Guest Editors introduce the Special Section on Photodynamic Therapy for the Journal of Biomedical Optics, Volume 25, Issue 6.

Significance: While clinical treatment of actinic keratosis by photodynamic therapy (PDT) is widely practiced, there is a well-known variability in response, primarily caused by heterogeneous accumulation of the photosensitizer protoporphyrin IX (PpIX) between patients and between lesions, but measurement of this is rarely done.

Aim: Develop a smartphone-based fluorescence imager for simple quantitative photography of the lesions and their PpIX levels that can be used in a new clinical workflow to guide the reliability of aminolevulinic acid (ALA) application for improved lesion clearance.

Approach: The smartphone fluorescence imager uses an iPhone and a custom iOS application for image acquisition, a 3D-printed base for measurement standardization, an emission filter for PpIX fluorescence isolation, and a 405-nm LED ring for optical excitation. System performance was tested to ensure measurement reproducibility and the ability to capture photosensitizer accumulation and photobleaching in pre-clinical and clinical settings.

Results: PpIX fluorescence signal from tissue-simulating phantoms showed linear sensitivity in the 0.01 to 2.0 μM range. Murine studies with Ameluz^® aminolevulinic acid (ALA) gel and initial human testing with Levulan^® ALA cream verified that in-vivo imaging was feasible, including that PpIX production over 1 h is easily captured and that photobleaching from the light treatment could be quantified.

Conclusions: The presented device is the first quantitative wide-field fluorescence imaging system for PDT dosimetry designed for clinical skin use and for maximal ease of translation into clinical workflow. The results lay the foundation for using the system in clinical studies to establish treatment thresholds for the individualization of PDT treatment.

Interstitial photodynamic therapy (iPDT) with 5-aminolevulinic acid (ALA) is a possible alternative treatment for malignant brain tumors. Further evaluation is, however, required before it can be clinically applied. Computational simulation of the photophysical process in ALA-iPDT can offer a quantitative tool for understanding treatment outcomes, which depend on various variables related to clinical treatment conditions. We propose a clinical simulation method of ALA-iPDT for malignant brain tumors using a singlet oxygen (O1₂) model and O1₂ threshold to induce cell death. In this method, the amount of O1₂ generated is calculated using a photosensitizer photobleaching coefficient and O1₂ quantum yield, which have been measured in several previous studies. Results of the simulation using clinical magnetic resonance imaging data show the need to specify the insertion positions of cylindrical light diffusers and the level of light fluence. Detailed analysis with a numerical brain tumor model demonstrates that ALA-iPDT treatment outcomes depend on combinations of photobleaching and threshold values. These results indicate that individual medical procedures, including pretreatment planning and treatment monitoring, will greatly benefit from simulation of ALA-iPDT outcomes.

Two pronounced absorption peaks in blue and red ranges of the chlorin-based photosensitizer (PS) absorption spectrum provide additional benefits in photodynamic therapy (PDT) performance. Differing optical properties of biological tissues in these ranges allow for both dual-wavelength diagnostics and PDT performance. We provide a comparative analysis of different PDT regimes performed with blue and red lights and their combination, with doses varying from 50 to 150  J  /  cm². The study was performed on the intact skin of a rabbit ear inner surface, with the use of chlorin e6 as a PS. PDT procedure protocol included monitoring of the treated site with fluorescence imaging technique to evaluate PS accumulation and photobleaching, as well as with optical coherence tomography (OCT) to register morphological and functional responses of the tissue. Optical diagnostic observations were compared with the results of histopathology examination. We demonstrated that PDT procedures with the considered regimes induce weaker organism reaction manifested by edema in normal tissue as compared to irradiation-only exposures with the same light doses. The light doses delivered with red light induce weaker tissue reaction as compared to the same doses delivered with blue light only or with a combination of red and blue lights in equal parts. Results of in-vivo OCT monitoring of tissue reaction are in agreement with the results of histopathology study.

Photodynamic therapy (PDT) is a well-established treatment modality for cancer and other malignant diseases; however, quantities such as light fluence and PDT dose do not fully account for all of the dynamic interactions between the key components involved. In particular, fluence rate (ϕ) effects, which impact the photochemical oxygen consumption rate, are not accounted for. In this preclinical study, reacted reactive oxygen species ([ROS]_rx) was investigated as a dosimetric quantity for PDT outcome. The ability of [ROS]_rx to predict the cure index (CI) of tumor growth, CI  =  1  −  k  /  k_ctr, where k and k_ctr are the growth rate of tumor under PDT study and the control tumor without PDT, respectively, for benzoporphyrin derivative (BPD)-mediated PDT, was examined. Mice bearing radiation-induced fibrosarcoma (RIF) tumors were treated with different in-air fluences (Φ  =  22.5 to 166.7  J  /  cm²) and in-air fluence rates (ϕ_air  =  75 to 250   mW  /  cm²) with a BPD dose of 1  mg  /  kg and a drug-light interval (DLI) of 15 min. Treatment was delivered with a collimated laser beam of 1-cm-diameter at 690 nm. Explicit measurements of in-air light fluence rate, tissue oxygen concentration, and BPD concentration were used to calculate for [ROS]_rx. Light fluence rate at 3-mm depth (ϕ_3  mm), determined based on Monte-Carlo simulations, was used in the calculation of [ROS]_rx at the base of tumor. CI was used as an endpoint for three dose metrics: light fluence, PDT dose, and [ROS]_rx. PDT dose was defined as the product of the time-integral of photosensitizer concentration and ϕ_3  mm. Preliminary studies show that [ROS]_rx best correlates with CI and is an effective dosimetric quantity that can predict treatment outcome. The threshold dose for [ROS]_rx for vascular BPD-mediated PDT using DLI of 15 min is determined to be 0.26 mM and is about 3.8 times smaller than the corresponding value for conventional BPD-mediated PDT using DLI of 3 h.

Significance: Singlet oxygen is a key cytotoxic agent in photodynamic therapy (PDT). As such, its imaging is highly desirable, but existing direct imaging methods are still limited by the exceptionally low yield of the luminescence signal. Singlet oxygen feedback delayed fluorescence (SOFDF) of the photosensitizer is a higher yield alternative for indirect measurement of this signal.

Aim: The aim was to explore feasibility of SOFDF imaging in vivo in tumor-bearing mice during PDT and investigate how SOFDF images can be transformed into images of singlet oxygen. In addition, we study whether lysosome permeabilization can be visualized through fluorescence lifetime.

Approach: Mice were intravenously injected with 2.5 mg/kg of photosensitizer aluminum(III) phthalocyanine tetrasulfonate (AlPcS₄) 20 h prior to experiments, having subcutaneous BxPC3 pancreas tumors. Time-resolved delayed fluorescence and prompt fluorescence (PF) were imaged using an intensified time-gated camera with 10-Hz pulsed laser excitation at 690 nm.

Results: Delayed emission from AlPcS₄ was detected with lifetimes 7 to 11  μs, which was attributed to SOFDF and shown to be oxygen-dependent. Singlet oxygen images were approximated by the ratio of SOFDF/PF at each pixel. SOFDF images of a good quality could be captured within several seconds with a radiant exposure of ∼20  mJ  /  cm². In addition, lifetime images of AlPcS₄ PF in ns-time domain enabled us to visualize the event of lysosome permeabilization, as the lifetime increased from ∼4.7 to 5.2 ns.

Conclusions: Imaging of SOFDF in vivo in mouse tumor during PDT with AlPcS₄ is feasible, and it is a promising method for singlet molecular oxygen monitoring. Moreover, the time-gated approach also enables visualization of the lysosome permeabilization that alters the PF lifetime.

Significance: Side effects of many cancer treatments are associated with the production of reactive oxygen species (ROS) in normal tissues. This explains why patients treated by photodynamic therapy (PDT) often suffer from skin photosensitization, whereas those subject to radiotherapies frequently experience damages in various organs, including the skin.

Aim: Catechin, which belongs to the natural flavanols family, is well known for its antioxidant properties. Hence, our main objective was to investigate whether catechin can reduce damages induced by PDT using protoporphyrin IX (PpIX-PDT), an endogenous photosensitizer commonly used in dermatology.

Approach: An in vivo model, the chick embryo chorioallantoic membrane (CAM), was used for this study. An amount of 20  μl of a solution containing 5-aminolevulinic acid, a natural precursor of PpIX, was applied topically on the CAM 4 h before PDTs (405 nm, 2.9  mW  /  cm², 1.2  J  /  cm²). Catechin was applied at different concentrations (1 to 50  μM) and times (0 to 240 min) before PDT. In addition, we assessed the potency of catechin to reduce the PpIX fluorescence photobleaching induced by PDT.

Results: We observed that catechin significantly reduces the vascular damages generated by PpIX-PDT. Moreover, we have shown that catechin inhibits PpIX photobleaching.

Conclusions: These observations suggest that catechin significantly reduces the level of ROS produced by PpIX-PDT.

Photodynamic therapy (PDT), a spatially localized phototoxic therapy that involves irradiation of a photosensitizer (PS) with specific wavelengths of light, has shown exceptional promise in impacting cancer treatment outcomes, particularly oral cancer. To reduce PDT outcome variability, attempts toward image-guided personalized PDT are being pursued by monitoring PS uptake either via fluorescence or photoacoustic imaging (PAI), a nonionizing modality dependent on optical absorption properties of the tissue. PAI-guided PDT requires a near-infrared contrast agent for deep tissue imaging with minimal photobleaching effect. We evaluate the impact of PDT agent, benzoporphyrin derivative (BPD), on PAI agent indocyanine green (ICG) and vice versa, given that they have different optical absorption properties and singlet oxygen quantum yields for PDT. Specifically, we demonstrate in two oral squamous cell carcinoma lines (FaDu and SCC4) that ICG has minimal effect on BPD PDT efficacy when irradiated with either a continuous or pulsed laser. Furthermore, the impact of BPD on ICG photodegradation was monitored with PAI in tissue-mimicking phantoms. These studies inform us that the combination of BPD and ICG can be utilized for PAI-guided PDT. However, researchers need to consider the photodegradation effects of ICG in the presence of BPD when designing their drug delivery strategies for PAI-guided PDT.

Significance: The diagnostic depth of photodynamic diagnosis (PDD) for gastric cancer with protoporphyrin IX (PpIX) is limited, which leads to missing intramucosal cancers in screening and surgery.

Aim: The reason is that the excitation light, whose wavelength is determined by the highest absorption peak of PpIX (∼405  nm), is strongly attenuated by mucosal tissues. We investigated an excitation wavelength that can extend the diagnostic depth of PpIX fluorescence at the mucosal subsurface.

Approach: By calculating the depth-dependent intensity of the excitation light in porcine gastric mucosa for each wavelength, relationships among the wavelength, fluorophore depth, and fluorescence intensity were assessed and fluorescence images of PpIX pellets located at different fluorophore depths were compared experimentally by changing the excitation wavelength.

Results: The numerical calculation showed that a 505-nm excitation light provided the highest fluorescence intensities at a fluorophore depth deeper than 1.1 mm. In the fluorescence observation, the fluorescence intensities at fluorophore depths of 0 and 1.0 mm at 405 nm were 5.4  ×  10³ and 1.0  ×  10³  arb. units, whereas those at 505 nm were 5.3  ×  10¹ and 1.9  ×  10²  arb. units, respectively.

Conclusion: The experimental results suggest that the diagnosis depth of PDD with PpIX for intramucosal cancer can be extended by 505-nm excitation light.

Significance: Photodynamic therapy (PDT) involves complex light-drug-pathophysiology interactions that can be affected by multiple parameters and often leads to large variations in treatment outcome from patient to patient. Direct PDT dosimetry technologies have been sought to optimize the control variables (e.g., light dose, drug administration, tissue oxygenation, and patient conditioning) for best patient outcomes. In comparison, singlet oxygen (O₂1) dosimetry has been tested in various forms to provide an accurate and perhaps comprehensive prediction of the treatment efficacy.

Aim: We discuss an advanced version of this approach provided by a noninvasive, continuous wave dosimeter that can measure near-infrared spectrally resolved luminescence of both photosensitizer (PS) and O₂1 generated during PDT cancer treatment.

Approach: This dosimetry technology uses an amplified, high quantum efficiency InGaAs detector with spectroscopic decomposition during the light treatment to continuously extract the maximum signal of O₂1 phosphorescence while suppressing the strong PS luminescence background by spectrally fitting the data points across nine narrow band wavelengths. O₂1 and PS luminescence signals were measured in vivo in FaDu xenograft tumors grown in mice during PDT treatment using Verteporfin as the PS and a continuous laser treatment at 690 nm wavelength.

Results: A cohort of 19 mice was used and observations indicate that the tumor growth rate inhibition showed a stronger correlation with O₂1 than with just the PS signal.

Conclusions: These results suggest that O₂1 measurement may be a more direct dosimeter of PDT damage, and it has potential value as a definitive diagnostic for PDT treatment, especially with spectral separation of the background luminescence and online estimation of the PS concentration.

Significance: Commercial lasers, lamps, and light-emitting diode (LED) light sources have stimulated the clinical translation of photodynamic therapy (PDT). Yet, the continued exploration of new photosensitizers (PSs) for PDT often requires separate activation wavelengths for each agent being investigated. Customized light sources for such research frequently come at significant financial or technical cost, especially when compounded over many agents and wavelengths.

Aim: LEDs offer potential as a cost-effective tool for new PS and multi-PS photodynamic research. A low-cost-per-wavelength tool leveraging high-power LEDs to facilitate efficient and versatile research is needed to further accelerate research in the field.

Approach: We developed and validated a high-power LED array system for benchtop PDT with a modular design for efficient switching between wavelengths that overcome many challenges in light source design. We describe the assembly of a low-cost LED module plus the supporting infrastructure, software, and protocols to streamline typical in vitro PDT experimentation.

Results: The LED array system is stable at intensities in excess of 100  mW  /  cm² with 2.3% variation across the illumination field, competitive with other custom and commercial devices. To demonstrate efficacy and versatility, a primary ovarian cancer cell line was treated with two widely used PSs, aminolevulinic acid and verteporfin, using the LED modules at a clinically relevant 50  J  /  cm² light dose that induced over 90% cell death for each treatment.

Conclusions: Our work provides the community with a tool for new PS and multi-PS benchtop photodynamic research that, unlike most commercial light sources, affords the user a low barrier to entry and low-cost-per-wavelength with the goal of illuminating new insights at the forefront of PDT.

Evaluating the optical properties of biological tissues is needed to achieve accurate dosimetry during photodynamic therapy (PDT). Currently, accurate assessment of the photosensitizer (PS) concentration by fluorescence measurements during PDT is typically hindered by the lack of information about tissue optical properties. In the present work, a hand-held fiber-optic probe instrument monitoring fluorescence and reflectance is used for assessing blood volume, reduced scattering coefficient, and PS concentration facilitating accurate dosimetry for PDT. System validation was carried out on tissue phantoms using nonlinear least squares support machine regression analysis. It showed a high correlation coefficient (>0.99) in the prediction of the PS concentration upon a large variety of phantom optical properties. In vivo measurements were conducted in a PDT chlorine e6 dose escalating trial involving 36 male Swiss mice with Ehrlich solid tumors in which fluences of 5, 15, and 40  J cm^−  2 were delivered at two fluence rates (100 and 40  mW cm^−  2). Remarkably, quantitative measurement of fluorophore concentration was achieved in the in vivo experiment. Diffuse reflectance spectroscopy (DRS) system was also used to independently measure the physiological properties of the target tissues for result comparisons. Then, blood volume and scattering coefficient measured by the fiber-optic probe system were compared with the corresponding result measured by DRS and showed agreement. Additionally, tumor hemoglobin oxygen saturation was measured using the DRS system. Overall, the system is capable of assessing the implicit photodynamic dose to predict the PDT outcome.

Significance: India has one of the highest rates of oral cancer incidence in the world, accounting for 30% of reported cancers. In rural areas, a lack of adequate medical infrastructure contributes to unchecked disease progression and dismal mortality rates. Photodynamic therapy (PDT) has emerged as an effective modality with potential for treating early stage disease in resource-limited settings, while photosensitizer fluorescence can be leveraged for treatment guidance.

Aim: Our aim was to assess the capability of a simple smartphone-based device for imaging 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence for treatment guidance and monitoring as part of an ongoing clinical study evaluating low-cost technology for ALA-based PDT treatment of early oral cancer.

Approach: A total of 29 subjects with <2  cm diameter moderately/well-differentiated microinvasive (  <  5  mm depth) oral squamous cell carcinoma lesions (33 lesions total, mean area ∼1.23  cm²) were administered 60  mg  /  kg ALA in oral solution and imaged before and after delivery of 100  J  /  cm² total light dose to the lesion surface. Smartphone-based fluorescence and white light (WL) images were analyzed and compared with ultrasound (US) imaging of the same lesions.

Results: We present a comparative analysis of pre- and post-treatment fluorescence, WL, and US images of oral lesions. There was no significant difference in the distribution of lesion widths measured by fluorescence and US (mean widths of 14.5 and 15.3 mm, respectively) and linear regression shows good agreement (R²  =  0.91). In general, PpIX fluorescence images obtained prior to therapeutic light delivery are able to resolve lesion margins while dramatic photobleaching (∼42  %  ) is visible post-treatment. Segmentation of the photobleached area confirms the boundaries of the irradiated zone.

Conclusions: A simple smartphone-based approach for imaging oral lesions is shown to agree in most cases with US, suggesting that this approach may be a useful tool to aid in PDT treatment guidance and monitoring photobleaching as part of a low-cost platform for intraoral PDT.

Significance: Low-frequency oscillations (LFOs) ranging from 0.01 to 0.15 Hz are common in functional imaging studies. Some of these LFOs are non-neuronal and are correlated with autonomic physiological processes.

Aim: We investigate the relationships between systemic low-frequency oscillations (sLFOs) measured at different peripheral sites during resting states in ischemic stroke patients.

Approach: Twenty-seven ischemic stroke patients (ages 44 to 90; 20 male and 7 female) were recruited for the study. During the experiments, fluctuations in oxyhemoglobin concentration were measured in the left and right toes, fingertips, and earlobes using a multichannel near-infrared spectroscopy instrument. We applied cross-correlation and frequency component analyses on the sLFO data.

Results: The results showed that embolization broke the symmetry of the sLFO transmission and that the damage was not limited to the local area but spread throughout the body. Among six peripheral sites, the power spectrum width of the earlobes was significantly larger than that of the fingers and toes. This indicates that the earlobes may contain more physiological information. Finally, the results of fuzzy clustering verified that sLFOs can serve as perfusion biomarkers to differentiate stroke from healthy subjects.

Conclusions: The high correlation values and corresponding delays in sLFOs support the hypothesis that (1) the correlation characteristics of sLFOs in stroke patients are different from those of healthy subjects. These characteristics can reflect patient condition, to an extent. Embolization in ischemic stroke patients breaks the symmetry of the body’s sLFO transmission, disrupting the balance of blood circulation. (2) sLFOs can be used as perfusion biomarkers to differentiate ischemic stroke patients from healthy subjects. Studying these signals can explicate the overall feedback/influence of pericentral interactions. Finally, peripheral sLFOs have been shown to be an effective and accurate tool for assessing peripheral blood circulation and vascular integrity in ischemic stroke patients.

Significance: The use of optofluidic time-stretch flow cytometry enables extreme-throughput cell imaging but suffers from the difficulties of capturing and processing a large amount of data. As significant amounts of continuous image data are generated, the images require identification with high speed.

Aim: We present an intelligent cell phenotyping framework for high-throughput optofluidic time-stretch microscopy based on the XGBoost algorithm, which is able to classify obtained cell images rapidly and accurately. The applied image recognition consists of density-based spatial clustering of applications with noise outlier detection, histograms of oriented gradients combining gray histogram fused feature, and XGBoost classification.

Approach: We tested the ability of this framework against other previously proposed or commonly used algorithms to phenotype two groups of cell images. We quantified their performances with measures of classification ability and computational complexity based on AUC and test runtime. The tested cell image datasets were acquired from high-throughput imaging of over 20,000 drug-treated and untreated cells with an optofluidic time-stretch microscope.

Results: The framework we built beats other methods with an accuracy of over 97% and a classification frequency of 3000  cells  /  s. In addition, we determined the optimal structure of training sets according to model performances under different training set components.

Conclusions: The proposed XGBoost-based framework acts as a promising solution to processing large flow image data. This work provides a foundation for future cell sorting and clinical practice of high-throughput imaging cytometers.

Significance: The effects of varying the indocyanine green injection dose, injection rate, physiologic dispersion of dye, and intravenous tubing volume propagate into the shape and magnitude of the arterial input function (AIF) during intraoperative fluorescence perfusion assessment, thereby altering the observed kinetics of the fluorescence images in vivo.

Aim: Numerical simulations are used to demonstrate the effect of AIF on metrics derived from tissue concentration curves such as peak fluorescence, time-to-peak (TTP), and egress slope.

Approach: Forward models of tissue concentration were produced by convolving simulated AIFs with the adiabatic approximation to the tissue homogeneity model using input parameters representing six different tissue examples (normal brain, glioma, normal skin, ischemic skin, normal bone, and osteonecrosis).

Results: The results show that AIF perturbations result in variations in estimates of total intensity of up to 80% and TTP error of up to 200%, with the errors more dominant in brain, less in skin, and less in bone. Interestingly, error in ingress slope was as high as 60% across all tissue types. These are key observable parameters used in fluorescence imaging either implicitly by viewing the image or explicitly through intensity fitting algorithms. Correcting by deconvolving the image with a measured subject-specific AIF provides an intuitive means of visualizing the data while also removing the source of variance and allowing intra- and intersubject comparisons.

Conclusions: These results suggest that intraoperative fluorescence perfusion assessment should be corrected by patient-specific AIFs measured by pulse dye densitometry.

Significance: The development of ultralow energy photoacoustic microscopy (PAM) on the clinically relevant pigmented rabbit eye model paves a road toward translation of the emerging PAM technology in ophthalmology clinics.

Aim: Since the eye is particularly vulnerable to laser damage, we aim to develop an ultralow energy PAM system to significantly improve the laser safety of PAM by increasing the sensitivity of the system and reducing the incident laser energy for imaging.

Approach: A multichannel data acquisition circuit with two-stage signal amplification was specially designed, which, in combination with the application of 3 by 3 median filter in the spatial domain, significantly improved the signal-to-noise ratio of the PAM system. The safety of this system was validated by histopathology, fluorescein angiography, and fundus photography.

Results: Experiments performed on pigmented rabbits demonstrated that, when using this ultralow energy PAM system, satisfactory image quality can be achieved in the eye with an incident laser fluence that is only 1% of the American National Standards Institute safety limit. Fundus photography, fluorescein angiography, and histopathology were performed after the imaging procedure, and no retinal or ocular damage was observed.

Conclusions: The proposed ultralow energy PAM system has excellent safety and holds potential to be developed into a clinical tool for ocular imaging.

Significance: The rates of melanoma and nonmelanoma skin cancer are rising across the globe. Due to a shortage of board-certified dermatologists, the burden of dermal lesion screening and erythema monitoring has fallen to primary care physicians (PCPs). An adjunctive device for lesion screening and erythema monitoring would be beneficial because PCPs are not typically extensively trained in dermatological care.

Aim: We aim to examine the feasibility of using a smartphone-camera-based dermascope and a USB-camera-based dermascope utilizing polarized white-light imaging (PWLI) and polarized multispectral imaging (PMSI) to map dermal chromophores and erythema.

Approach: Two dermascopes integrating LED-based PWLI and PMSI with both a smartphone-based camera and a USB-connected camera were developed to capture images of dermal lesions and erythema. Image processing algorithms were implemented to provide chromophore concentrations and redness measures.

Results: PWLI images were successfully converted to an alternate colorspace for erythema measures, and the spectral bandwidth of the PMSI LED illumination was sufficient for mapping of deoxyhemoglobin, oxyhemoglobin, and melanin chromophores. Both types of dermascopes were able to achieve similar relative concentration results.

Conclusion: Chromophore mapping and erythema monitoring are feasible with PWLI and PMSI using LED illumination and smartphone-based cameras. These systems can provide a simpler, more portable geometry and reduce device costs compared with interference-filter-based or spectrometer-based clinical-grade systems. Future research should include a rigorous clinical trial to collect longitudinal data and a large enough dataset to train and implement a machine learning-based image classifier.

Significance: Hyperspectral imaging (HSI) is an emerging optical technique that has a double function of spectroscopy and imaging.

Aim: Near-infrared hyperspectral imaging (NIR-HSI) (900 to 1700 nm) with the help of chemometrics was investigated for gastric cancer diagnosis.

Approach: Mean spectra and standard deviation of normal and cancerous pixels were extracted. Principal component analysis (PCA) was used to compress the dimension of hypercube data and select the optimal wavelengths. Moreover, spectral angle mapper (SAM) was utilized as chemometrics to discriminate gastric cancer from normal.

Results: Major spectral difference of cancerous and normal gastric tissue was observed around 975, 1215, and 1450 nm by comparison. A total of six wavelengths (i.e., 975, 1075, 1215, 1275, 1390, and 1450 nm) were then selected as optimal wavelengths by PCA. The accuracy using SAM is up to 90% according to hematoxylin–eosin results.

Conclusions: These results suggest that NIR-HSI has the potential as a cutting-edge optical diagnostic technique for gastric cancer diagnosis with suitable chemometrics.

Significance: The initial dip in hemoglobin-oxygenation response to stimulations is a spatially confined endogenous indicator that is faster than the blood flow response, making it a desired label-free contrast to map the neural activity. A fundamental question is whether a single-impulse stimulus, much shorter than the response delay, could produce an observable initial dip without repeated stimulation.

Aim: To answer this question, we report high-speed functional photoacoustic (PA) microscopy to investigate the initial dip in mouse brains.

Approach: We developed a Raman-laser-based dual-wavelength functional PA microscope that can image capillary-level blood oxygenation at a 1-MHz one-dimensional imaging rate. This technology was applied to monitor the hemodynamics of mouse cerebral vasculature after applying an impulse stimulus to the forepaw.

Results: We observed a transient initial dip in cerebral microvessels starting as early as 0.13 s after the onset of the stimulus. The initial dip and the subsequent overshoot manifested a wave pattern propagating across different microvascular compartments.

Conclusions: We quantified both spatially and temporally the single-impulse-stimulated microvascular hemodynamics in mouse brains at single-vessel resolution. Fast label-free imaging of single-impulse response holds promise for real-time brain–computer interfaces.

Significance: Stimulated Raman scattering (SRS) and pump-probe microscopy are implementations of multiphoton microscopy that acquire high-resolution, label-free images of live samples encoded with molecular contrast. Most commercial multiphoton microscopes cannot access these techniques since they require sample illumination by two temporally synchronized ultrafast pulse trains. We present a compact and robust way of synchronizing an additional Ti:sapphire laser with a conventional single-beam multiphoton microscope to realize an instrument that can acquire images with enhanced molecular specificity.

Aim: A passive optical synchronization scheme for a pair of commercially available, unmodified modelocked Ti:sapphire lasers was developed. The suitability of this synchronization scheme for advanced biomedical microscopy was investigated.

Approach: A pair of modelocked Ti:sapphire lasers were aligned in master–slave configuration. Five percent of the master laser output was used to seed the modelocking in the slave laser cavity. The timing jitter of the master and slave pulse trains was characterized using an optical autocorrelator. The synchronized output of both lasers was coupled into a laser scanning microscope and used to acquire spectral focusing SRS and pump-probe microscopy images from biological and nonbiological samples.

Results: A timing jitter between the modelocked pulse trains of 0.74 fs was recorded. Spectral focusing SRS allowed spectral discrimination of polystyrene and polymethyl methacrylate beads. Pump-probe microscopy was used to record excited state lifetime curves from hemoglobin in intact red blood cells.

Conclusion: Our work demonstrates a simple and robust method of upgrading single-beam multiphoton microscopes with an additional ultrafast laser. The resulting dual-beam instrument can be used to acquire label-free images of sample structure and composition with high biochemical specificity.

Significance: Photodynamic therapy (PDT) could become a treatment option for nonmuscle invasive bladder cancer when the current high morbidity rate associated with red light PDT and variable PDT dose can be overcome through a combination of intravesical instillation of the photosensitizer and the use of green light creating a steep PDT dose gradient.

Aim: To determine how a high PDT selectivity can be maintained throughout the bladder wall considering other efficacy determining parameters, in particular, the average optical properties of the mucosal layer governing the fluence rate multiplication factor, as well as the bladder shape and the position of the emitter in relationship to the bladder wall.

Approach: We present three irradiance monitoring systems and evaluate their ability to enable selective bladder PDT considering previously determined photodynamic threshold values for the bladder cancer, mucosa and urothelium in a preclinical model, and the photosensitizer’s specific uptake ratio. Monte Carlo-based light propagation simulations performed for six human bladders at the time of therapy for a range of tissue optical properties. The performance of one irradiance sensing device in a clinical phase 1B trial is presented to underline the impact of irradiance monitoring, and it is compared to the Monte Carlo-derived dose surface histogram.

Results: Monte Carlo simulations showed that irradiance monitoring systems need to comprise at least three sensors. Light scattering inside the bladder void needs to be minimized to prevent increased heterogeneity of the irradiance. The dose surface histograms vary significantly depending on the bladder shape and bladder volume but are less dependent on tissue optical properties.

Conclusions: We demonstrate the need for adequate irradiance monitoring independent of a photosensitizer’s specific uptake ratio.

Significance: Worldwide, ∼460  million people suffer from disabling hearing impairment. Many of these patients are still not sufficiently supplied with currently available auditory technologies. Optical stimulation of the hearing organ offers a promising alternative for a new generation of auditory prostheses.

Aim: To assess the biocompatibility margins of our laser pulse amplitude strategy in vitro, we designed a protocol and present the effects on normal human dermal fibroblasts, human chondrocytes, and human osteoblasts.

Approach: Laser pulses of 532 nm were applied over 120 s using our laser pulse amplitude modulation strategy. We then assessed cell viability and cytotoxicity through fluorescence staining and quantitative polymerase chain reaction-analysis regarding 84 key player-genes for cytotoxicity and stress response.

Results: The first in vitro biocompatibility margins for our stimulation parameters applied to cells of the peripheral hearing organ were between 200 and 223 mW (3348  J/cm²). After irradiation with a subphototoxic laser power of 199 mW (2988  J/cm²), only the fibroblasts showed a significant upregulation of GADD45G.

Conclusion: Further studies are underway to optimize parameters for the optoacoustic stimulation of the auditory system. Our protocol and results on laser–tissue interactions can be useful for translational laser applications in various other irradiated biological tissues.

The erratum corrects a misspelled author surname.

The erratum corrects an error in the y-axis labels of Fig. 4 and Fig. S2.