Monte Carlo based dosimetry of extraoral photobiomodulation for prevention of oral mucositis.

Photobiomodulation therapy (PBMT) is recommended for prevention and treatment of oral mucositis, a painful condition that occurs in cancer patients. Intraoral PBMT is limited to treating distal oral mucosa and oropharynx. Extraoral PBMT may provide a more efficient intervention. The goal of this study was to develop a clinically viable protocol for extraoral PBMT. Monte Carlo modeling was used to predict the distribution of 850 nm light for four treatment sites, using anatomical data obtained from MRI and optical properties from the literature. Simulated incident light power density was limited to 399 mW/cm2 to ensure treatment safety and to prevent tissue temperature increase. The results reveal that total tissue thickness determines fluence rate at the oral mucosa, whereas the thickness of individual tissue layers and melanin content are of minor importance. Due to anatomical differences, the fluence rate varied greatly among patients. Despite these variations, a universal protocol was established using a median treatment time methodology. The determined median treatment times required to deliver efficacious dose between 1 and 6 J/cm2 were within 15 min. The developed PBMT protocol can be further refined using the combination of pretreatment imaging and the Monte Carlo simulation approach implemented in this study.

Translational Potential of Fluorescence Polarization for Breast Cancer Cytopathology.

Breast cancer is the most common malignancy in women. The standard of care for diagnosis involves invasive core needle biopsy followed by time-consuming histopathological evaluation. A rapid, accurate, and minimally invasive method to diagnose breast cancer would be invaluable. Therefore, this clinical study investigated the fluorescence polarization (Fpol) of the cytological stain methylene blue (MB) for the quantitative detection of breast cancer in fine needle aspiration (FNA) specimens. Cancerous, benign, and normal cells were aspirated from excess breast tissues immediately following surgery. The cells were stained in aqueous MB solution (0.05 mg/mL) and imaged using multimodal confocal microscopy. The system provided MB Fpol and fluorescence emission images of the cells. Results from optical imaging were compared to clinical histopathology. In total, we imaged and analyzed 3808 cells from 44 breast FNAs. Fpol images displayed quantitative contrast between cancerous and noncancerous cells, whereas fluorescence emission images showed the morphological features comparable to cytology. Statistical analysis demonstrated that MB Fpol is significantly higher (p < 0.0001) in malignant vs. benign/normal cells. It also revealed a correlation between MB Fpol values and tumor grade. The results indicate that MB Fpol could provide a reliable, quantitative diagnostic marker for breast cancer at the cellular level.

Investigation of silk as a phantom material for ultrasound and photoacoustic imaging.

Comprehensive characterization of biomedical imaging systems require phantoms that are easy to fabricate and can mimic human tissue. Additionally, with the arrival of engineered tissues, it is key to develop phantoms that can mimic bioengineered samples. In ultrasound and photoacoustic imaging, water-soluble phantom materials such as gelatin undergo rapid degradation while polymer-based materials such as polyvinyl alcohol are not conducive for generating bioengineered tissues that can incorporate cells. Here we propose silk protein-based hydrogels as an ultrasound and photoacoustic phantom material that has potential to provide a 3D environment for long-term sustainable cell growth. Common acoustic, optical, and biomechanical properties such as ultrasound attenuation, reduced scattering coefficient, and Young's modulus were measured. The results indicate that silk acoustically mimics many tissue types while exhibiting similar reduced optical scattering in the wavelength range of 400-1200 nm. Furthermore, silk-based materials can be stored long-term with no change in acoustic and optical properties, and hence can be utilized to assess the performance of ultrasound and photoacoustic systems.

Design and Validation of a Handheld Optical Polarization Imager for Preoperative Delineation of Basal Cell Carcinoma.

BACKGROUND: Accurate removal of basal cell carcinoma (BCC) is challenging due to the subtle contrast between cancerous and normal skin. A method aiding with preoperative delineation of BCC margins would be valuable. The aim of this study was to implement and clinically validate a novel handheld optical polarization imaging (OPI) device for rapid, noninvasive, in vivo assessment of skin cancer margins. METHODS: The handheld imager was designed, built, and tested. For clinical validation, 10 subjects with biopsy-confirmed BCC were imaged. Presumable cancer margins were marked by the study surgeon. The optical images were spectrally encoded to mitigate the impact of endogenous skin chromophores. The results of OPI and of the surgeon's preoperative visual assessment were compared to clinical intraoperative histopathology. RESULTS: As compared to the previous prototype, the handheld imager incorporates automated image processing and has 10-times shorter acquisition times. It is twice as light and provides twice as large a field of view. Clinical validation demonstrated that margin assessments using OPI were more accurate than visual assessment by the surgeon. The images were in good correlation with histology in 9 out of 10 cases. CONCLUSIONS: Handheld OPI could improve the outcomes of skin cancer treatments without impairing clinical workflows.

Fluorescence Polarization Imaging of Methylene Blue Facilitates Quantitative Detection of Thyroid Cancer in Single Cells.

BACKGROUND: Diagnostic accuracy of the standard of care fine-needle aspiration cytology (FNAC) remains a significant problem in thyroid oncology. Therefore, a robust and accurate method for reducing uncertainty of cytopathological evaluation would be invaluable. METHODS: In this double-blind study, we employed fluorescence emission and quantitative fluorescence polarization (Fpol) confocal imaging for sorting thyroid cells into benign/malignant categories. Samples were collected from malignant tumors, benign nodules, and normal thyroid epithelial tissues. RESULTS: A total of 32 samples, including 12 from cytologically indeterminate categories, were stained using aqueous methylene blue (MB) solution, imaged, and analyzed. Fluorescence emission images yielded diagnostically relevant information on cytomorphology. Significantly higher MB Fpol was measured in thyroid cancer as compared to benign and normal cells. The results obtained from 12 indeterminate samples revealed that MB Fpol accurately differentiated benign and malignant thyroid nodules. CONCLUSIONS: The developed imaging approach holds the potential to provide an accurate and objective biomarker for thyroid cancer, improve diagnostic accuracy of cytopathology, and decrease the number of lobectomy and near-total thyroidectomy procedures.

Validation of a Monte Carlo Modelling Based Dosimetry of Extraoral Photobiomodulation.

An in vivo validation study was performed to confirm the accuracy of extraoral photobiomodulation therapy (PBMT) dosimetry determined by modelling. The Monte Carlo technique was utilized to calculate the fluence rate and absorbed power of light delivered through multi-layered tissue. Optical properties used during Monte Carlo simulations were taken from the literature. Morphological data of four study volunteers were acquired using magnetic resonance imaging (MRI) scans. Light emitting diode (LED) coupled to a power meter were utilized to measure transmitted power through each volunteer's cheek, in vivo. The transmitted power determined by Monte Carlo modelling was compared to the in vivo measurements to determine the accuracy of the simulations. Experimental and simulation results were in good agreement for all four subjects. The difference between the mean values of the measured transmission was within 12% from the respective transmission obtained using Monte Carlo simulations. The results of the study indicate that Monte Carlo modelling is a robust and reliable method for light dosimetry.

Feasibility of dual-contrast fluorescence imaging of pathological breast tissues.

The combination of intravital dye, methylene blue (MB), with molecular cancer marker, pH low insertion peptide (pHLIP) conjugated with fluorescent Alexa532 (Alexa532-pHLIP), was evaluated for enhancing contrast of pathological breast tissue ex vivo. Fresh, thick breast specimens were stained sequentially with Alexa532-pHLIP and aqueous MB and imaged using dual-channel fluorescence microscopy. MB and Alexa532-pHLIP accumulated in the nuclei and cytoplasm of cancer cells, respectively. MB also stained nuclei of normal cells. Some Alexa532-pHLIP fluorescence emission was detected from connective tissue and benign cell membranes. Overall, Alexa532-pHLIP showed high affinity to cancer, while MB highlighted tissue morphology. The results indicate that MB and Alexa532-pHLIP provide complementary information and show promise for the detection of breast cancer.

Temperature induced changes in the optical properties of skin in vivo.

Knowledge of temperature-induced changes of skin optical properties is required for accurate dosimetry of photothermal treatments. We determined and compared in vivo optical properties of mouse ear skin at different temperatures. The diffuse reflectance, total and diffuse transmittance were measured in the spectral range from 400 to 1650 nm using an integrating sphere spectrometer at the temperatures of 25 °C, 36 °C and 60 °C. Target temperatures were attained and maintained using an automated heater equipped with a sensor for feed-back and control. Temperature and temperature induced morphological changes of skin were monitored using an infrared thermal camera and reflectance confocal microscopy, respectively. An inverse Monte Carlo technique was utilized to determine absorption, scattering, and anisotropy factors from the measured quantities. Our results indicate significant differences between the optical properties of skin at different temperatures. Absorption and scattering coefficients increased, whereas anisotropy factors decreased with increasing temperature. Changes in absorption coefficients indicate deoxygenation of hemoglobin, and a blue shift of water absorption bands. Confocal imaging confirmed that our observations can be explained by temperature induced protein denaturation and blood coagulation. Monitoring spectral responses of treated tissue may become a valuable tool for accurate dosimetry of light treatments.

The Path to an Evidence-Based Treatment Protocol for Extraoral Photobiomodulation Therapy for the Prevention of Oral Mucositis.

Oral mucositis is a painful complication of hematopoietic stem cell transplantation for which photobiomodulation therapy (PBMT) is a safe and effective intervention. Extraoral delivery of PBMT has clinical advantages over intraoral delivery but requires additional dosimetric considerations due to the external tissue layers through which the light must propagate before reaching the oral mucosa. Additionally, to date there has been no dose modeling study, a task essential to developing a justified treatment protocol. We review here some of the complexities surrounding extraoral photobiomodulation therapy and offer that may help guide researchers toward an evidence-based treatment protocol for the prevention of oral mucositis.

Dual-Wavelength Optical Polarization Imaging for Detecting Skin Cancer Margins.

Treatment of keratinocyte carcinomas requires an assessment of the extent of tumor spread. Visual delineation of tumor margins is error-prone owing to the limited contrast between cancerous and normal skin. In this contribution, we introduce spectrally-encoded optical polarization imaging and evaluate its performance for preoperative demarcation of keratinocyte carcinomas. Subjects with basal or squamous cell carcinoma, scheduled for Mohs surgery, were enrolled. The surgeon outlined the clinical boundary of each lesion preoperatively. Optical images of the lesions were then acquired at 440 and 640 nm. Spectral encoding of the experimental images minimized the impact of background pigmentation and vascularization. The surgeon was blinded to the imaging results. Margin assessments by imaging and by the surgeon were recorded and compared with the intraoperative histopathology. In total, 53 lesions were imaged in vivo. Thirteen cases required more than one Mohs stage. In all these cases, images accurately visualized the tumor. For cases negative following the first Mohs stage, margin assessments correlated with histopathology in 39 out of 40 cases. Imaging demonstrated 100% sensitivity and 98% specificity. Spectrally-encoded optical polarization imaging may prove valuable for real-time noninvasive preoperative delineation of skin cancer.

Multimodal quantitative imaging of brain cancer in cultured cells.

Fluorescence emission, polarization and subcellular localization of methylene blue (MB) were studied in four cancerous and two normal human brain cell lines. Fluorescence emission and polarization images were acquired and analyzed. The co-localization of MB with mitochondria, lysosomes and nuclei of the cells was evaluated. Glioblastoma cells exhibited significantly higher MB fluorescence polarization compared to normal astrocytes. Preferential accumulation of MB in mitochondria of glioblastoma cells may explain higher fluorescence polarization values in cancer cells as compared to normal. These findings may lead to the development of a quantitative method for the detection of brain cancer in single cells.

Fluorescence Polarization of Methylene Blue as a Quantitative Marker of Breast Cancer at the Cellular Level.

A quantitative technique to detect cancer in single cells could transform cancer diagnosis. Current cancer diagnosis utilizes histopathology, which requires tissue acquisition, extensive processing and, in most cases, relies on the qualitative morphological analysis of tissues and cells. Molecular biomarkers are only available for a few specific tumor subtypes. We discovered that the fluorescence polarization (Fpol) of Methylene Blue (MB) is significantly higher in cancer than in normal human breast tissues and cells. We confirmed that fluorescence polarization imaging did not affect the viability of the cells and yielded highly significant differences between cancer and normal cells using MB concentrations as low as 0.05 and 0.01 mg/ml. To explain this phenomenon we examined intracellular localization of MB and its fluorescence lifetime. We determined that higher fluorescence polarization of MB occurs due to its increased accumulation in mitochondria of cancer cells, as well as shorter fluorescence lifetime in cancer relative to normal cells. As quantitative MB Fpol imaging can be performed in vivo and in real time, it holds the potential to provide an accurate quantitative marker of cancer at the cellular level.

Collagen disruption as a marker for basal cell carcinoma in presurgical margin detection.

INTRODUCTION AND OBJECTIVE: Nonmelanoma skin cancers (NMSCs) are the most common malignancies in the United States. Surgery is the most common treatment for these tumors, but pre-operative identification of surgical margins is challenging. The objective in this study was to determine whether optical polarization imaging (OPI) could be used prior to surgery to detect the extent of subclinical tumor spread by monitoring disruption in collagen. MATERIALS AND METHODS: OPI is a non-invasive and rapid imaging modality that highlights the structure of dermal collagen. OPI was used preoperatively at wavelengths of 440 and 640 nm to perform imaging of NMSCs on six patients scheduled to undergo Mohs surgery for biopsy-proven basal cell carcinoma. This pilot study did not alter the course of routine MMS for any of the patients. The surgeon was blinded from the preoperative imaging results and completed the entire procedure without relying on the new technology. The study was conducted in an outpatient surgical setting. Patients over 18 years of age with biopsy-proven basal cell carcinoma participated. RESULTS AND CONCLUSION: OPI accurately predicted the presence or absence of tumor at the surgical margin in six out of six cases, as confirmed on histology. OPI may allow efficient surgical planning by identifying tumor extension beyond visibly involved skin. Lasers Surg. Med. 50:902-907, 2018. © 2018 Wiley Periodicals, Inc.

Optical mapping of nonmelanoma skin Cancers-A pilot clinical study.

BACKGROUND AND OBJECTIVE: Nonmelanoma skin cancer (NMSC) is the most common human cancer. Because these tumors often affect the face, there is a strong need for both accurate removal of these neoplasms to prevent recurrence and maximal tissue preservation to prevent cosmetic or functional deformity. Polarization-enhanced reflectance and fluorescence imaging (PERFI) is a new bedside method that uses fluorescent chromophores to image NMSC. While the feasibility of the technique has been successfully demonstrated in ex vivo studies, this is the first pilot study to extend the use of PERFI to in vivo intraoperative imaging of NMSC. MATERIALS AND METHODS: Subjects were recruited from a population of patients with biopsy-confirmed NMSC, scheduled for Mohs micrographic surgery. Eight cases were studied. Sterile methylene blue (MB) was diluted in anaesthetic solution and infused into the peritumoral space. Digital photographs of the lesion were taken and Mohs surgery was performed. Then, the surgical bed was re-imaged. Each excision was also imaged ex vivo and processed for routine histopathology. Optical images were processed and compared with histopathology. RESULTS AND CONCLUSIONS: The injection of MB was well tolerated. We observed a transient blue staining of the treated area, which disappeared completely within 1 week in all of the patients. In all subjects, the contrast agent, MB, was preferentially retained in the tumor. The ex vivo images correlated well with histopathology. In vivo images qualitatively delineated the tumor margins. The results of our pilot trial indicate that PERFI may be useful for accurate and rapid delineation of NMSC during surgery. Lasers Surg. Med. 49:803-809, 2017. © 2017 Wiley Periodicals, Inc.

Multimodal imaging for nonmelanoma skin cancer margin delineation.

BACKGROUND AND OBJECTIVE: Nonmelanoma skin cancer (NMSC) is the most common form of cancer worldwide. The most effective form of treating this cancer is its surgical removal. As NMSC rarely metastasize, its complete excision is often curative. We investigated the potential of combining Terahertz Pulsed Imaging (TPI) with polarization enhanced reflectance optical imaging for the accurate intraoperative delineation of NMSC. MATERIALS AND METHODS: Fresh thick samples with residual cancer were obtained from surgeries. The samples were imaged within two hours using polarization optical and TPI systems without remounting. Analysis of the TPI results was performed in the frequency domain. Co- and cross-polarized optical images were acquired at 410 nm. Superficial optical images were obtained by subtracting cross-polarized from the respective co-polarized images. Terahertz, optical, and histological images were overlaid and compared. RESULTS AND CONCLUSIONS: Our results show that the frequency powers of diseased and normal skin tissues differ significantly at 0.47 THz. While TPI has demonstrated contrast between diseased and normal tissue, it can also highlight normal structures. As TPI alone lacks the resolution necessary to distinguish between tissue types morphologically, polarization optical imaging was used for the inspection of the suspicious areas highlighted by TPI. Combined TPI and optical imaging has the potential for quick intraoperative delineation of cancers. Lasers Surg. Med. 49:319-326, 2017. © 2016 Wiley Periodicals, Inc.

Polarization enhanced wide-field imaging for evaluating dermal changes caused by non-ablative fractional laser treatment.

BACKGROUND AND OBJECTIVE: Laser non-ablative fractional treatment (NAFT) is an important part of armamentarium of modern dermatology. Recently, such treatments have become available in at-home setting due to advent of self-application NAFT devices. Safety and clinical efficacy of NAFT are well established in multiple studies. Less information is available on morphological and functional changes in tissue occurring as a result of NAFT. Polarization-enhanced multispectral wide-field imaging device allows for in vivo real time visualization of dermal structures. The objective of this study is to use this imaging modality to monitor early effects of the home-use NAFT on collagen networks. MATERIALS AND METHODS: Eight subjects (skin types I-III) used a commercially available NAFT device (wavelength 1410 nm, energy per pulse up to 15 mJ) to treat peri-orbital wrinkles in standard recommended mode, that is daily, for a period of two weeks. In each session, subjects applied a pre-treatment gel to the peri-orbital areas and then used the device, delivering 8-10 applications to each side of the face without overlap. Subjects were asked to use the highest device setting. Cross-polarized 440 nm wide-field images were acquired from peri-orbital areas before and two weeks after the onset of the treatment regimen. Wide-field images were normalized and thresholded to a level of 40% brightness to emphasize collagen structure. Collagen content was quantitatively determined from thresholded collagen images. Improvement in collagen content at two weeks of daily treatments was assessed. RESULTS: Eight subjects (age 24-53 years) completed the study. Cross-polarized 440 nm wide-field images clearly delineated collagen networks. Quantitative assessment of collagen images revealed statistically significant (P < 0.05) improvement of collagen content at a time point of two weeks. Seven out of eight subjects showed varying degree of improvement. The increase of collagen content in responders ranged from 1-26%, with the mean improvement of 11%. Subjects in their early 40s showed the best improvement in comparison to younger and older age groups. CONCLUSIONS: Polarization-enhanced multispectral wide-field reflectance imaging method is a suitable technique for noninvasive in vivo assessment of dermal structures. Post-treatment images, taken three days after the last treatment session, demonstrate that non-ablative fractional treatment resulted in increased dermal collagen content as measured by the polarization-enhanced technique as early as two weeks post onset of the treatments. However, further studies with a larger number of subjects and longer treatment period are required to determine the optimal regimen and how long the results will last.

Demeclocycline as a contrast agent for detecting brain neoplasms using confocal microscopy.

Complete resection of brain tumors improves life expectancy and quality. Thus, there is a strong need for high-resolution detection and microscopically controlled removal of brain neoplasms. The goal of this study was to test demeclocycline as a contrast enhancer for the intraoperative detection of brain tumors. We have imaged benign and cancerous brain tumors using multimodal confocal microscopy. The tumors investigated included pituitary adenoma, meningiomas, glioblastomas, and metastatic brain cancers. Freshly excised brain tissues were stained in 0.75 mg ml(-1) aqueous solution of demeclocyline. Reflectance images were acquired at 402 nm. Fluorescence signals were excited at 402 nm and registered between 500 and 540 nm. After imaging, histological sections were processed from the imaged specimens and compared to the optical images. Fluorescence images highlighted normal and cancerous brain cells, while reflectance images emphasized the morphology of connective tissue. The optical and histological images were in accordance with each other for all types of tumors investigated. Demeclocyline shows promise as a contrast agent for intraoperative detection of brain tumors.

Wavelength optimized cross-polarized wide-field imaging for noninvasive and rapid evaluation of dermal structures.

Changes in the morphology of dermal collagen may indicate aging or pathological processes. At present, there is no technology for in vivo real-time assessment of collagen structures. Our goal was to introduce and validate polarization optical imaging for noninvasive quantitative evaluation of dermal collagen. Seventeen volunteers participated in the study. Cross-polarized 440 nm images were acquired noninvasively from facial skin of the study subjects. Collagen content and intensity histogram were computed from the optical images. Quantitative results showed a decrease in the collagen content with increasing age of the subjects. Analysis of the collagen image histogram parameters demonstrated decreasing mean pixel value and increasing full width at half maximum (FWHM) with increasing age. Polarization optical imaging has the potential for rapid noninvasive in vivo evaluation of human dermis. Cross-polarized imaging at different wavelengths emphasizes different features of human skin. (A) Skin photograph. (B) In vivo cross-polarized image at 440 nm. (C) In vivo cross-polarized image at 570 nm. (D) In vivo cross-polarized image at 690 nm.

Polarization-sensitive multimodal imaging for detecting breast cancer.

Intraoperative delineation of breast cancer is a significant problem in surgical oncology. A reliable method for demarcation of malignant breast tissue during surgery would reduce the re-excision rate due to positive margins. We present a novel method of identifying breast cancer margins using combined dye-enhanced wide-field fluorescence polarization imaging for en face cancer margins and polarization-sensitive (PS) optical coherence tomography (OCT) for cross-sectional evaluation. Tumor specimens were collected following breast surgery, stained with methylene blue, and imaged. Wide-field fluorescence polarization images were excited at 640 nm and registered between 660 and 750 nm. Standard and PS OCT images were acquired using a commercial 1,310-nm swept-source system. The imaging results were validated against histopathology. Statistically significant higher fluorescence polarization of cancer as compared with both normal and fibrocystic tumor tissue was measured in all the samples. Fluorescence polarization delineated lateral breast cancer margins with contrast superior to that provided by OCT. However, OCT complemented fluorescence polarization imaging by facilitating cross-sectional inspection of tissue. PS OCT yielded higher contrast between cancer and connective tissue, as compared with standard OCT. Combined PS OCT and fluorescence polarization imaging shows promise for intraoperative delineation of breast cancer.

Imaging of ex vivo nonmelanoma skin cancers in the optical and terahertz spectral regions optical and terahertz skin cancers imaging.

We tested the hypothesis that polarization sensitive optical and terahertz imaging may be combined for accurate nonmelanoma skin cancer (NMSC) delineation. Nine NMSC specimens were imaged. 513 µm and 440 nm wavelengths were used for terahertz and optical imaging, respectively. Histopathology was processed for evaluation. Terahertz reflectance of NMSC was quantified. Our results demonstrate that cross-polarized terahertz images correctly identified location of the tumours, whereas cross-polarized and polarization difference optical images accurately presented morphological features. Cross-polarized terahertz images exhibited lower reflectivity values in cancer as compared to normal tissue. Combination of optical and terahertz imaging shows promise for intraoperative delineation of NMSC.