Measurement of Cyanine Dye Photobleaching in Photosensitizer Cyanine Dye Conjugates Could Help in Optimizing Light Dosimetry for Improved Photodynamic Therapy of Cancer.

Photodynamic therapy (PDT) of cancer is dependent on three primary components: photosensitizer (PS), light and oxygen. Because these components are interdependent and vary during the dynamic process of PDT, assessing PDT efficacy may not be trivial. Therefore, it has become necessary to develop pre-treatment planning, on-line monitoring and dosimetry strategies during PDT, which become more critical for two or more chromophore systems, for example, PS-CD (Photosensitizer-Cyanine dye) conjugates developed in our laboratory for fluorescence-imaging and PDT of cancer. In this study, we observed a significant impact of variable light dosimetry; (i) high light fluence and fluence rate (light dose: 135 J/cm², fluence rate: 75 mW/cm²) and (ii) low light fluence and fluence rate (128 J/cm² and 14 mW/cm² and 128 J/cm² and 7 mW/cm²) in photobleaching of the individual chromophores of PS-CD conjugates and their long-term tumor response. The fluorescence at the near-infrared (NIR) region of the PS-NIR fluorophore conjugate was assessed intermittently via fluorescence imaging. The loss of fluorescence, photobleaching, caused by singlet oxygen from the PS was mapped continuously during PDT. The tumor responses (BALB/c mice bearing Colon26 tumors) were assessed after PDT by measuring tumor sizes daily. Our results showed distinctive photobleaching kinetics rates between the PS and CD. Interestingly, compared to higher light fluence, the tumors exposed at low light fluence showed reduced photobleaching and enhanced long-term PDT efficacy. The presence of NIR fluorophore in PS-CD conjugates provides an opportunity of fluorescence imaging and monitoring the photobleaching rate of the CD moiety for large and deeply seated tumors and assessing PDT tumor response in real-time.

A prospective study of pain control by a 2-step irradiance schedule during topical photodynamic therapy of nonmelanoma skin cancer.

BACKGROUND: Topical photodynamic therapy (PDT) for selected nonmelanoma skin cancer using 5-aminolevulinic acid (ALA) or methyl aminolevulinate (MAL) has yielded high long-term complete response rates with very good cosmesis. Pain during light activation of the photosensitizer can be a serious adverse event. A 2-step irradiance protocol has previously been shown to minimize ALA-PDT pain. OBJECTIVE: To determine the irradiance-dependent pain threshold for MAL-PDT, to adapt the 2-step protocol to a light-emitting diode (LED) light source, and assess clinical response. METHODS: In this prospective study, 25 superficial basal cell carcinoma (sBCC) received an initial irradiance by laser at 40 or 50 mW/cm², or LED at 35 mW/cm² followed by an irradiance at 70 mW/cm² for a total of 75 J/cm². Pain levels were recorded for both irradiance steps. Efficacy was assessed at 6, 12, or 24 months. RESULTS: Pain was mild in the 40/70 mW/cm² laser cohort. Three instances of irradiance-limiting pain occurred at 50/70 mW/cm². Pain was minimal in the 35/70 mW/cm² LED cohort. Clinical response rates were 80% in the 50/70 mW/cm² laser cohort and 90% in the 35/70 mW/cm² LED cohort. CONCLUSION: Topical PDT can be effectively delivered to sBCC with minimal treatment-related pain by a 2-step irradiance protocol.

Non-Invasive Continuous Optical Monitoring of Cerebral Blood Flow after Traumatic Brain Injury in Mice Using Fiber Camera-Based Speckle Contrast Optical Spectroscopy.

Neurocritical care focuses on monitoring cerebral blood flow (CBF) to prevent secondary brain injuries before damage becomes irreversible. Thus, there is a critical unmet need for continuous neuromonitoring methods to quantify CBF within the vulnerable cortex continuously and non-invasively. Animal models and imaging biomarkers can provide valuable insights into the mechanisms and kinetics of head injury, as well as insights for potential treatment strategies. For this purpose, we implemented an optical technique for continuous monitoring of blood flow changes after a closed head injury in a mouse model, which is based on laser speckle contrast imaging and a fiber camera-based approach. Our results indicate a significant decrease (~10%, p-value < 0.05) in blood flow within 30 min of a closed head injury. Furthermore, the low-frequency oscillation analysis also indicated much lower power in the trauma group compared to the control group. Overall, blood flow has the potential to be a biomarker for head injuries in the early phase of a trauma, and the system is useful for continuous monitoring with the potential for clinical translation.

Chemophototherapeutic Ablation of Doxorubicin-Resistant Human Ovarian Tumor Cells.

Porphyrin-phospholipid (PoP) liposomes loaded with Doxorubicin (Dox) have been demonstrated to be an efficient vehicle for chemophototherapy (CPT). Multidrug resistance (MDR) of cancer cells is a problematic phenomenon in which tumor cells develop resistance to chemotherapy. Herein, we report that Dox-resistant tumor cells can be ablated using our previously described formulation termed long-circulating Dox loaded in PoP liposomes (LC-Dox-PoP), which is a PEGylated formulation containing 2 mol. % of the PoP photosensitizer. In vitro studies using free Dox and LC-Dox-PoP showed that human ovarian carcinoma A2780 cells were more susceptible to Dox compared to the corresponding Dox-resistant A2780-R cells. When CPT was applied with LC-Dox-PoP liposomes, effective killing of both nonresistant and resistant A2780 cell lines was observed. An in vivo study to assess the efficiency of LC-Dox-PoP showed effective tumor shrinkage and prolonged survival of athymic nude mice bearing subcutaneous Dox-resistant A2780-R tumor xenografts when they were irradiated with a red laser. Biodistribution analysis demonstrated enhanced tumoral drug uptake in Dox-resistant tumors with CPT, suggesting that increased drug delivery was sufficient to induce ablation of resistant tumor cells.

Folate-Targeted Nanoliposomal Chemophototherapy.

Light-responsive liposomes have been developed for the on-demand release of drugs. However, efficient delivery of chemotherapeutic drugs to tumor for cancer theranostics remains a challenge. Herein, folic acid (FA), an established ligand for targeted drug delivery, was used to decorate light-sensitive porphyrin-phospholipid (PoP) liposomes, which were assessed for FA-targeted chemophototherapy (CPT). PoP liposomes and FA-conjugated PoP liposomes were loaded with Doxorubicin (Dox), and physical properties were characterized. In vitro, FA-PoP liposomes that were incubated with FA receptor-overexpressing human KB cancer cells showed increased uptake compared to non-targeted PoP liposomes. Dox and PoP contributed towards chemophototherapy (CPT) in vitro, and PoP and FA-PoP liposomes induced cell killing. In vivo, mice bearing subcutaneous KB tumors treated with PoP or FA-PoP liposomes loaded with Dox, followed by 665 nm laser treatment, had delayed tumor growth and improved survival. Dox delivery to tumors increased following laser irradiation for both PoP and FA-PoP liposomes. Thus, while Dox-FA-PoP liposomes were effective following systemic administration and local light irradiation in this tumor model, the FA targeting moiety did not appear essential for anti-tumor responses.

First-in-clinical application of a time-gated diffuse correlation spectroscopy system at 1064†nm using superconducting nanowire single photon detectors in a neuro intensive care unit.

Recently proposed time-gated diffuse correlation spectroscopy (TG-DCS) has significant advantages compared to conventional continuous wave (CW)-DCS, but it is still in an early stage and clinical capability has yet to be established. The main challenge for TG-DCS is the lower signal-to-noise ratio (SNR) when gating for the deeper traveling late photons. Longer wavelengths, such as 1064 nm have a smaller effective attenuation coefficient and a higher power threshold in humans, which significantly increases the SNR. Here, we demonstrate the clinical utility of TG-DCS at 1064 nm in a case study on a patient with severe traumatic brain injury admitted to the neuro-intensive care unit (neuroICU). We showed a significant correlation between TG-DCS early (ρ = 0.67) and late (ρ = 0.76) gated against invasive thermal diffusion flowmetry. We also analyzed TG-DCS at high temporal resolution (50 Hz) to elucidate pulsatile flow data. Overall, this study demonstrates the first clinical translation capability of the TG-DCS system at 1064 nm using a superconducting nanowire single-photon detector.

Quantification of perfusion and metabolism in an autism mouse model assessed by diffuse correlation spectroscopy and near-infrared spectroscopy.

There is a need for quantitative biomarkers for early diagnosis of autism. Cerebral blood flow and oxidative metabolism parameters may show superior contrasts for improved characterization. Diffuse correlation spectroscopy (DCS) has been shown to be reliable method to obtain cerebral blood flow contrast in animals and humans. Thus, in this study, we evaluated the combination of DCS and fNIRS in an established autism mouse model. Our results indicate that autistic group had significantly (P = .001) lower (~40%) blood flow (1.16 ± 0.26) × 10-8  cm2 /s), and significantly (P = .015) lower (~70%) oxidative metabolism (52.4 ± 16.6 µmol/100 g/min) compared to control group ([1.93 ± 0.74] × 10-8  cm2 /s, 177.2 ± 45.8 µmol/100 g/min, respectively). These results suggest that the combination of DCS and fNIRS can provide hemodynamic and metabolic contrasts for in vivo assessment of autism pathological conditions noninvasively.

Mapping fluorescence resonance energy transfer parameters of a bifunctional agent using time-domain fluorescence diffuse optical tomography.

We present a method to map fluorescence resonance energy transfer (FRET) parameters of a bifunctional photodynamic therapy agent, (2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a)-cyanine dye (HPPH-CD) conjugate, which consists of a photosensitizer (HPPH) and a fluorescent agent CD. We utilized time-domain fluorescence diffuse optical tomography, the normalized Born ratio model in the Fourier-domain, and an iterative algorithm to map depth-resolved spatial heterogeneities of FRET parameters. Our results exhibited depth-resolved changes of fluorophore's lifetime and the distance maps due to FRET between HPPH and CD. Our model suggests a potential approach of using FRET parameters to monitor efficacies of multifunctional photodynamic therapy agents in deep tissue.

Noninvasive Optical Monitoring of Cerebral Blood Flow and EEG Spectral Responses after Severe Traumatic Brain Injury: A Case Report.

Survivors of severe brain injury may require care in a neurointensive care unit (neuro-ICU), where the brain is vulnerable to secondary brain injury. Thus, there is a need for noninvasive, bedside, continuous cerebral blood flow monitoring approaches in the neuro-ICU. Our goal is to address this need through combined measurements of EEG and functional optical spectroscopy (EEG-Optical) instrumentation and analysis to provide a complementary fusion of data about brain activity and function. We utilized the diffuse correlation spectroscopy method for assessing cerebral blood flow at the neuro-ICU in a patient with traumatic brain injury. The present case demonstrates the feasibility of continuous recording of noninvasive cerebral blood flow transients that correlated well with the gold-standard invasive measurements and with the frequency content changes in the EEG data.

Monitoring photobleaching and hemodynamic responses to HPPH-mediated photodynamic therapy of head and neck cancer: a case report.

We present initial results obtained during the course of a Phase I clinical trial of 2-1[hexyloxyethyl]-2-devinylpyropheophorbide-a (HPPH)-mediated photo-dynamic therapy (PDT) in a head and neck cancer patient. We quantified blood flow, oxygenation and HPPH drug photobleaching before and after therapeutic light treatment by utilizing fast, non-invasive diffuse optical methods. Our results showed that HPPH-PDT induced significant drug photobleaching, and reduction in blood flow and oxygenation suggesting significant vascular and cellular reaction. These changes were accompanied by cross-linking of the signal transducer and activator of transcription 3 (STAT3), a molecular measure for the oxidative photoreaction. These preliminary results suggest diffuse optical spectroscopies permit non-invasive monitoring of PDT in clinical settings of head and neck cancer patients.

Deep learning model for ultrafast quantification of blood flow in diffuse correlation spectroscopy.

Diffuse correlation spectroscopy (DCS) is increasingly used in the optical imaging field to assess blood flow in humans due to its non-invasive, real-time characteristics and its ability to provide label-free, bedside monitoring of blood flow changes. Previous DCS studies have utilized a traditional curve fitting of the analytical or Monte Carlo models to extract the blood flow changes, which are computationally demanding and less accurate when the signal to noise ratio decreases. Here, we present a deep learning model that eliminates this bottleneck by solving the inverse problem more than 2300% faster, with equivalent or improved accuracy compared to the nonlinear fitting with an analytical method. The proposed deep learning inverse model will enable real-time and accurate tissue blood flow quantification with the DCS technique.

Cerebral Blood Flow-Based Resting State Functional Connectivity of the Human Brain using Optical Diffuse Correlation Spectroscopy.

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a key hemodynamic parameter related to cerebral oxygen supply. Resting state low frequency fluctuations based on oxygenation contrast have been shown to provide correlations between functionally connected regions. The presented protocol uses optical diffuse correlation spectroscopy (DCS) to assess blood flow-based resting state functional connectivity (RSFC) in the human brain. Results of CBF-based RSFC in human frontal cortex indicate that intra-regional RSFC is significantly higher in the left and right cortices compared to inter-regional RSFC in both cortices. This protocol should be of interest to researchers who employ multi-modal imaging techniques to study human brain function, especially in the pediatric population.

Inter- and Intra-physician variation in quantifying actinic keratosis skin photodamage.

We investigated the variations in physician evaluation of skin photodamage based on a published photodamage scale. Of interest is the utility of a 10-level scale ranging from none and mild photodamage to actinic keratosis (AK). The dorsal forearms of 55 adult subjects with various amounts of photodamage were considered. Each forearm was independently evaluated by 15 board-certified dermatologists according to the Global Assessment Severity Scale ranging from 0 (less severe) to 9 (the most progressed stage of skin damage). Dermatologists rated the levels of photodamage based upon the photographs in blinded fashion. Results show substantial disagreement amongst the dermatologists on the severity of photodamage. Our results indicate that ratings could be more consistent if using a scale of less levels (5-levels or 3-levels). Ultimately, clinicians can use this knowledge to provide better interpretation of inter-rater evaluations and provide more reliable assessment and frequent monitoring of high-risk populations.

In vivo simultaneous monitoring of two fluorophores with lifetime contrast using a full-field time domain system.

Optical molecular imaging of small animals in vivo has witnessed dramatic growth during the past decade. Most commercial systems are based on continuous wave technology and measure solely bioluminescence or fluorescence intensity. Time domain (TD) technology enables the measurement of both intensity and fluorescence lifetime as an additional imaging metric. We have developed a novel, in-house, full-field TD system with dramatically faster acquisition times than available from a commercial TD system. Recent in vivo data from a mouse imaged with the full-field TD system has demonstrated the potential to monitor and discriminate two fluorophores injected simultaneously based on their fluorescence lifetime contrast.

Quantifying skin photodamage with spatial frequency domain imaging: statistical results.

We investigated the change in optical properties and vascular parameters to characterize skin tissue from mild photodamage to actinic keratosis (AK) with comparison to a published photodamage scale. Multi-wavelength spatial frequency domain imaging (SFDI) measurements were performed on the dorsal forearms of 55 adult subjects with various amounts of photodamage. Dermatologists rated the levels of photodamage based upon the photographs in blinded fashion to allow comparison with SFDI data. For characterization of statistical data, we used artificial neural networks. Our results indicate that optical and vascular parameters can be used to quantify photodamage and can discriminate between the stages as low, medium, and high grades, with the best performance of ∼70%, ∼76% and 80% for characterization of low- medium- and high-grade lesions, respectively. Ultimately, clinicians can use this noninvasive approach for risk assessment and frequent monitoring of high-risk populations.

Resting-state functional connectivity measured by diffuse correlation spectroscopy.

Near-infrared diffuse correlation spectroscopy (DCS) is used to record spontaneous cerebral blood flow fluctuations in the frontal cortex. Nine adult subjects participated in the experiments, in which 8-minute spontaneous fluctuations were simultaneously recorded from the left and right dorsolateral and inferior frontal regions. Resting-state functional connectivity (RSFC) was measured by the temporal correlation of the low frequency fluctuations. Our data shows the RSFC within the dorsolateral region is significantly stronger than that between the inferior and dorsolateral regions, in line with previous observations with functional near-infrared spectroscopy. This indicates that DCS is capable of investigating brain functional connectivity in terms of cerebral blood flow.

Review of structured light in diffuse optical imaging.

Diffuse optical imaging probes deep living tissue enabling structural, functional, metabolic, and molecular imaging. Recently, due to the availability of spatial light modulators, wide-field quantitative diffuse optical techniques have been implemented, which benefit greatly from structured light methodologies. Such implementations facilitate the quantification and characterization of depth-resolved optical and physiological properties of thick and deep tissue at fast acquisition speeds. We summarize the current state of work and applications in the three main techniques leveraging structured light: spatial frequency-domain imaging, optical tomography, and single-pixel imaging. The theory, measurement, and analysis of spatial frequency-domain imaging are described. Then, advanced theories, processing, and imaging systems are summarized. Preclinical and clinical applications on physiological measurements for guidance and diagnosis are summarized. General theory and method development of tomographic approaches as well as applications including fluorescence molecular tomography are introduced. Lastly, recent developments of single-pixel imaging methodologies and applications are reviewed.

Early assessment of burn severity in human tissue ex vivo with multi-wavelength spatial frequency domain imaging.

Early knowledge about burn severity and depth can lead to improved outcome for patients. In this study, we investigated the change in optical properties in ex vivo human skin following thermal burn injuries. Human skin removed during body contouring procedures was subjected to thermal burn injury for either 10 or 60 s. Multi-wavelength spatial frequency domain imaging (SFDI) measurements were performed on each sample and the optical properties (absorption and scattering parameters) were obtained at each wavelength. Multi-wavelength fitting was used to quantify absorption and scattering parameters, and these parameters were compared to histologic assessments of burn depth related to burn severity. Our results indicated substantial changes in optical scattering parameters and these changes correlated well with the burn severity and depth, and fit closely with previously reported studies using porcine in vivo models. This study provides the characterization of thermal burn injury on human skin ex vivo by using the optical method of SFDI with high sensitivity and specificity. This preclinical human model system without live animals could have uses in testing the imaging parameters of other skin injuries, including from caustic agents.

Hemodynamic responses to antivascular therapy and ionizing radiation assessed by diffuse optical spectroscopies.

Diffuse optical methods were used to monitor two different therapies in K1735 malignant mouse melanoma tumor models: anti-vascular therapy and radiation therapy. Anti-vascular therapy induced acute variation in hemodynamic parameters within an hour, and radiation therapy induced longitudinal changes within 2 weeks. During anti-vascular therapy, the drug Combretastatin A-4 3-O-Phosphate (CA4P, 2.5 mg/200 mul PBS/mouse) significantly decreased tissue blood flow (65%) and blood oxygenation (38%) one hour after injection. In the longitudinal study, single-fraction ionizing radiation (12 Gy x 1) induced significant reduction of tissue blood flow (36%) and blood oxygenation (24%) 14 days after radiation. The results correlated well with contrast enhanced ultrasound, tumor histology, and a nitroimidazole hypoxia marker (EF5). The research provides further evidence that noninvasive diffuse optical spectroscopies can be useful tools for monitoring cancer therapy in vivo.