Significance: 5-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.

Aim: We investigate the ability of pulse sampling, fiber-based FLIm to simultaneously image PpIX and NAD(P)H fluorescence of glioma infiltrative margins in patients.

Approach: A mesoscopic fiber-based point-scanning FLIm device (355 nm pulses) was used to simultaneously resolve the fluorescence decay of PpIX (629/53 nm) and NAD(P)H (470/28 nm). The FLIm device enabled data acquisition at room light and rapid (<33  ms) augmentation of FLIm parameters on the surgical field-of-view. FLIm measurements from superficial tumors and tissue areas around the resection margins were performed on three glioblastoma patients in vivo following inspection of PpIX visible fluorescence with a conventional neurosurgical microscope. Microbiopsies were collected from FLIm imaged areas for histopathological evaluation.

Results: The average lifetime from PpIX and NAD(P)H fluorescence distinguished between tumor and surrounding tissue. FLIm measurements of resection margins presented a range of PpIX and NAD(P)H lifetime values (τ_PpIX  ∼  3 to 14 ns, τ_NAD(P)H  =  3 to 6 ns) associated with unaffected tissue and areas of low-density tumor infiltration.

Conclusions: Intraoperative FLIm could simultaneously detect the emission of PpIX and NAD(P)H from patients in vivo during craniotomy procedures. This approach doubles as a clinical tool to identify tumor areas while performing tissue resection and as a research tool to study tumor microenvironmental changes in vivo. Intraoperative FLIm of 5-ALA-induced PpIX and tissue autofluorescence makes a promising surgical adjunct to guide tumor resection surgery.

Significance: Biomedical optics system design, image formation, and image analysis have primarily been guided by classical physical modeling and signal processing methodologies. Recently, however, deep learning (DL) has become a major paradigm in computational modeling and has demonstrated utility in numerous scientific domains and various forms of data analysis.

Aim: We aim to comprehensively review the use of DL applied to macroscopic diffuse optical imaging (DOI).

Approach: First, we provide a layman introduction to DL. Then, the review summarizes current DL work in some of the most active areas of this field, including optical properties retrieval, fluorescence lifetime imaging, and diffuse optical tomography.

Results: The advantages of using DL for DOI versus conventional inverse solvers cited in the literature reviewed herein are numerous. These include, among others, a decrease in analysis time (often by many orders of magnitude), increased quantitative reconstruction quality, robustness to noise, and the unique capability to learn complex end-to-end relationships.

Conclusions: The heavily validated capability of DL’s use across a wide range of complex inverse solving methodologies has enormous potential to bring novel DOI modalities, otherwise deemed impractical for clinical translation, to the patient’s bedside.

Significance: Diffuse reflectance spectroscopy (DRS) allows discrimination of tissue type. Its application is limited by the inability to mark the scanned tissue and the lack of real-time measurements.

Aim: This study aimed to develop a real-time tracking system to enable localization of a DRS probe to aid the classification of tumor and non-tumor tissue.

Approach: A green-colored marker attached to the DRS probe was detected using hue-saturation-value (HSV) segmentation. A live, augmented view of tracked optical biopsy sites was recorded in real time. Supervised classifiers were evaluated in terms of sensitivity, specificity, and overall accuracy. A developed software was used for data collection, processing, and statistical analysis.

Results: The measured root mean square error (RMSE) of DRS probe tip tracking was 1.18  ±  0.58  mm and 1.05  ±  0.28  mm for the x and y dimensions, respectively. The diagnostic accuracy of the system to classify tumor and non-tumor tissue in real time was 94% for stomach and 96% for the esophagus.

Conclusions: We have successfully developed a real-time tracking and classification system for a DRS probe. When used on stomach and esophageal tissue for tumor detection, the accuracy derived demonstrates the strength and clinical value of the technique to aid margin assessment in cancer resection surgery.

Significance: The primary method of COVID-19 detection is reverse transcription polymerase chain reaction (RT-PCR) testing. PCR test sensitivity may decrease as more variants of concern arise and reagents may become less specific to the virus.

Aim: We aimed to develop a reagent-free way to detect COVID-19 in a real-world setting with minimal constraints on sample acquisition. The machine learning (ML) models involved could be frequently updated to include spectral information about variants without needing to develop new reagents.

Approach: We present a workflow for collecting, preparing, and imaging dried saliva supernatant droplets using a non-invasive, label-free technique—Raman spectroscopy—to detect changes in the molecular profile of saliva associated with COVID-19 infection.

Results: We used an innovative multiple instance learning-based ML approach and droplet segmentation to analyze droplets. Amongst all confounding factors, we discriminated between COVID-positive and COVID-negative individuals yielding receiver operating coefficient curves with an area under curve (AUC) of 0.8 in both males (79% sensitivity and 75% specificity) and females (84% sensitivity and 64% specificity). Taking the sex of the saliva donor into account increased the AUC by 5%.

Conclusion: These findings may pave the way for new rapid Raman spectroscopic screening tools for COVID-19 and other infectious diseases.

Significance: Functional near-infrared spectroscopy (fNIRS) is a promising optical neuroimaging technique, measuring the hemodynamic signals from the cortex. However, improving signal quality and reducing artifacts arising from oscillation and baseline shift (BS) are still challenging up to now for fNIRS applications.

Aim: Considering the advantages and weaknesses of the different algorithms to reduce the artifact effect in fNIRS signals, we propose a hybrid artifact detection and correction approach.

Approach: First, distinct artifact detection was realized through an fNIRS detection strategy. Then the artifacts were divided into three categories: BS, slight oscillation, and severe oscillation. A comprehensive correction was applied through three main steps: severe artifact correction by cubic spline interpolation, BS removal by spline interpolation, and slight oscillation reduction by dual-threshold wavelet-based method.

Results: Using fNIRS data acquired during whole night sleep monitoring, we compared the performance of our approach with existing algorithms in signal-to-noise ratio (SNR) and Pearson’s correlation coefficient (R). We found that the proposed method showed improvements in performance in SNR and R with strong stability.

Conclusions: These results suggest that the new hybrid artifact detection and correction method enhances the viability of fNIRS as a functional neuroimaging modality.

Significance: Refractive surgery in ophthalmology uses pulsed lasers at 193, 210, or 213 nm. The reason is that most molecular constituents of cornea absorb strongly in this wavelength range. Precise refractive surgery via ablation requires an accurate knowledge of the absorption coefficient at the relevant wavelengths. Yet, the absorption coefficients of corneal tissue reported in literature vary by almost an order of magnitude; moreover, they were measured mostly at the wavelengths mentioned earlier.

Aim: By measuring the corneal absorption coefficient of intact eyeballs stored at different environmental conditions, prepared by following different procedures, and as a function of postmortem time, we determine the absorption coefficient for the entire wavelength range between 185 and 250 nm for as close as possible to in-vivo conditions.

Approach: We use a specially designed UV ellipsometer to measure refractive index and absorption coefficient. Specifically, we investigate the temporal evolution of refractive index and absorption coefficient after enucleation of the eyeballs under different environmental conditions and preparation procedures.

Results: Our measurements provide accurate values for refractive index as well as absorption coefficient of cornea in the wavelength range between 185 and 250 nm. We find that the absorption coefficient decreases with time and that neither storage conditions nor preparation procedures but a continuous degeneration of the cornea is responsible for the observed time evolution. We use the measured time evolution to extrapolate refractive index and absorption coefficient to in-vivo conditions.

Conclusion: Our measurements of the close to in-vivo absorption coefficient of cornea between 185 and 250 nm allow for a better understanding and modeling of refractive cornea surgery, also at other than the three commonly used wavelengths. In the future, this may be relevant when new pulsed laser sources with other wavelengths become available.

Significance: Our work demonstrates in preclinical models that continuous-wave transrectal diffuse optical tomography (TRDOT) can be used to accurately monitor photothermal therapy (PTT) and, in particular, the progression of the photocoagulation boundary toward the rectum. When used in patients, this should prevent rectal damage during PTT, thereby achieving maximum treatment efficacy while ensuring safety, using a technology platform suitable for wide dissemination.

Aim: We aim to validate that TRDOT measurements analyzed using a shape-based image-reconstruction algorithm (SBDOT) allow localization of the photocoagulation boundary during PTT within ±1  mm toward the rectum in the transverse plane.

Approach: TRDOT measurements were performed in tissue-simulating phantoms, ex vivo tissues, and an in vivo canine prostate model. The accuracy and sensitivity of reconstructing the size and location of the coagulation zone were determined, based on changes in the tissue absorption and reduced scattering coefficients upon photocoagulation. The reconstruction also yields the native and coagulated tissue optical properties.

Results: The TRDOT measurements and SBDOT reconstruction algorithm were confirmed to perform sufficiently well for clinical translation in PTT monitoring, recovering the location of the coagulation boundary within ±1  mm compared to the true value as determined by direct visualization postexcision and/or MRI.

Conclusions: Implementing previously described TRDOT instrumentation and SBDOT image reconstruction in different tissue models confirms the potential for clinincal translation, including required refinements of the system and reconstruction algorithm.

Significance: Full-field optical angiography is critical for vascular disease research and clinical diagnosis. Existing methods struggle to improve the temporal and spatial resolutions simultaneously.

Aim: Spatiotemporal absorption fluctuation imaging (ST-AFI) is proposed to achieve dynamic blood flow imaging with high spatial and temporal resolutions.

Approach: ST-AFI is a dynamic optical angiography based on a low-coherence imaging system and U-Net. The system was used to acquire a series of dynamic red blood cell (RBC) signals and static background tissue signals, and U-Net is used to predict optical absorption properties and spatiotemporal fluctuation information. U-Net was generally used in two-dimensional blood flow segmentation as an image processing algorithm for biomedical imaging. In the proposed approach, the network simultaneously analyzes the spatial absorption coefficient differences and the temporal dynamic absorption fluctuation.

Results: The spatial resolution of ST-AFI is up to 4.33  μm, and the temporal resolution is up to 0.032 s. In vivo experiments on 2.5-day-old chicken embryos were conducted. The results demonstrate that intermittent RBCs flow in capillaries can be resolved, and the blood vessels without blood flow can be suppressed.

Conclusions: Using ST-AFI to achieve convolutional neural network (CNN)-based dynamic angiography is a novel approach that may be useful for several clinical applications. Owing to their strong feature extraction ability, CNNs exhibit the potential to be expanded to other blood flow imaging methods for the prediction of the spatiotemporal optical properties with improved temporal and spatial resolutions.

Significance: Corneal cross-linking (CXL) is a well-known procedure for treating certain eye disorders such as keratoconus. However, characterization of the biomechanical changes in the cornea as a result of this procedure is still under active research. Specifically, there is a clinical need for high-resolution characterization of individual corneal layers.

Aim: A high-resolution elastography method in conjunction with a custom optical coherence tomography system is used to track these biomechanical changes in individual corneal layers. Pre- and post-treatment analysis for both low-dose and high-dose CXL experiments are performed.

Approach: A recently developed elastography technique that utilizes the theory of reverberant shear wave fields, with optical coherence tomography as the modality, is applied to pig corneas ex vivo to evaluate elasticity changes associated with corneal CXL. Sets of low-dose and high-dose CXL treatments are evaluated before and after treatments with three pairs of pig corneas per experiment.

Results: The reverberant three-dimensional (3D) optical coherence elastography (OCE) technique can identify increases in elasticity associated with both low-dose and high-dose CXL treatments. There is a notable graphical difference between low-dose and high-dose treatments. In addition, the technique is able to identify which layers of the cornea are potentially affected by the CXL procedure and provides insight into the nonlinearity of the elasticity changes.

Conclusions: The reverberant 3D OCE technique can identify depth-resolved changes in elasticity of the cornea associated with CXL procedures. This method could be translated to assess and monitor CXL efficacy in various clinical settings.

Significance: Multispectral imaging enables mapping of chromophore content changes in skin neoplasms, which helps to diagnose a pathology. Different types of light sources can be used for the imaging. Design of laser-based illuminators is more complicated and, consequently, they are more expensive than LED-based illuminators. On the other hand, spectral line illumination has the advantage of less complicated calculations, since only the discrete maximum wavelengths need to be considered. Spectral band and spectral line approaches for multispectral skin diagnostics have not been compared so far. This can help to evaluate the accuracy and effectiveness of both approaches.

Aim: To compare two specific illumination modalities—spectral band and spectral line illumination—from the point of performance for mapping of in vivo skin chromophores.

Approach: Three spectral images of the same skin malformations were captured by a smartphone RGB camera with two different add-on illuminators comprising LED emitters and laser emitters, respectively. Five types of benign skin neoplasms were included in our study. Concentrations of skin melanin, oxy- and deoxy-hemoglobin at image pixel groups were calculated using the Beer–Lambert law.

Results: Skin chromophore maps and statistical analysis of mean concentrations’ changes in the neoplasms compared to the surrounding skin are presented and discussed. The data of the laser emitters led to significantly higher (∼10 times) increase of the oxy-hemoglobin values in vascular neoplasms and much lower deoxy-hemoglobin values, if compared to the data obtained by the LED emitters.

Conclusions: Analysis of the obtained chromophore distribution maps and concentration variations in neoplasms led to conclusion that the spectral line illumination approach is more appropriate for this application. Considering only the peak wavelengths of illumination spectral bands leads to essentially different results if compared to those obtained by spectral line illumination and may cause misinterpretations in the clinical assessment of skin neoplasms.

Significance: Intrinsic optical signals (IOS) generated in the cortical tissue as a result of various interacting metabolic processes are used extensively to elucidate the underlying mechanisms that govern neurovascular coupling. However, current IOS measurements still often rely on bulky, tabletop imaging systems, and there remains a dearth of studies in freely moving subjects. Lightweight, miniature head-mounted imaging devices provide unique opportunities for investigating cortical dynamics in small animals under a variety of naturalistic behavioral settings.

Aim: The aim of this work was to monitor IOS in the somatosensory cortex of wild-type mice by developing a lightweight, biocompatible imaging device that readily lends itself to animal experiments in freely moving conditions.

Approach: Herein we describe a method for realizing long-term IOS imaging in mice using a 0.54-g, compact, CMOS-based, head-mounted imager. The two-part module, consisting of a tethered sensor plate and a base plate, allows facile assembly prior to imaging sessions and disassembly when the sensor is not in use. LEDs integrated into the device were chosen to illuminate the cortical mantle at two different wavelengths in the visible regime (λ_center: 535 and 625 nm) for monitoring volume- and oxygenation state-dependent changes in the IOS, respectively. To test whether the system can detect robust cortical responses, we recorded sensory-evoked IOS from mechanical stimulation of the hindlimbs (HL) of anesthetized mice in both acute and long-term implantation conditions.

Results: Cortical IOS recordings in the primary somatosensory cortex hindlimb receptive field (S1HL) of anesthetized mice under green and red LED illumination revealed robust, multiphasic profiles that were time-locked to the mechanical stimulation of the contralateral plantar hindpaw. Similar intrinsic signal profiles observed in S1HL at 40 days postimplantation demonstrated the viability of the approach for long-term imaging. Immunohistochemical analysis showed that the brain tissue did not exhibit appreciable immune response due to the device implantation and operation. A proof-of-principle imaging session in a freely behaving mouse showed minimal locomotor impediment for the animal and also enabled estimation of blood flow speed.

Conclusions: We demonstrate the utility of a miniature cortical imaging device for monitoring IOS and related hemodynamic processes in both anesthetized and freely moving mice, cueing potential for applications to some neuroscientific studies of sensation and naturalistic behavior.