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.

Significance: Optical coherence tomography (OCT) provides cross-sectional and volumetric images of backscattering from biological tissue that reveal the tissue morphology. The strength of the scattering, characterized by an attenuation coefficient, represents an alternative and complementary tissue optical property, which can be characterized by parametric imaging of the OCT attenuation coefficient. Over the last 15 years, a multitude of studies have been reported seeking to advance methods to determine the OCT attenuation coefficient and developing them toward clinical applications.

Aim: Our review provides an overview of the main models and methods, their assumptions and applicability, together with a survey of preclinical and clinical demonstrations and their translation potential.

Results: The use of the attenuation coefficient, particularly when presented in the form of parametric en face images, is shown to be applicable in various medical fields. Most studies show the promise of the OCT attenuation coefficient in differentiating between tissues of clinical interest but vary widely in approach.

Conclusions: As a future step, a consensus on the model and method used for the determination of the attenuation coefficient is an important precursor to large-scale studies. With our review, we hope to provide a basis for discussion toward establishing this consensus.

Significance: 2019 SPIE Photonics West conference hosted over 5000 presentations. Some important presentations in the Industrial Laser, Laser Source and Application (LASE) and Optoelectronics, Photonic Materials and Devices (OPTO) sections of the SPIE Photonics West conference have a risk of being overlooked by the biomedical community despite their implications for the field of biophotonics. We review some recent advances in the area of development coherent radiation sources in the infrared (IR), ultraviolet (UV), and terahertz (THz) regimes.

Aim: Recent advances in coherent radiation sources in the IR, deep UV, and THz regimes were outlined, and the importance of each presentation to one or more promising biomedical applications was assessed.

Approach: Presentations and proceedings from the LASE and OPTO sections were reviewed for inclusion. Emphasis was placed on talks from the Nonlinear Frequency Generation and Conversion: Materials and Devices XVIII conference, and the Terahertz, RF, Millimeter, and Submillimeter-Wave Technology and Applications XII conference. Conference sections that directly focused on biomedical applications were excluded.

Results: Enhanced IR supercontinuum generation with compact supercontinuum sources may allow for real-time biomarker detection and create new opportunities for imaging tissues using the third biological window (1600 to 1850 nm). Efficient methods to generate deep UV (200 to 260 nm) radiation allow for the study of biologically important molecules through techniques such as resonance Raman spectroscopy while avoiding fluorescence overlap. Likewise, novel and improved THz generation methods seek to bridge the “THz gap” that has previously limited biomedical applications.

Conclusions: Advances in coherent radiation sources in the IR, UV, and THz regimes have created new opportunities for biomedical optics research.

Significance. Quantifying human milk composition is important for daily nutritional management in neonatal intensive cares worldwide. Photonic solutions based on visible light can potentially aid in this analysis, as energy content of human milk depends largely on fat content, and the optical scattering properties of human milk predominantly depend on the size and concentration of fat globules. However, it is expected that human milk scattering changes upon homogenization, routinely done before analysis, which may affect fat globule size.

Aim. The first aim of this study was to investigate how the most common homogenization methods (gently inverting by hand, vortexing, and sonication) affect the optical properties of human milk. The second aim was to estimate the scattering contribution of casein micelles, the second most dominant scatterers in human milk.

Approach. We combined diffuse reflectance spectroscopy with spectroscopic optical coherence tomography to measure the scattering coefficient μ_s, reduced scattering coefficient μs′, and anisotropy g between 450 and 600 nm.

Results. Sonication induced the strongest changes in μ_s, μs′, and g compared to the gently inverted samples (203%, 202%, and 7%, respectively, at 550 nm), but also vortexing changed μs′ with 20%. Although casein micelles only showed a modest contribution to μ_s and g at 550 nm (7% and 1%, respectively), their contribution to μs′ was 29%.

Conclusions. The scattering properties of human milk strongly depend on the homogenization method that is employed, and gentle inversion should be the preferred method. The contribution of casein micelles was relatively small for μ_s and g but considerably larger for μs′.

Significance: In laser therapy and diagnosis of skin diseases, the irradiated light distribution, which is determined by the absorption coefficient μ_a and reduced scattering coefficient μs′ of the epidermis, dermis, and subcutaneous fat, affects the treatment outcome and diagnosis accuracy. Although values for μ_a and μs′ have been reported, detailed analysis for Asian skin tissues is still lacking.

Aim: We present μ_a and μs′ measurements of Asian skin tissues in the 400- to 1100-nm wavelength range for evaluating optical penetration depth and energy deposition.

Approach: The measurements with Asian human skin samples are performed employing a double integrating sphere spectrometric system and an inverse Monte Carlo technique. Using the measured parameters, the optical penetration depth and energy deposition are quantitatively analyzed.

Results: The μ_a of the epidermis layer varies among different ethnic groups, while the μ_a of the other layers and the μs′ of all of the layers exhibit almost no differences. The analysis reveals that the optical penetration depth and the energy deposition affect the photodynamic therapy treatment depth and the heat production in skin tissue, respectively.

Conclusions: The experimentally measured values of μ_a and μs′ for Asian skin tissues are presented, and the light behavior in Asian skin tissues is analyzed using a layered tissue model.

Significance: We present a Monte Carlo (MC) computational framework that simulates near-infrared (NIR) hyperspectral imaging (HSI) aimed at assisting quantification of the in vivo hemodynamic and metabolic states of the exposed cerebral cortex in small animal experiments. This can be done by targeting the NIR spectral signatures of oxygenated (HbO₂) and deoxygenated (HHb) hemoglobin for hemodynamics as well as the oxidative state of cytochrome-c-oxidase (oxCCO) for measuring tissue metabolism.

Aim: The aim of this work is to investigate the performances of HSI for this specific application as well as to assess key factors for the future design and operation of a benchtop system.

Approach: The MC framework, based on Mesh-based Monte Carlo (MMC), reproduces a section of the exposed cortex of a mouse from an in vivo image and replicates hyperspectral illumination and detection at multiple NIR wavelengths (up to 121).

Results: The results demonstrate: (1) the fitness of the MC framework to correctly simulate hyperspectral data acquisition; (2) the capability of HSI to reconstruct spatial changes in the concentrations of HbO₂, HHb, and oxCCO during a simulated hypoxic condition; (3) that eight optimally selected wavelengths between 780 and 900 nm provide minimal differences in the accuracy of the hyperspectral results, compared to the “gold standard” of 121 wavelengths; and (4) the possibility to mitigate partial pathlength effects in the reconstructed data and to enhance quantification of the hemodynamic and metabolic responses.

Conclusions: The MC framework is proved to be a flexible and useful tool for simulating HSI also for different applications and targets.

Significance: Understanding how the brain recovers from cerebral tissue and vascular damage after an ischemic event can help develop new therapeutic strategies for the treatment of stroke.

Aim: We investigated cerebral tissue repair and microvasculature regeneration and function after a targeted ischemic stroke.

Approach: Following photothrombosis occlusion of microvasculature, chronic optical coherence tomography (OCT)-based angiography was used to track ischemic tissue repair and microvasculature regeneration at three different cortical depths and up to 28 days in awake animals. Capillary network orientation analysis was performed to study the structural pattern of newly formed microvasculature. Based on the time-resolved OCT-angiography, we also investigated capillary stalling, which is likely related to ischemic stroke-induced inflammation.

Results: Deeper cerebral tissue was found to have a larger ischemic area than shallower regions at any time point during the course of poststroke recovery, which suggests that cerebral tissue located deep in the cortex is more vulnerable. Regenerated microvasculature had a highly organized pattern at all cortical depths with a higher degree of structural reorganization in deeper regions. Additionally, capillary stalling event analysis revealed that cerebral ischemia augmented stalling events considerably.

Conclusion: Longitudinal OCT angiography reveals that regenerated capillary network has a highly directional pattern and an increased density and incidence of capillary stalling event.

Significance: Optical coherence tomography (OCT) has proven useful for detecting various oral maxillofacial abnormalities. To apply it to clinical applications including biopsy guidance and routine screening, a handheld imaging probe is indispensable. OCT probes reported for oral maxillofacial imaging were either based on a bulky galvanometric mirror pair (not compact or long enough) or a distal-end microelectromechanical systems (MEMS) scanner (raised safety concerns), or adapted from fiber-optic catheters (ill-suited for oral cavity geometry).

Aim: To develop a handheld probe featuring great compactness and excellent maneuverability for oral maxillofacial tissue imaging.

Approach: A dual-axis MEMS scanner was deployed at the proximal end of the probe and the scanned beam was relayed to the distal end through a 4f configuration. Such design provides both a perfect dual-axis telecentric scan and excellent compactness.

Results: A handheld probe with a rigid part 70 mm in length and 7 mm in diameter and weighing 25 g in total was demonstrated through both ex vivo and in vivo experiments, including structural visualization of various oral maxillofacial tissues and monitoring the recovery process of an oral mucosa canker sore.

Conclusions: The proposed probe exhibits excellent maneuverability and imaging performance showing great potential in clinical applications.

Significance: Green-fluorescent protein (GFP)-like fluorescent proteins are used extensively as genetic reporters in fluorescence imaging due to their distinctive ability to form chromophores independent of external enzymes or cofactors. However, their use for photoacoustic (PA) imaging has not been demonstrated in mammalian tissues because they possess low PA signal generation efficiency in their native state. By engineering them to become nonfluorescent (NF), their PA generation efficiency was increased. This enabled the generation of in vivo contrast in mice, making it possible for GFP-like proteins to be used as PA genetic reporters in mammalian tissues.

Aim: The aim was to develop a darkened GFP-like protein reporter by modifying E2 crimson fluorescent protein (FP) in order to generate NF mutant proteins with high PA signal generation efficiency for in vivo imaging.

Approach: The absorbance, fluorescence, and PA amplitude spectra of purified protein solutions of the FP and engineered NF mutants were measured in order to identify the mutant with the highest PA signal generation efficiency. This mutant, referred to as NFA, and the native FP were then stably expressed in LS174T human colorectal tumor cells using a retroviral vector and tested for photostability under continuous pulsed illumination. To demonstrate the improvement in PA signal generation in vivo, cells expressing the FP and NFA mutant were injected subcutaneously in mice and imaged using a Fabry–Perot based PA scanner.

Results: The NF mutants of E2 crimson exhibited fluorescence that was 2 orders of magnitude lower than the FP and a higher PA signal generation efficiency; the NFA-generated PA signal was approximately three times higher than the FP. Tumor cells expressing the NFA mutant provided sufficient image contrast to be visualized in vivo against a background of strong vascular contrast, whereas the FP-expressing cells did not generate visible contrast.

Conclusion: A GFP-like protein has been demonstrated as a genetic reporter for PA imaging in mammalian tissue for the first time. This was achieved by a mutation, which darkened the FP and increased the PA signal generation efficiency. The approach taken suggests that GFP-like proteins could be a promising addition to the current cohort of genetic reporters available for in vivo PA imaging.

Significance: Quantitative optoacoustic (OA) imaging has the potential to provide blood oxygen saturation (SO₂) estimates due to the proportionality between the measured signal and the blood’s absorption coefficient. However, due to the wavelength-dependent attenuation of light in tissue, a spectral correction of the OA signals is required, and a prime challenge is the validation of both the optical characterization of the tissue and the SO₂.

Aim: We propose to assess the reliability of SO₂ levels retrieved from spectral fitting by measuring the similarity of OA spectra to the fitted blood absorption spectra.

Approach: We introduce a metric that quantifies the trends of blood spectra by assigning a pair of spectral slopes to each spectrum. The applicability of the metric is illustrated with in vivo measurements on a human forearm.

Results: We show that physiologically sound SO₂ values do not necessarily imply a successful spectral correction and demonstrate how the metric can be used to distinguish SO₂ values that are trustworthy from unreliable ones.

Conclusions: The metric is independent of the methods used for the OA data acquisition, image reconstruction, and spectral correction, thus it can be readily combined with existing approaches, in order to monitor the accuracy of quantitative OA imaging.

Significance: Accurate and objective identification of Alzheimer’s disease (AD) and dementia with Lewy bodies (DLB) is of major clinical importance due to the current lack of low-cost and noninvasive diagnostic tools to differentiate between the two. Developing an approach for such identification can have a great impact in the field of dementia diseases as it would offer physicians a routine objective test to support their diagnoses. The problem is especially acute because these two dementias have some common symptoms and characteristics, which can lead to misdiagnosis of DLB as AD and vice versa, mainly at their early stages.

Aim: The aim is to evaluate the potential of mid-infrared (IR) spectroscopy in tandem with machine learning algorithms as a sensitive method to detect minor changes in the biochemical structures that accompany the development of AD and DLB based on a simple peripheral blood test, thus improving the diagnostic accuracy of differentiation between DLB and AD.

Approach: IR microspectroscopy was used to examine white blood cells and plasma isolated from 56 individuals: 26 controls, 20 AD patients, and 10 DLB patients. The measured spectra were analyzed via machine learning.

Results: Our encouraging results show that it is possible to differentiate between dementia (AD and DLB) and controls with an ∼86  %   success rate and between DLB and AD patients with a success rate of better than 93%.

Conclusions: The success of this method makes it possible to suggest a new, simple, and powerful tool for the mental health professional, with the potential to improve the reliability and objectivity of diagnoses of both AD and DLB.

Significance: Recent evidence suggests that hydroxyapatite (HAP) in sub-retinal pigment epithelial (sub-RPE) deposits in aged human eyes may act to nucleate and contribute to their growth to clinically detectable size. Sub-RPE deposits such as drusen are clinical hallmarks of age-related macular degeneration (AMD), therefore enhanced and earlier detection is a clinical need. We found that tetracycline-family antibiotics, long known to stain HAP in teeth and bones, can also label the HAP in sub-RPE deposits. However, HAP-bound tetracycline fluorescence excitation and emission spectra overlap with the well-known autofluorescence of outer retinal tissues, making them difficult to resolve.

Aim: In this initial study, we sought to determine if the HAP-bound tetracyclines also exhibit enhanced fluorescence lifetimes, providing a useful difference in lifetime compared with the short lifetimes observed in vivo in the human retina by the pioneering work of Schweitzer, Zinkernagel, Hammer, and their colleagues, and thus a large enough effect size to resolve the HAP from background by fluorescence lifetime imaging.

Approach: We stained authentic HAP with tetracyclines and measured the lifetime(s) by phase fluorometry, and stained aged, fixed human cadaver retinas with drusen with selected tetracyclines and imaged them by fluorescence lifetime imaging microscopy (FLIM).

Results: We found that chlortetracycline and doxycycline exhibited substantial increase in fluorescence lifetime compared to the free antibiotics and the retinal background, and the drusen were easily resolvable from the retinal background in these specimens by FLIM.

Conclusions: These findings suggest that FLIM imaging of tetracycline (and potentially other molecules) binding to HAP could become a diagnostic tool for the development and progression of AMD.

Significance: Currently, various scaffolds with immobilized cells are widely used in tissue engineering and regenerative medicine. However, the physiological activity and cell viability in such constructs might be impaired due to a lack of oxygen and nutrients. Photobiomodulation (PBM) is a promising method of preconditioning cells to increase their metabolic activity and to activate proliferation or differentiation.

Aim: Investigation of the potential of PBM for stimulation of cell activities in hydrogels.

Approach: Mesenchymal stromal cells (MSCs) isolated from human gingival mucosa were encapsulated in modified fibrin hydrogels with different thicknesses and concentrations. Constructs with cells were subjected to a single-time exposure to red (630 nm) and near-infrared (IR) (840 nm) low-intensity irradiation. After 3 days of cultivation, the viability and physiological activity of the cells were analyzed using confocal microscopy and a set of classical tests for cytotoxicity.

Results: The cell viability in fibrin hydrogels depended both on the thickness of the hydrogels and the concentration of gel-forming proteins. The PBM was able to improve cell viability in hydrogels. The most pronounced effect was achieved with near-IR irradiation at the 840-nm wavelength.

Conclusions: PBM using near-IR light can be applied for stimulation of MSCs metabolism and proliferation in hydrogel-based constructs with thicknesses up to 3 mm.

The erratum corrects a misspelled author surname.

The erratum corrects an error in the published article.

The erratum notes a correction to Fig. 5 for the published article.