitative assessment of hyperemia in joints by PAI demonstrated statistically significant differences among the three conditions. The imaging results from the subclinically active arthritis joints also suggested that the LED-based PAI has a higher sensitivity to angiogenic microvascularity compared to US Doppler imaging. This initial clinical study on arthritis patients validates that PAI can be a potential imaging modality for the diagnosis of inflammatory arthritis.

It is hypothesized that the local, viscoelastic (time-dependent) properties of the airway are important to accurately model and ultimately predict dynamic airway collapse in airway obstruction. Toward this end, we present a portable, endoscopic, swept-source anatomical optical coherence tomography (aOCT) system combined with a pressure catheter to capture local airway dynamics in vivo during respiration. aOCT scans were performed in the airways of a mechanically ventilated pig under paralysis with dynamic and static ventilation protocols. Validation of dynamic aOCT luminal cross-sectional area (CSA) measurements against Cine CT, obtained during the same exam, showed an aggregate difference of 15 % ± 3 % . aOCT-derived CSA obtained in the in vivo trachea also exhibited hysteresis as a function of pressure, depicting the viscoelastic nature of the airway wall. The volumetric imaging capabilities were validated by comparing aOCT- and CT-derived geometries of the porcine airway spanning nine generations from the trachea to the bronchioles. The ability to delineate regional differences in airway viscoelastic properties, by measuring airway deformation using aOCT combined with intraluminal pressure, paves the way to patient-specific models of dynamic airway collapse.

We present a highly scalable Monte Carlo (MC) three-dimensional photon transport simulation platform designed for heterogeneous computing systems. Through the development of a massively parallel MC algorithm using the Open Computing Language (OpenCL) framework, this research extends our existing graphics processing unit (GPU)-accelerated MC technique to a highly scalable vendor-independent heterogeneous computing environment, achieving significantly improved performance and software portability. A number of parallel computing techniques are investigated to achieve portable performance over a wide range of computing hardware. Furthermore, multiple thread-level and device-level load-balancing strat- egies are developed to obtain efficient simulations using multiple central processing units (CPUs) and GPUs., Accepted for Publication in Journal of Biomedical Optics Letters on Jan 4, 2018, to appear in Volume 23, Issue 2

Rare-earth-doped nanocomposites have appealing optical properties for use as biomedical contrast agents, but few systems exist for imaging these materials. We describe the design and characterization of (i) a preclinical system for whole animal in vivo imaging and (ii) an integrated optical coherence tomography/confocal microscopy system for high-resolution imaging of ex vivo tissues. We demonstrate these systems by administering erbium-doped nanocomposites to a murine model of metastatic breast cancer. Short-wave infrared emissions were detected in vivo and in whole organ imaging ex vivo. Visible upconversion emissions and tissue autofluorescence were imaged in biopsy specimens, alongside optical coherence tomography imaging of tissue microstructure. We anticipate that this work will provide guidance for researchers seeking to image these nanomaterials across a wide range of biological models.

Docosanol is an over-the-counter topical agent that has proved to be one of the most effective therapies for treating herpes simplex labialis. However, the mechanism by which docosanol suppresses lesion formation remains poorly understood. To elucidate its mechanism of action, we investigated the uptake of docosanol in living cells using coherent anti-Stokes Raman scattering microscopy. Based on direct visualization of the deuterated docosanol, we observed highly concentrated docosanol inside living cells 24 h after drug treatment. In addition, different spatial patterns of drug accumulation were observed in different cell lines. In keratinocytes, which are the targeted cells of docosanol, the drug molecules appeared to be docking at the periphery of the cell membrane. In contrast, the drug molecules in fibroblasts appeared to accumulate in densely packed punctate regions throughout the cytoplasm. These results suggest that this molecular imaging approach is suitable for the longitudinal tracking of drug molecules in living cells to identify cell-specific trafficking and may also have implications for elucidating the mechanism by which docosanol suppresses lesion formation.

Visualization and assessment of the cellular structure and function require localized delivery of the molecules into specific cells in restricted spatial regions of the tissue and may necessitate subcellular delivery and localization. Earlier, we have shown ultrafast near-infrared laser beam-assisted optoporation of actin-staining molecules into cortical neurons with single-cell resolution and high efficiency. However, diffusion of optoporated molecules in soma degrades toward the growth cone, leading to difficulties in visualization of the actin network in the growth cone in cases of long axons. Here, we demonstrate optoporation of impermeable molecules to functional cortical neurons by precise laser subaxotomy near the growth cone, leading to visualization of the actin network in the growth cone. Further, we demonstrate patterned delivery of impermeable molecules into targeted retinal cells in the rat eye. The development of optoporation as a minimally invasive approach to reliably deliver exogenous molecules into targeted axons and soma of retinal neurons in vivo will enable enhanced visualization of the structure and function of the retina.

We have developed compressed sensing single pixel spatial frequency domain imaging (cs-SFDI) to characterize tissue optical properties over a wide field of view ( 35 mm × 35 mm ) using multiple near-infrared (NIR) wavelengths simultaneously. Our approach takes advantage of the relatively sparse spatial content required for mapping tissue optical properties at length scales comparable to the transport scattering length in tissue ( l tr ∼ 1 mm ) and the high bandwidth available for spectral encoding using a single-element detector. cs-SFDI recovered absorption ( μ a ) and reduced scattering ( μ s ′ ) coefficients of a tissue phantom at three NIR wavelengths (660, 850, and 940 nm) within 7.6% and 4.3% of absolute values determined using camera-based SFDI, respectively. These results suggest that cs-SFDI can be developed as a multi- and hyperspectral imaging modality for quantitative, dynamic imaging of tissue optical and physiological properties.

Current clinical tools provide critical information about ocular health such as intraocular pressure (IOP). However, they lack the ability to quantify tissue material properties, which are potent markers for ocular tissue health and integrity. We describe a single instrument to measure the eye-globe IOP, quantify corneal biomechanical properties, and measure corneal geometry with a technique termed applanation optical coherence elastography (Appl-OCE). An ultrafast OCT system enabled visualization of corneal dynamics during noncontact applanation tonometry and direct measurement of micro air-pulse induced elastic wave propagation. Our preliminary results show that the proposed Appl-OCE system can be used to quantify IOP, corneal biomechanical properties, and corneal geometry, which builds a solid foundation for a unique device that can provide a more complete picture of ocular health.

Lymph node biopsy is a primary means of staging breast cancer, yet standard pathological techniques are time-consuming and typically sample less than 1% of the total node volume. A low-cost fluorescence optical projection tomography (OPT) protocol is demonstrated for rapid imaging of whole lymph nodes in three dimensions. The relatively low scattering properties of lymph node tissue can be leveraged to significantly improve spatial resolution of lymph node OPT by employing angular restriction of photon detection. It is demonstrated through porcine lymph node metastases models that simple filtered-backprojection reconstruction is sufficient to detect and localize 200-μm-diameter metastases (the smallest clinically significant) in 1-cm-diameter lymph nodes.

We propose a handheld projective imaging device for orthotopic projection of near-infrared fluorescence images onto target biological tissue at visible wavelengths without any additional visual aid. The device integrates a laser diode light source module, a camera module, a projector, an ultrasonic distance sensor, a Raspberry Pi single-board computer, and a battery module in a rugged handheld unit. It is calibrated at the detected working distance for seamless coregistration between fluorescence emission and projective imaging at the target tissue site. The proposed device is able to achieve a projection resolution higher than 314 μm and a planar projection bias less than 1 mm at a projection field of view of 58×108 mm2 and a working distance of 27 cm. Technical feasibility for projective imaging is verified in an ex vivo model of chicken breast tissue using indocyanine green as a fluorescence agent. Clinical utility for image-guided surgery is demonstrated in a clinical trial where sentinel lymph nodes in breast cancer patients are identified and resected under the guidance of projective imaging. Our ex vivo and in vivo experiments imply the clinical utility of deploying the proposed device for image-guided surgical interventions in resource-limited settings.

We acquired multiphoton images of normal and lung adenocarcinoma cell lines in three dimensions. Image stacks of the cells were then processed to obtain nucleus-to-cytoplasm (N/C) ratios in two and three dimensions. While N/C ratios in three dimensions can be unambiguously determined from the volumetric ratios of the nucleus and cytoplasm, two-dimensional (2-D) N/C can vary depending on the axial plane selected for N/C ratio determination. We determined 2-D N/C ratios from three criteria: (1) axial position at which the nuclear area is the largest; (2) the largest 2-D N/C ratio value; and (3) axial position at the midpoint of nuclear axial position. We found that different definitions of 2-D N/C ratio will significantly affect its value. Furthermore, in general, larger variance was found in 2-D rather than three-dimensional (3-D) N/C ratios. Lack of ambiguity in definition and reduced variance suggest that 3-D N/C ratio is a better parameter for characterizing tumor cells in the clinical setting.

Rare-earth-doped nanoparticles are one of the emerging probes for bioimaging due to their visible-to-near-infrared (NIR) upconversion emission via sequential single-photon absorption at NIR wavelengths. The NIR-excited upconversion property and high photostability make this probe appealing for deep tissue imaging. So far, upconversion nanoparticles include ytterbium ions (Yb3 + ) codoped with other rare earth ions, such as erbium (Er3 + ) and thulium (Tm3 + ). In these types of upconversion nanoparticles, through energy transfer from Yb3 + excited with continuous wave light at a wavelength of 980 nm, upconversion emission of the other rare earth dopants is induced. We have found that the use of the excitation of Er3 + in the 1550-nm wavelength region allows us to perform deep tissue imaging with reduced degradation of spatial resolution. In this excitation–emission process, three and four photons of 1550-nm light are sequentially absorbed, and Er3 + emits photons in the 550- and 660-nm wavelength regions. We demonstrate that, compared with the case using 980-nm wavelength excitation, the use of 1550-nm light enables us to moderate degradation of spatial resolution in deep tissue imaging due to the lower light scattering coefficient compared with 980-nm light. We also demonstrate that live cell imaging is feasible with this 1550 nm excitation.

Early stage porcine parthenogenetic embryos were evaluated for metabolic activity using a biodynamic microscope (BDM) that images dynamic light scattering using low-coherence digital holography. The microscope has a 45-deg illumination configuration that reduces specular background for the imaging of small translucent samples. The off-axis illumination is compatible with coherence-gated imaging because of volumetric light scattering in which the coherence plane is tilted at half the illumination angle in a three-dimensional tissue target. The BDM was used to profile the viability of porcine parthenotes with normal and with inhibited mitochondrial adenosine triphosphate (ATP) production using Doppler fluctuation spectroscopy. The ATP concentrations in the parthenotes, which are indicative of developmental potential, were validated by a conventional bioluminescence assay. Biodynamic classifications achieved ∼80% accuracy correlating sample ATP treatment, providing a quick, label-free surrogate measurement to replace invasive metabolic assays as a candidate for evaluating quality of early embryos in the assisted reproductive technology setting.

Optical clearing agents (OCAs) and many chemicals are widely used in functional diagnosis of skin tissues. Numerous studies are associated with the transcutaneous diffusion of OCA in epidermal, dermal, and hypodermal tissues, which results in changing their optical properties. In addition, an objective approach that is suitable for screening the influence of utilized OCA, as well as various chemical agents, synthetics, and nanomaterials, on blood and lymph flows is highly desirable. In our study, a highly sensitive laser speckle imaging (LSI) system and fluorescent intravital microscopy (FIM) were used team-wise to inspect the acute skin vascular permeability reaction in mouse ear during the local application of OCA on the skin surface. Fluorescent contrast material administrated intravenously was used for quantitatively assessing the intensity of vascular permeability reaction and the strength of skin irritation. The obtained results suggest that a combined use of LSI and FIM is highly effective for monitoring the cutaneous vascular permeability reaction, with great potential for assessment of allergic reactions of skin in response to interactions with chemical substances.

The mesh-based Monte Carlo (MMC) method is an efficient algorithm to model light propagation inside tissues with complex boundaries, but choosing appropriate mesh density can be challenging. A fine mesh improves the spatial resolution of the output but requires more computation. We propose an improved MMC—dual-grid mesh-based Monte Carlo (DMMC)—to accelerate photon simulations using a coarsely tessellated tetrahedral mesh for ray-tracing computation and an independent voxelated grid for output data storage. The decoupling between ray-tracing and data storage grids allows us to simultaneously achieve faster simulations and improved output spatial accuracy. Furthermore, we developed an optimized ray-tracing technique to eliminate unnecessary ray–tetrahedron intersection tests in optically thick mesh elements. We validate the proposed algorithms using a complex heterogeneous domain and compare the solutions with those from MMC and voxel-based Monte Carlo. We found that DMMC with an unrefined constrained Delaunay tessellation of the boundary nodes yielded the highest speedup, ranging from 1.3× to 2.9× for various scattering settings, with nearly no loss in accuracy. In addition, the optimized ray-tracing technique offers excellent acceleration in high-scattering media, reducing the ray–tetrahedron test count by over 100-fold. Our DMMC software can be downloaded at http://mcx.space/mmc.

Phase wrapping is a crucial issue in Doppler optical coherence tomography (OCT) and restricts its automatic implementation for clinical applications that quantify total retinal blood flow. We propose an automated phase-unwrapping technique that takes advantage of the parabolic profile of blood flow velocity in vessels. Instead of inspecting the phase shift manually, the algorithm calculates the gradient magnitude of the phase shift on the cross-sectional image and automatically detects the presence of phase wrapping. The voxels affected by phase wrapping are corrected according to the determined flow direction adjacent to the vessel walls. We validated this technique in the rodent retina using a prototype visible-light OCT and in the human retina with a commercial infrared OCT system. We believe this signal processing method may well accelerate clinical applications of Doppler OCT in ophthalmology.

High resolution is important for sensitive detection of subtle distortions of retinal morphology at an early stage of eye diseases. We demonstrate virtually structured detection (VSD) as a feasible method to achieve in vivo super-resolution ophthalmoscopy. A line-scanning strategy was employed to achieve a super-resolution imaging speed up to 127 ?? frames / s with a frame size of 512 × 512 ?? pixels . The proof-of-concept experiment was performed on anesthetized frogs. VSD-based super-resolution images reveal individual photoreceptors and nerve fiber bundles unambiguously. Both image contrast and signal-to-noise ratio are significantly improved due to the VSD implementation.

This study demonstrates the feasibility of using the Rayleigh wave model (RWM) in combination with optical coherence elastography (OCE) technique to assess the viscoelasticity of soft tissues. Dispersion curves calculated from the spectral decomposition of OCE-measured air-pulse induced elastic waves were used to quantify the viscoelasticity of samples using the RWM. Validation studies were first conducted on 10% gelatin phantoms with different concentrations of oil. The results showed that the oil increased the viscosity of the gelatin phantom samples. This method was then used to quantify the viscoelasticity of chicken liver. The Young’s modulus of the chicken liver tissues was estimated as E=2.04±0.88??kPa with a shear viscosity ?=1.20±0.13??Pa?s. The analytical solution of the RWM correlated very well with the OCE-measured phased velocities (R2=0.96±0.04). The results show that the combination of the RWM and OCE is a promising method for noninvasively quantifying the biomechanical properties of soft tissues and may be a useful tool for detecting disease.

Imaging technologies working in the spatial frequency domain are becoming increasingly popular for generating wide-field maps of optical properties, enabling rapid analysis of tissue parameters. While acquisition methods have become faster and are now performing in real-time, processing methods remain slow, precluding real-time display of information. We present solutions that rapidly solve the inverse problem for extracting optical properties by use of advanced lookup tables (LUTs). We present methods and results based on a dense, linearly sampled lookup table and an analytical representation that generate maps of absorption and reduced scattering in ?10??ms, which is 100× faster than the standard method, with ?4% error compared to the Monte-Carlo simulation. Combined with real-time acquisition methods, the proposed techniques enable video-rate feedback of real-time property maps, enabling full video-rate guidance in the clinic.

Quantifying tissue biomechanical properties can assist in detection of abnormalities and monitoring disease progression and/or response to a therapy. Optical coherence elastography (OCE) has emerged as a promising technique for noninvasively characterizing tissue biomechanical properties. Several mechanical loading techniques have been proposed to induce static or transient deformations in tissues, but each has its own areas of applications and limitations. This study demonstrates the combination of Lorentz force excitation and phase-sensitive OCE at ∼1.5 million A-lines per second to quantify the elasticity of tissue by directly imaging Lorentz force-induced elastic waves. This method of tissue excitation opens the possibility of a wide range of investigations using tissue biocurrents and conductivity for biomechanical analysis.

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

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.

Angiogenesis in a tumor region creates arteriovenous (AV) shunts that cause an abnormal venous blood oxygen saturation (sO_2) distribution. Here, we applied optical-resolution photoacoustic microscopy to study the AV shunting in vivo. First, we built a phantom to image sO_2 distribution in a vessel containing converged flows from two upstream blood vessels with different sO_2 values. The phantom experiment showed that the blood from the two upstream vessels maintained a clear sO_2 boundary for hundreds of seconds, which is consistent with our theoretical analysis using a diffusion model. Next, we xenotransplanted O-786 tumor cells in mouse ears and observed abnormal sO_2 distribution in the downstream vein from the AV shunts in vivo. Finally, we identified the tumor location by tracing the sO_2 distribution. Our study suggests that abnormal sO_2 distribution induced by the AV shunts in the vessel network may be used as a new functional benchmark for early tumor detection.

Total internal reflection microscopy (TIRF) has been a powerful tool in biological research. The most valuable feature of the method has been the ability to image 100- to 200-nm-thick layer of cell features adjacent to a coverslip, such as membrane lipids, membrane receptors, and structures proximal-to-basal membranes. Here, we demonstrate an alternative method of imaging thin-layer proximal-to-basal membranes by placing a sample on a high refractive index coverslip covered by a thin layer of gold. The sample is illuminated using the Kretschmann method (i.e., from the top to an aqueous medium). Fluorophores that are close to the metal surface induce surface plasmons in the metal film. Fluorescence from fluorophores near the metal surface couple with surface plasmons allowing them to penetrate the metal surface and emerge at a surface plasmon coupled emission angle. The thickness of the detection layer is further reduced in comparison with TIRF by metal quenching of fluorophores at a close proximity (below 10 nm) to a surface. Fluorescence is collected by a high NA objective and imaged by EMCCD or converted to a signal by avalanche photodiode fed by a single-mode optical fiber inserted in the conjugate image plane of the objective. The system avoids complications of through-the-objective TIRF associated with shared excitation and emission light path, has thin collection thickness, produces excellent background rejection, and is an effective method to study molecular motion.

The enamel scattering coefficient decreases markedly with increasing wavelength from the visible to the near-infrared (NIR). However, beyond 1300 nm, the scattering coefficient is difficult to measure, and it is not known whether light scattering continues to decrease significantly at longer wavelengths. It is hypothesized that water absorption is a major contributor to the contrast between sound and demineralized enamel beyond 1300 nm since deeply penetrating photons in sound enamel are likely absorbed by water. Reflectance images of demineralization on tooth surfaces were acquired at wavelengths near 1450, 1860, 1880, and 1950 nm. The magnitude of water absorption is similar at 1450 and 1880 nm but varies markedly between 1860, 1880, and 1950 nm. Multispectral comparisons of lesion contrast provide insight into the mechanism responsible for higher contrast at longer NIR wavelengths. The highest contrast was at 1950 nm; however, the markedly higher contrast at 1880 compared to 1450 nm and similar contrast between 1860 and 1880 nm suggests that the enamel scattering coefficient continues to decrease beyond 1300 nm, and that reduced light scattering in sound enamel is most responsible for the higher lesion contrast at longer NIR wavelengths. This has important implications for the choice of wavelengths for caries detection and diagnostic devices, including the performance of optical coherence tomography beyond 1300 nm.

Rod-dominated transient retinal phototropism (TRP) has been observed in freshly isolated retinas, promising a noninvasive biomarker for objective assessment of retinal physiology. However, in vivo mapping of TRP is challenging due to its subcellular signal magnitude and fast time course. We report here a virtually structured detection-based super-resolution ophthalmoscope to achieve subcellular spatial resolution and millisecond temporal resolution for in vivo imaging of TRP. Spatiotemporal properties of in vivo TRP were characterized corresponding to variable light intensity stimuli, confirming that TRP is tightly correlated with early stages of phototransduction.

Radiation therapy produces Cherenkov optical emission in tissue, and this light can be utilized to activate molecular probes. The feasibility of sensing luminescence from a tissue molecular oxygen sensor from within a human body phantom was examined using the geometry of the axillary lymph node region. Detection of regions down to 30-mm deep was feasible with submillimeter spatial resolution with the total quantity of the phosphorescent sensor PtG4 near 1 nanomole. Radiation sheet scanning in an epi-illumination geometry provided optimal coverage, and maximum intensity projection images provided illustration of the concept. This work provides the preliminary information needed to attempt this type of imaging in vivo.

Normal development of the visual system in infants relies on clear images being projected onto the retina, which can be disrupted by lens opacity caused by congenital cataract. This disruption, if uncorrected in early life, results in amblyopia (permanently decreased vision even after removal of the cataract). Doctors are able to prevent amblyopia by removing the cataract during the first several weeks of life, but this surgery risks a host of complications, which can be equally visually disabling. Here, we investigated the feasibility of focusing light noninvasively through highly scattering cataractous lenses to stimulate the retina, thereby preventing amblyopia. This approach would allow the cataractous lens removal surgery to be delayed and hence greatly reduce the risk of complications from early surgery. Employing a wavefront shaping technique named time-reversed ultrasonically encoded optical focusing in reflection mode, we focused 532-nm light through a highly scattering ex vivo adult human cataractous lens. This work demonstrates a potential clinical application of wavefront shaping techniques.

Rare-earth (RE) doped nanocomposites emit visible luminescence when illuminated with continuous wave near-infrared light, making them appealing candidates for use as contrast agents in biomedical imaging. However, the emission lifetime of these materials is much longer than the pixel dwell times used in scanning intravital microscopy. To overcome this limitation, we have developed a line-scanning confocal microscope for high-resolution, optically sectioned imaging of samples labeled with RE-based nanomaterials. Instrument performance is quantified using calibrated test objects. NaYF4 : Er,Yb nanocomposites are imaged in vitro, and in ex vivo tissue specimens, with direct comparison to point-scanning confocal microscopy. We demonstrate that the extended pixel dwell time of line-scanning confocal microscopy enables subcellular-level imaging of these nanomaterials while maintaining optical sectioning. The line-scanning approach thus enables microscopic imaging of this emerging class of contrast agents for preclinical studies, with the potential to be adapted for real-time in vivo imaging in the clinic.

Oxygen supplementation [hyperoxia, increased fraction of inspired oxygen (FiO 2 )] is an indispensable treatment in the intensive care unit for patients in respiratory failure. Like other treatments or drugs, hyperoxia has a risk-benefit profile that guides its clinical use. While hyperoxia is known to damage respiratory epithelium, it is unknown if damage can result in impaired capacity to generate cilia-driven fluid flow. Here, we demonstrate that quantifying cilia-driven fluid flow velocities in the sub-100 μm/s regime (sub-0.25 in./min regime) reveals hyperoxia-mediated damage to the capacity of ciliated respiratory mucosa to generate directional flow. Flow quantification was performed using particle tracking velocimetry optical coherence tomography (PTV-OCT) in ex vivo mouse trachea. The ability of PTV-OCT to detect biomedically relevant flow perturbations in the sub-100 μm/s regime was validated by quantifying temperature- and drug-mediated modulation of flow performance in ex vivo mouse trachea. Overall, PTV-OCT imaging of cilia-driven fluid flow in ex vivo mouse trachea is a powerful and straightforward approach for studying factors that modulate and damage mammalian respiratory ciliary physiology.

Efficient phenotyping of developmental defects in model organisms is critical for understanding the genetic specification of normal development and congenital abnormalities in humans. We previously reported that optical coherence tomography (OCT) combined with live embryo culture is a valuable tool for mouse embryo imaging and four-dimensional (4-D) cardiodynamic analysis; however, its capability for analysis of mouse mutants with cardiac phenotypes has not been previously explored. Here, we report 4-D (three-dimensional+time) OCT imaging and analysis of the embryonic heart in a Wdr19 mouse mutant, revealing a heart looping defect. Quantitative analysis of cardiac looping revealed a statistically significant difference between mutant and control embryos. Our results indicate that live 4-D OCT imaging provides a powerful phenotyping approach to characterize embryonic cardiac function in mouse models.

Fourier transform infrared imaging (FTIRI) combined with chemometrics algorithm has strong potential to obtain complex chemical information from biology tissues. FTIRI and partial least squares-discriminant analysis (PLS-DA) were used to differentiate healthy and osteoarthritic (OA) cartilages for the first time. A PLS model was built on the calibration matrix of spectra that was randomly selected from the FTIRI spectral datasets of healthy and lesioned cartilage. Leave-one-out cross-validation was performed in the PLS model, and the fitting coefficient between actual and predicted categorical values of the calibration matrix reached 0.95. In the calibration and prediction matrices, the successful identifying percentages of healthy and lesioned cartilage spectra were 100% and 90.24%, respectively. These results demonstrated that FTIRI combined with PLS-DA could provide a promising approach for the categorical identification of healthy and OA cartilage specimens.