Significance: Diffuse light is ubiquitous in biomedical optics and imaging. Understanding the process of migration of an initial photon population entering tissue to a completely randomized, diffusely scattered population provides valuable insight to the interpretation and design of optical measurements.

Aim: The goal of this perspective is to present a brief, unifying analytical framework to describe how properties of light transition from an initial state to a distributed state as light diffusion occurs.

Approach: First, measurement parameters of light are introduced, and Monte Carlo simulations along with a simple analytical expression are used to explore how these individual parameters might exhibit diffusive behavior. Second, techniques to perform optical measurements are considered, highlighting how various measurement parameters can be leveraged to subsample photon populations.

Results: Simulation results reinforce the fact that light undergoes a transition from a non-diffuse population to one that is first subdiffuse and then fully diffuse. Myriad experimental methods exist to isolate subpopulations of photons, which can be broadly categorized as source- and/or detector-encoded techniques, as well as methods of tagging the tissue of interest.

Conclusions: Characteristic properties of light progressing to diffusion can be described by some form of Gaussian distribution that grows in space, time, angle, wavelength, polarization, and coherence. In some cases, these features can be approximated by simpler exponential behavior. Experimental methods to subsample features of the photon distribution can be achieved or theoretical methods can be used to better interpret the data with this framework.

Significance: Optical trapping is a technique capable of applying minute forces that has been applied to studies spanning single molecules up to microorganisms. Aim: The goal of this perspective is to highlight some of the main advances in the last decade in this field that are pertinent for a biomedical audience. Approach: First, the direct determination of forces in optical tweezers and the combination of optical and acoustic traps, which allows studies across different length scales, are discussed. Then, a review of the progress made in the direct trapping of both single-molecules, and even single-viruses, and single cells with optical forces is outlined. Lastly, future directions for this methodology in biophotonics are discussed. Results: In the 21st century, optical manipulation has expanded its unique capabilities, enabling not only a more detailed study of single molecules and single cells but also of more complex living systems, giving us further insights into important biological activities. Conclusions: Optical forces have played a large role in the biomedical landscape leading to exceptional new biological breakthroughs. The continuous advances in the world of optical trapping will certainly lead to further exploitation, including exciting in-vivo experiments.

Significance: Fluorescence lifetime imaging microscopy (FLIM) measures the decay rate of fluorophores, thus providing insights into molecular interactions. FLIM is a powerful molecular imaging technique that is widely used in biology and medicine. Aim: This perspective highlights some of the major advances in FLIM instrumentation, analysis, and biological and clinical applications that we have found impactful over the last year. Approach: Innovations in FLIM instrumentation resulted in faster acquisition speeds, rapid imaging over large fields of view, and integration with complementary modalities such as single-molecule microscopy or light-sheet microscopy. There were significant developments in FLIM analysis with machine learning approaches to enhance processing speeds, fit-free techniques to analyze images without a priori knowledge, and open-source analysis resources. The advantages and limitations of these recent instrumentation and analysis techniques are summarized. Finally, applications of FLIM in the last year include label-free imaging in biology, ophthalmology, and intraoperative imaging, FLIM of new fluorescent probes, and lifetime-based Förster resonance energy transfer measurements. Conclusions: A large number of high-quality publications over the last year signifies the growing interest in FLIM and ensures continued technological improvements and expanding applications in biomedical research.

Significance: Despite remarkable advances in the core modalities used in combating cancer, malignant diseases remain the second largest cause of death globally. Interstitial photodynamic therapy (IPDT) has emerged as an alternative approach for the treatment of solid tumors.

Aim: The aim of our study is to outline the advancements in IPDT in recent years and provide our vision for the inclusion of IPDT in standard-of-care (SoC) treatment guidelines of specific malignant diseases.

Approach: First, the SoC treatment for solid tumors is described, and the attractive properties of IPDT are presented. Second, the application of IPDT for selected types of tumors is discussed. Finally, future opportunities are considered.

Results: Strong research efforts in academic, clinical, and industrial settings have led to significant improvements in the current implementation of IPDT, and these studies have demonstrated the unique advantages of this modality for the treatment of solid tumors. It is envisioned that further randomized prospective clinical trials and treatment optimization will enable a wide acceptance of IPDT in the clinical community and inclusion in SoC guidelines for well-defined clinical indications.

Conclusions: The minimally invasive nature of this treatment modality combined with the relatively mild side effects makes IPDT a compelling alternative option for treatment in a number of clinical applications. The adaptability of this technique provides many opportunities to both optimize and personalize the treatment.

Significance: The Mueller matrix decomposition method is widely used for the analysis of biological samples. However, its presumed sequential appearance of the basic optical effects (e.g., dichroism, retardance, and depolarization) limits its accuracy and application.

Aim: An approach is proposed for detecting and classifying human melanoma and non-melanoma skin cancer lesions based on the characteristics of the Mueller matrix elements and a random forest (RF) algorithm.

Approach: In the proposal technique, 669 data points corresponding to the 16 elements of the Mueller matrices obtained from 32 tissue samples with squamous cell carcinoma (SCC), basal cell carcinoma (BCC), melanoma, and normal features are input into an RF classifier as predictors.

Results: The results show that the proposed model yields an average precision of 93%. Furthermore, the classification results show that for biological tissues, the circular polarization properties (i.e., elements m₄₄, m₃₄, m₂₄, and m₁₄ of the Mueller matrix) dominate the linear polarization properties (i.e., elements m₁₃, m₃₁, m₂₂, and m₄₁ of the Mueller matrix) in determining the classification outcome of the trained classifier.

Conclusions: Overall, our study provides a simple, accurate, and cost-effective solution for developing a technique for classification and diagnosis of human skin cancer.

Significance: Seborrheic keratoses (SKs) are harmless pigmented skin lesions (PSLs) that may be confused clinically not only with other benign conditions but also with cutaneous melanoma (CM). As SKs are one of the most common neoplasms in adults, the importance of their correct diagnosis is high. Misclassifying SK as malignant is not rare and leads to a high number of unnecessary biopsies. On the other hand, misdiagnosing CM as SK may have a large impact on prognosis or therapy.

Aim: In the non-invasive technique of dermatofluoroscopy, the fluorophores in melanocytes and keratinocytes are excited in vivo with nanosecond laser pulses and the resulting spectrally resolved, melanin-dominated fluorescence signals are used to differentiate between pigmented benign lesions and CM.

Approach: In this single-center, non-interventional study, 33 PSLs of 20 patients were scanned with dermatofluoroscopy in vivo. For all included cases, dermatofluoroscopic signals were compared to pathology classification.

Results: The characteristic spectral features of SK were identified, where the signals are dominated by keratin, NAD(P)H, and melanin. The fluorescence spectra of SKs differed substantially from those of CM: a characteristic spectrum of SK has been identified in 27 of 28 SKs.

Conclusions: The high-accuracy differential diagnosis between CM and SK is possible with dermatofluoroscopy.

Significance: Most cases of high-grade serous ovarian carcinoma originate as serous tubal intraepithelial carcinoma (STIC) lesions in the fallopian tube epithelium (FTE), enabling early endoscopic detection.

Aim: The cell-acquiring fallopian endoscope (CAFE) was built to meet requirements for locating potentially pathological tissue indicated by an alteration in autofluorescence or presence of a targeted fluorophore. A channel was included for directed scrape biopsy of cells from regions of interest.

Approach: Imaging resolution and fluorescence sensitivity were measured using a standard resolution target and fluorescence standards, respectively. A prototype was tested in ex vivo tissue, and collected cells were counted and processed.

Results: Measured imaging resolution was 88  μm at a 5-mm distance, and full field of view was ∼45  deg in air. Reflectance and fluorescence images in ex vivo porcine reproductive tracts were captured, and fit through human tracts was verified. Hemocytometry counts showed that on the order of 10⁵  cells per scrape biopsy could be collected from ex vivo porcine tissue.

Conclusions: All requirements for viewing STIC in the FTE were met, and collected cell counts exceeded input requirements for relevant analyses. Our benchtop findings suggest the potential utility of the CAFE device for in vivo imaging and cell collection in future clinical trials.

Significance: Three-dimensional (3D) vascular and metabolic imaging (VMI) of whole organs in rodents provides critical and important (patho)physiological information in studying animal models of vascular network.

Aim: Autofluorescence metabolic imaging has been used to evaluate mitochondrial metabolites such as nicotinamide adenine dinucleotide (NADH) and flavine adenine dinucleotide (FAD). Leveraging these autofluorescence images of whole organs of rodents, we have developed a 3D vascular segmentation technique to delineate the anatomy of the vasculature as well as mitochondrial metabolic distribution.

Approach: By measuring fluorescence from naturally occurring mitochondrial metabolites combined with light-absorbing properties of hemoglobin, we detected the 3D structure of the vascular tree of rodent lungs, kidneys, hearts, and livers using VMI. For lung VMI, an exogenous fluorescent dye was injected into the trachea for inflation and to separate the airways, confirming no overlap between the segmented vessels and airways.

Results: The kidney vasculature from genetically engineered rats expressing endothelial-specific red fluorescent protein TdTomato confirmed a significant overlap with VMI. This approach abided by the “minimum work” hypothesis of the vascular network fitting to Murray’s law. Finally, the vascular segmentation approach confirmed the vascular regression in rats, induced by ionizing radiation.

Conclusions: Simultaneous vascular and metabolic information extracted from the VMI provides quantitative diagnostic markers without the confounding effects of vascular stains, fillers, or contrast agents.

Significance: The use of the transscleral illumination approach has the potential to simplify the optical design of fundus cameras. In particular, this approach could allow the use of smaller and cheaper cameras that are easier to use by non-specialists, thereby facilitating a wider spread of eye disease screening programs.

Aim: Our aim was to investigate the suitability of transscleral illumination in a fundus camera system. In particular, we explored the impact of the illumination spectrum and the eye pigmentation on the quality of the image. These factors have never been systematically investigated before in the literature on transscleral illumination.

Approach: A fundus camera was constructed using transscleral illumination. We studied the influence of eye pigmentation and choice of illumination spectra on the image quality for a group of 10 individuals with varied skin pigmentation, ranging from pale white (North-European) to darkest brown (African). The influence of the light source spectrum on the image quality was assessed using wavelength filters.

Results: There was a difference of a factor of 100 in the signal level of retinal images between individuals with low and high skin pigmentation. The image contrast was highest using illumination wavelengths of 500 to 600 nm. The illumination level can be adjusted to obtain high-quality images for highly pigmented eyes while keeping the system eye-safe.

Conclusions: We have demonstrated that a fundus camera with transscleral illumination can provide high-quality images. However, the variations observed in scleral and retinal pigmentation in a practical setting require a system that must be able to adapt illumination and/or exposure to the individual patient.

Significance: In neurosurgery, it is essential to differentiate between tumor and healthy brain regions to maximize tumor resection while minimizing damage to vital healthy brain tissue. However, conventional intraoperative imaging tools used to guide neurosurgery are often unable to distinguish tumor margins, particularly in infiltrative tumor regions and low-grade gliomas.

Aim: The aim of this work is to assess the feasibility of a label-free molecular imaging tool called stimulated Raman scattering-spectroscopic optical coherence tomography (SRS-SOCT) to differentiate between healthy brain tissue and tumor based on (1) structural biomarkers derived from the decay rate of signals as a function of depth and (2) molecular biomarkers based on relative differences in lipid and protein composition extracted from the SRS signals.

Approach: SRS-SOCT combines the molecular sensitivity of SRS (based on vibrational spectroscopy) with the spatial and spectral multiplexing capabilities of SOCT to enable fast, spatially and spectrally resolved molecular imaging. SRS-SOCT is applied to image a 9L gliosarcoma rat tumor model, a well-characterized model that recapitulates human high-grade gliomas, including high proliferative capability, high vascularization, and infiltration at the margin. Structural and biochemical signatures acquired from SRS-SOCT are extracted to identify healthy and tumor tissues.

Results: Data show that SRS-SOCT provides light-scattering-based signatures that correlate with the presence of tumors, similar to conventional OCT. Further, nonlinear phase changes from the SRS interaction, as measured with SRS-SOCT, provide an additional measure to clearly separate tumor tissue from healthy brain regions. We also show that the nonlinear phase signals in SRS-SOCT provide a signal-to-noise advantage over the nonlinear amplitude signals for identifying tumors.

Conclusions: SRS-SOCT can distinguish both spatial and spectral features that identify tumor regions in the 9L gliosarcoma rat model. This tool provides fast, label-free, nondestructive, and spatially resolved molecular information that, with future development, can potentially assist in identifying tumor margins in neurosurgery.

Significance: Chronic lung allograft dysfunction (CLAD) is the leading cause of death in transplant patients who survive past the first year post-transplant. Current diagnosis is based on sustained decline in lung function; there is a need for tools that can identify CLAD onset.

Aim: Endoscopic optical coherence tomography (OCT) can visualize structural changes in the small airways, which are of interest in CLAD progression. We aim to identify OCT features in the small airways of lung allografts that correlate with CLAD status.

Approach: Imaging was conducted with an endoscopic rotary pullback OCT catheter during routine bronchoscopy procedures (n  =  54), collecting volumetric scans of three segmental airways per patient. Six features of interest were identified, and four blinded raters scored the dataset on the presence and intensity of each feature.

Results: Airway dilation (AD) was the only feature found to significantly (p  <  0.003) correlate with CLAD diagnosis (R  =  0.40 to 0.61). AD could also be fairly consistently scored between raters (κ_inter-rater  =  0.48, κ_intra-rater  =  0.64). There is a stronger relationship between AD and the combined obstructive and restrictive (BOS + RAS) phenotypes than the obstructive-only (BOS) phenotype for two raters (R  =  0.92  ,  0.94).

Conclusions: OCT examination of small AD shows potential as a diagnostic indicator for CLAD and CLAD phenotype and merits further exploration.

Significance: Simulations have the potential to be a powerful tool when planning the placement of photoacoustic imaging system components for surgical guidance. While elastic simulations (which include both compressional and shear waves) are expected to more accurately represent the physical transcranial acoustic wave propagation process, these simulations are more time-consuming and memory-intensive than the compressional-wave-only simulations that our group previously used to identify optimal acoustic windows for transcranial photoacoustic imaging.

Aim: We present qualitative and quantitative comparisons of compressional and elastic wave simulations to determine which option is more suitable for preoperative surgical planning.

Approach: Compressional and elastic photoacoustic k-Wave simulations were performed based on a computed tomography volume of a human cadaver head. Photoacoustic sources were placed in the locations of the internal carotid arteries and likely positions of neurosurgical instrument tips. Transducers received signals from three previously identified optimal acoustic windows (i.e., the ocular, nasal, and temporal regions). Target detectability, image-based target size estimates, and target-to-instrument distances were measured using the generalized contrast-to-noise ratio (gCNR), resolution, and relative source distances, respectively, for each simulation method.

Results: The gCNR was equivalent between compressional and elastic simulations. The areas of the −6  dB contours of point spread functions utilized to measure resolution differed by 0.33 to 3.35  mm². Target-to-instrument distance measurements were within 1.24 mm of the true distances.

Conclusions: These results indicate that it is likely sufficient to utilize the less time-consuming, less memory-intensive compressional wave simulations for presurgical planning.

Significance: Hyperspectral and multispectral imaging (HMSI) in medical applications provides information about the physiology, morphology, and composition of tissues and organs. The use of these technologies enables the evaluation of biological objects and can potentially be applied as an objective assessment tool for medical professionals. Aim: Our study investigates HMSI systems for their usability in medical applications. Approach: Four HMSI systems (one hyperspectral pushbroom camera and three multispectral snapshot cameras) were examined and a spectrometer was used as a reference system, which was initially validated with a standardized color chart. The spectral accuracy of the cameras reproducing chemical properties of different biological objects (porcine blood, physiological porcine tissue, and pathological porcine tissue) was analyzed using the Pearson correlation coefficient. Results: All the HMSI cameras examined were able to provide the characteristic spectral properties of blood and tissues. A pushbroom camera and two snapshot systems achieve Pearson coefficients of at least 0.97 compared to the ground truth, indicating a very high positive correlation. Only one snapshot camera performs moderately to high positive correlation (0.59 to 0.85). Conclusion: The knowledge of the suitability of HMSI cameras for accurate measurement of chemical properties of biological objects offers a good opportunity for the selection of the optimal imaging tool for specific medical applications, such as organ transplantation.

Significance: Collagen is the most abundant protein in vertebrates and is found in tissues that regularly experience tension, compression, and shear forces. However, the underlying mechanism of collagen fibril formation and remodeling is poorly understood.

Aim: We explore how a collagen monomer is visualized using fluorescence microscopy and how its spatial orientation is determined. Defining the orientation of collagen monomers is not a trivial problem, as the monomer has a weak contrast and is relatively small. It is possible to attach fluorescence tags for contrast, but the size is still a problem for detecting orientation using fluorescence microscopy.

Approach: We present two methods for detecting a monomer and classifying its orientation. A modified Gabor filter set and an automatic classifier trained by convolutional neural network based on a synthetic dataset were used.

Results: By evaluating the performance of these two approaches with synthetic and experimental data, our results show that it is possible to determine the location and orientation with an error of ∼37  deg of a single monomer with fluorescence microscopy.

Conclusions: These findings can contribute to our understanding of collagen monomers interaction with collagen fibrils surface during fibril formation and remodeling.

Significance: Measurements of auditory ossicles displacement are commonly carried out by means of laser-Doppler vibrometry (LDV), which is considered to be a gold standard. The limitation of the LDV method, especially for in vivo measurements, is the necessity to expose an object in a straight line to a laser beam operating from a distance. An alternative to this approach is the use of a handheld laser-fiber vibrometry probe (HLFVP) with a curved tip.

Aim: We evaluate the feasibility of an HLFVP with a curved tip for measuring sound-induced displacement of the auditory ossicles.

Approach: A handheld vibrometer probe guiding the laser beam with a fiber-optic cable was used for displacement measurements of the incus body and the posterior crus of the stapes. Tonal stimuli at frequencies of 0.5, 1, 2, and 4 kHz were presented by means of an insert earphone positioned in the outer ear canal. The probe was fixed at the measurement site using a tripod or hand-held by one of the two surgeons.

Results: The measurements were carried out on six fresh temporal bones. Multivariate analysis of variance showed statistically significant differences for stimulus frequency (F3,143  =  29.37, p  <  0.001, and η2  =  0.35), bone (F5,143  =  4.61, p  =  0.001, and η2  =  0.01), and measurement site (F1,143  =  4.74, p  =  0.03, and η2  =  0.02) in the absence of statistically significant differences for the probe fixation method (F2,143  =  0.15, p  =  0.862, and η2  =  0.001). Standard deviations of the means were 6.9, 2.6, 1.9, and 0.6  nm  /  Pa for frequency, bone, site, and fixation, respectively. Ear transfer functions were found to be consistent with literature data.

Conclusions: The feasibility of applying HLFVP to measure the displacement of auditory ossicles has been confirmed. HLFVP offers the possibility of carrying out measurements at various angles; however, this needs to be standardized taking into account anatomical limitations and surgical convenience.

Significance: The light dose in photodynamic therapy (PDT) has a considerable influence on its treatment effect, and irradiance uniformity is an issue of much concern for researchers. However, achieving intelligent and personalized dosimetry adjustments remains a challenge for current PDT instruments.

Aim: To meet the requirements of intelligent and personalized dosimetry adjustments for the light dose on an irregular surface, a new PDT device with its optimal control method is proposed.

Approach: This research introduces a new PDT device that includes a 3D scanner, a light-emitting diode (LED) array, and a computer. The 3D scanner is proposed to generate the point cloud of the lesion and the LED array light source, and obtain the relative position and rotation parameters between them. Then, an image segmentation algorithm is used to segment the lesion point cloud into several cluster regions. Last, the current of each LED unit is adjusted separately to achieve the expected irradiance on each cluster.

Results: Compared with the general light source, the optimized light source increases the effective irradiance area by 9% to 15% and improves its uniformity by ∼9  %   on a human port-wine stain head model.

Conclusions: The device and its optimal method may be used for optimizing the light dosimetry to realize intelligent and personalized treatment.

The erratum corrects an author affiliation.