The guest editorial introduces the Journal of Biomedical Optics special collection, Perspectives in Biophotonics, https://www.spiedigitallibrary.org/biophotonics-tutorials.

Significance: Fast and reliable detection of infectious SARS-CoV-2 virus loads is an important issue. Fluorescence spectroscopy is a sensitive tool to do so in clean environments. This presumes a comprehensive knowledge of fluorescence data.

Aim: We aim at providing fully featured information on wavelength and time-dependent data of the fluorescence of the SARS-CoV-2 spike protein S1 subunit, its receptor-binding domain (RBD), and the human angiotensin-converting enzyme 2, especially with respect to possible optical detection schemes.

Approach: Spectrally resolved excitation-emission maps of the involved proteins and measurements of fluorescence lifetimes were recorded for excitations from 220 to 295 nm. The fluorescence decay times were extracted by using a biexponential kinetic approach. The binding process in the SARS-CoV-2 RBD was likewise examined for spectroscopic changes.

Results: Distinct spectral features for each protein are pointed out in relevant spectra extracted from the excitation-emission maps. We also identify minor spectroscopic changes under the binding process. The decay times in the biexponential model are found to be   (  2.0  ±  0.1  )   ns and   (  8.6  ±  1.4  )    ns.

Conclusions: Specific material data serve as an important background information for the design of optical detection and testing methods for SARS-CoV-2 loaded media.

Significance: Full-field optical coherence tomography (FF-OCT) enables en face views of scattering samples at a given depth with subcellular resolution, similar to biopsy without the need of sample slicing or other complex preparation. This noninvasive, high-resolution, three-dimensional (3D) imaging method has the potential to become a powerful tool in biomedical research, clinical applications, and other microscopic detection.

Aim: Our review provides an overview of the disruptive innovations and key technologies to further improve FF-OCT performance, promoting FF-OCT technology in biomedical and other application scenarios.

Approach: A comprehensive review of state-of-the-art accomplishments in OCT has been performed. Methods to improve performance of FF-OCT systems are reviewed, including advanced phase-shift approaches for imaging speed improvement, methods of denoising, artifact reduction, and aberration correction for imaging quality optimization, innovations for imaging flux expansion (field-of-view enlargement and imaging-depth-limit extension), new implementations for multimodality systems, and deep learning enhanced FF-OCT for information mining, etc. Finally, we summarize the application status and prospects of FF-OCT in the fields of biomedicine, materials science, security, and identification.

Results: The most worth-expecting FF-OCT innovations include combining the technique of spatial modulation of optical field and computational optical imaging technology to obtain greater penetration depth, as well as exploiting endogenous contrast for functional imaging, e.g., dynamic FF-OCT, which enables noninvasive visualization of tissue dynamic properties or intracellular motility. Different dynamic imaging algorithms are compared using the same OCT data of the colorectal cancer organoid, which helps to understand the disadvantages and advantages of each. In addition, deep learning enhanced FF-OCT provides more valuable characteristic information, which is of great significance for auxiliary diagnosis and organoid detection.

Conclusions: FF-OCT has not been completely exploited and has substantial growth potential. By elaborating the key technologies, performance optimization methods, and application status of FF-OCT, we expect to accelerate the development of FF-OCT in both academic and industry fields. This renewed perspective on FF-OCT may also serve as a road map for future development of invasive 3D super-resolution imaging techniques to solve the problems of microscopic visualization detection.

Significance: Optical polarimetry is an emerging modality that effectively quantifies the bulk optical properties that correlate with the anisotropic structural properties of cardiac tissues. We demonstrate the application of a polarimetric tool for characterizing healthy and fibrotic human myocardial tissues efficiently with a high degree of accuracy.

Aim: The study was aimed to characterize the myocardial tissues from the left ventricle and right ventricle of N  =  7 control and N  =  10 diseased subjects. The diseased subjects were composed of two groups: N  =  7 with rheumatic heart disease (RHD) and N  =  3 with myxomatous valve (MV) disease.

Approach: A portable, affordable, and accurate linear polarization-based diagnostic tool is developed to measure the degree of linear polarization (DOLP) of the myocardial tissues while working at a wavelength of 850 nm.

Results: The sensitivity, specificity, and accuracy of the polarimetric tool in distinguishing the control group from the RHD group were found to be 73.33%, 76.92%, and 75%, respectively, and from the MV group were 91.6%, 62.5%, and 80%, respectively, which demonstrates the efficacy of the polarimetric tool to distinguish the healthy myocardial tissues from diseased tissues.

Conclusions: We have successfully developed a polarimetric tool that can aid cardiologists in characterizing the myocardial tissues in conjunction with endomyocardial biopsy. This work should be followed up with experiments on a large cohort of control and diseased subjects. We intend to create and develop a probe to quantify the DOLP of in vivo heart tissue during surgery.

Significance: The imaging of objects hidden in light-scattering media is a vital practical task in a wide range of applications, including biological imaging. Deep-learning-based methods have been used to reconstruct images behind scattering media under complex scattering conditions, but improvements in the quality of the reconstructed images are required.

Aim: To investigate the effect of image plane on the accuracy of reconstructed images.

Approach: Light reflected from an object passing through glass diffusers is captured by changing the image plane of an optical imaging system. Images are reconstructed by deep learning, and evaluated in terms of structural similarity index measure, classification accuracy of digital images, and training and testing error curves.

Results: The reconstruction accuracy was improved for the case in which the diffuser was imaged, compared to the case where the object was imaged. The training and testing error curves show that the loss converged to lower values in fewer epochs when the diffuser was imaged.

Conclusions: The proposed approach demonstrates an improvement in the accuracy of the reconstruction of objects hidden through glass diffusers by imaging glass diffuser surfaces, and can be applied to objects at unknown locations in a scattering medium.

Significance: Quantitative differential phase contrast (qDPC) microscopy enhances phase contrast by asymmetric illumination using partially coherent light and multiple intensity measurements. However, for live cell imaging, motion artifacts and image acquisition time are important issues. For live cell imaging, a large number of intensity measurements can limit the imaging quality and speed. The minimum number of intensity measurements in qDPC can greatly enhance performance for live imaging.

Aim: To obtain high-contrast, isotropic qDPC images with two intensity measurements and perform time-lapse imaging of biological samples.

Approach: Based on the color-coded design, a dual-color linear-gradient pupil is proposed to achieve isotropic phase contrast response with two intensity measurements. In our method, the purpose of designing a dual-color coded pupil is twofold: first, to obtain a linear amplitude gradient for asymmetric illumination, which is required to get a circular symmetry of transfer function, and second, to reduce the required number of frames for phase retrieval.

Results: To demonstrate the imaging performance of our system, standard microlens arrays were used as samples. We performed time-lapse quantitative phase imaging of rat astrocytes under a low-oxygen environment. Detailed morphology and dynamic changes such as the apoptosis process and migration of cells were observed.

Conclusions: It is shown that dual-color linear-gradient pupils in qDPC can outperform half-circle and vortex pupils, and isotropic phase transfer function can be achieved with only two-axis measurements. The reduced number of frames helps in achieving faster imaging speed as compared to the typical qDPC system. The imaging performance of our system is evaluated by time-lapse imaging of rat astrocytes. Different morphological changes in cells during their life cycle were observed in terms of quantitative phase change values.

Significance: There is a scarcity of published research on the potential role of thermal imaging in the remote detection of respiratory issues due to coronavirus disease-19 (COVID-19). This is a comprehensive study that explores the potential of this imaging technology resulting from its convenient aspects that make it highly accessible: it is contactless, noninvasive, and devoid of harmful radiation effects, and it does not require a complicated installation process.

Aim: We aim to investigate the role of thermal imaging, specifically thermal video, for the identification of SARS-CoV-2-infected people using infrared technology and to explore the role of breathing patterns in different parts of the thorax for the identification of possible COVID-19 infection.

Approach: We used signal moment, signal texture, and shape moment features extracted from five different body regions of interest (whole upper body, chest, face, back, and side) of images obtained from thermal video clips in which optical flow and super-resolution were used. These features were classified into positive and negative COVID-19 using machine learning strategies.

Results: COVID-19 detection for male models [receiver operating characteristic (ROC) area under the ROC curve (AUC) = 0.605 95% confidence intervals (CI) 0.58 to 0.64] is more reliable than for female models (ROC AUC = 0.577 95% CI 0.55 to 0.61). Overall, thermal imaging is not very sensitive nor specific in detecting COVID-19; the metrics were below 60% except for the chest view from males.

Conclusions: We conclude that, although it may be possible to remotely identify some individuals affected by COVID-19, at this time, the diagnostic performance of current methods for body thermal imaging is not good enough to be used as a mass screening tool.

Significance: To achieve early detection of osteoporosis, a simple bone densitometry method using optics was proposed. However, individual differences in soft tissue structure and optical properties can cause errors in quantitative bone densitometry. Therefore, developing optical bone densitometry that is robust to soft tissue variations is important for the early detection of osteoporosis.

Aim: The purpose of this study was to develop an optical bone densitometer that is insensitive to soft tissue, using Monte Carlo simulation and machine learning techniques, and to verify its feasibility.

Approach: We propose a method to measure spatially resolved diffuse light from three directions of the biological tissue model and used machine learning techniques to predict bone density from these data. The three directions are backward, forward, and lateral to the direction of ballistic light irradiation. The method was validated using Monte Carlo simulations using synthetic biological tissue models with 1211 different random structural and optical properties.

Results: The results were computed after a 10-fold cross-validation. From the simulated optical data, the machine learning model predicted bone density with a coefficient of determination of 0.760.

Conclusions: The optical bone densitometry method proposed in this study was found to be robust against individual differences in soft tissue.

Significance: Photoacoustic imaging has shown advantages over the periodontal probing method in measuring the periodontal probing depth, but the large size of conventional photoacoustic transducers prevents imaging of the more posterior teeth.

Aim: Our aim is to develop a photoacoustic imaging system to image the more posterior periodontal pocket.

Approach: We report a clinical “hockey-stick”-style transducer integrated with fibers for periodontal photoacoustic imaging. Cuttlefish ink labeled the periodontal pocket as the photoacoustic contrast agent.

Results: We characterized the imaging system and then measured the pocket depth of 35 swine teeth. Three raters evaluated the performance of the hockey-stick transducer. The measurements between the Williams probing (gold standard) and the photoacoustic methods were blinded but highly correlated. We showed a bias of ∼0.3  mm for the imaging-based technique versus Williams probing. The minimum inter-reliability was over 0.60 for three different raters of varying experience, suggesting that this approach to measure the periodontal pocket is reproducible. Finally, we imaged three pre-molars of a human subject. We could access more upper and posterior teeth than conventional linear transducers.

Conclusions: The unique angle shape of the hockey-stick transducer allows it to image more posterior teeth than regular linear transducers. This study demonstrated the ability of a hockey-stick transducer to measure the periodontal pocket via photoacoustic imaging.

Significance: Means for quantitation of myelinated fibers in peripheral nerve may guide diagnosis and clinical decision making in management of peripheral nerve disorders. Multiphoton microscopy techniques such as the third-harmonic generation enable label-free in vivo imaging of peripheral nerves.

Aim: Develop a multiphoton microscope based on a custom high-power infrared fiber laser for label-free imaging of peripheral nerve.

Approach: A cost-effective multiphoton microscope employing a single fiber laser source at 1300 nm was designed and used for stain-free multicolor imaging of murine and human peripheral nerve.

Results: Second-harmonic generation signal from collagen centered about 650-nm delineated neural connective tissue, whereas third-harmonic general signal centered about 433-nm delineated myelin and other lipids. In sciatic nerve from transgenic reporter mice expressing yellow fluorescent protein within peripheral neurons, three-photon-excitation with emission peak at 527-nm delineated axoplasm. The signal obtained from unlabeled axially sectioned samples was adequate for segmentation of myelinated fibers using commercial image processing software. In unlabeled whole mount specimens, imaging depths over 100-μm were achieved.

Conclusions: A multiphoton microscope powered by a fiber laser enables stain-free histomorphometry of mammalian peripheral nerve. The simplicity of the microscope design carries potential for clinical translation to inform decision making in peripheral nerve disorders.

Significance: Hyperspectral imaging (HSI) provides rich spectral information for improved histopathological cancer detection. However, acquiring high-resolution HSI data for whole-slide imaging (WSI) can be time-consuming and requires a huge amount of storage space.

Aim: WSI using a color camera can be achieved with fast speed, high image resolution, and excellent image quality due to the established techniques. We aim to develop an RGB-guided unsupervised hyperspectral super-resolution reconstruction method that is hypothesized to improve image quality while maintaining the spectral characteristics.

Approach: High-resolution hyperspectral images of 32 histologic slides were obtained via automated WSI. High-resolution RGB histology images were registered to the hyperspectral images for RGB guidance. An unsupervised super-resolution network was trained to take the downsampled low-resolution hyperspectral patches (LR-HSI) and high-resolution RGB patches (HR-RGB) as inputs to reconstruct high-resolution hyperspectral patches (HR-HSI). Then, an Inception-based network was trained with the HR-RGB, original HR-HSI, and generated HR-HSI, respectively, for whole-slide histopathological cancer detection.

Results: Our super-resolution reconstruction network generated high-resolution hyperspectral images with well-maintained spectral characteristics and improved image quality. Image classification using the original hyperspectral data outperformed RGB because of the extra spectral information. The generated hyperspectral image patches further improved the results.

Conclusions: The proposed method potentially reduces image acquisition time, saves storage space without compromising image quality, and improves the image classification performance.

Significance: Confocal endoscopy images often suffer distortions, resulting in image quality degradation and information loss, increasing the difficulty of diagnosis and even leading to misdiagnosis. It is important to assess image quality and filter images with low diagnostic value before diagnosis.

Aim: We propose a no-reference image quality assessment (IQA) method for confocal endoscopy images based on Weber’s law and local descriptors. The proposed method can detect the severity of image degradation by capturing the perceptual structure of an image.

Approach: We created a new dataset of 642 confocal endoscopy images to validate the performance of the proposed method. We then conducted extensive experiments to compare the accuracy and speed of the proposed method with other state-of-the-art IQA methods.

Results: Experimental results demonstrate that the proposed method achieved an SROCC of 0.85 and outperformed other IQA methods.

Conclusions: Given its high consistency in subjective quality assessment, the proposed method can screen high-quality images in practical applications and contribute to diagnosis.

Significance: Needle biopsy (NB) procedures are important for the initial diagnosis of many types of cancer. However, the possibility of NB specimens being unable to provide diagnostic information, (i.e., non-diagnostic sampling) and the time-consuming histological evaluation process can cause delays in diagnoses that affect patient care.

Aim: We aim to demonstrate the advantages of this label-free multimodal nonlinear optical imaging (NLOI) technique as a non-destructive point-of-procedure evaluation method for NB tissue cores, for the visualization and characterization of the tissue microenvironment.

Approach: A portable, label-free, multimodal NLOI system combined second-harmonic generation (SHG) and third-harmonic generation and two- and three-photon autofluorescence (2PF, 3PF) microscopy. It was used for intraoperative imaging of fresh NB tissue cores acquired during canine cancer surgeries, which involved liver, lung, and mammary tumors as well as soft-tissue sarcoma; in total, eight canine patients were recruited. An added tissue culture chamber enabled the use of this NLOI system for longitudinal imaging of fresh NB tissue cores taken from an induced rat mammary tumor and healthy mouse livers.

Results: The intraoperative NLOI system was used to assess fresh canine NB specimens during veterinary cancer surgeries. Histology-like morphological features were visualized by the combination of four NLOI modalities at the point-of-procedure. The NLOI results provided quantitative information on the tissue microenvironment such as the collagen fiber orientation using Fourier-domain SHG analysis and metabolic profiling by optical redox ratio (ORR) defined by 2PF/(2PF + 3PF). The analyses showed that the canine mammary tumor had more randomly oriented collagen fibers compared to the tumor margin, and hepatocarcinoma had a wider distribution of ORR with a lower mean value compared to the liver fibrosis and the normal-appearing liver. Moreover, the loss of metabolic information during tissue degradation of fresh murine NB specimens was shown by overall intensity decreases in all channels and an increase of mean ORR from 0.94 (standard deviation 0.099) to 0.97 (standard deviation 0.077) during 1-h longitudinal imaging of a rat mammary tumor NB specimen. The tissue response to staurosporine (STS), an apoptotic inducer, from fresh murine liver NB specimens was also observed. The mean ORR decreased from 0.86 to 0.74 in the first 40 min and then increased to 0.8 during the rest of the hour of imaging, compared to the imaging results without the addition of STS, which showed a continuous increase of ORR from 0.72 to 0.75.

Conclusions: A label-free, multimodal NLOI platform reveals microstructural and metabolic information of the fresh NB cores during intraoperative cancer imaging. This system has been demonstrated on animal models to show its potential to provide a more comprehensive histological assessment and a better understanding of the unperturbed tumor microenvironment. Considering tissue degradation, or loss of viability upon fixation, this intraoperative NLOI system has the advantage of immediate assessment of freshly excised tissue specimens at the point of procedure.

Significance: The optical redox ratio (ORR) [autofluorescence intensity of the reduced form of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H)/flavin adenine dinucleotide (FAD)] provides a label-free method to quantify cellular metabolism. However, it is unclear whether changes in the ORR with B-cell lymphoma 2 (Bcl-2) family protein inhibition are due to metabolic stress alone or compromised cell viability.

Aim: Determine whether ABT-263 (navitoclax, Bcl-2 family inhibitor) changes the ORR due to changes in mitochondrial function that are independent of changes in cell viability.

Approach: SW48 colon cancer cells were used to investigate changes in ORR, mitochondrial membrane potential, oxygen consumption rates, and cell state (cell growth, viability, proliferation, apoptosis, autophagy, and senescence) with ABT-263, TAK-228 [sapanisertib, mammalian target of rapamycin complex 1/2 (mTORC 1/2) inhibitor], and their combination at 24 h.

Results: Changes in the ORR with Bcl-2 inhibition are driven by increases in both NAD(P)H and FAD autofluorescence, corresponding with increased basal metabolic rate and increased mitochondrial polarization. ABT-263 treatment does not change cell viability or induce autophagy but does induce a senescent phenotype. The metabolic changes seen with ABT-263 treatment are mitigated by combination with mTORC1/2 inhibition.

Conclusions: The ORR is sensitive to increases in mitochondrial polarization, energetic state, and cell senescence, which can change independently from cell viability.

Corrections to the published article include values in Table 1 and provision of an omitted reference.

The erratum corrects the name of the first author listed in a reference cited.