Investigating ionizing radiation-induced changes in breast cancer cells using stimulated Raman scattering microscopy.

Significance: Altered lipid metabolism of cancer cells has been implicated in increased radiation resistance. A better understanding of this phenomenon may lead to improved radiation treatment planning. Stimulated Raman scattering (SRS) microscopy enables label-free and quantitative imaging of cellular lipids but has never been applied in this domain. Aim: We sought to investigate the radiobiological response in human breast cancer MCF7 cells using SRS microscopy, focusing on how radiation affects lipid droplet (LD) distribution and cellular morphology. Approach: MCF7 breast cancer cells were exposed to either 0 or 30 Gy (X-ray) ionizing radiation and imaged using a spectrally focused SRS microscope every 24 hrs over a 72-hr time period. Images were analyzed to quantify changes in LD area per cell, lipid and protein content per cell, and cellular morphology. Cell viability and confluency were measured using a live cell imaging system while radiation-induced lipid peroxidation was assessed using BODIPY C11 staining and flow cytometry. Results: The LD area per cell and total lipid and protein intensities per cell were found to increase significantly for irradiated cells compared to control cells from 48 to 72 hrs post irradiation. Increased cell size, vacuole formation, and multinucleation were observed as well. No significant cell death was observed due to irradiation, but lipid peroxidation was found to be greater in the irradiated cells than control cells at 72 hrs. Conclusions: This pilot study demonstrates the potential of SRS imaging for investigating ionizing radiation-induced changes in cancer cells without the use of fluorescent labels.

Spectral focusing-based stimulated Raman scattering microscopy using compact glass blocks for adjustable dispersion.

Spectral focusing is a well-established technique for increasing spectral resolution in coherent Raman scattering microscopy. However, current methods for tuning optical chirp in setups using spectral focusing, such as glass rods, gratings, and prisms, are very cumbersome, time-consuming to use, and difficult to align, all of which limit more widespread use of the spectral focusing technique. Here, we report a stimulated Raman scattering (SRS) configuration which can rapidly tune optical chirp by utilizing compact adjustable-dispersion TIH53 glass blocks. By varying the height of the blocks, the number of bounces in the blocks and therefore path length of the pulses through the glass can be quickly modulated, allowing for a convenient method of adjusting chirp with almost no necessary realignment. To demonstrate the flexibility of this configuration, we characterize our system's signal-to-noise ratio and spectral resolution at different chirp values and perform imaging in both the carbon-hydrogen stretching region (MCF-7 cells) and fingerprint region (prostate cores). Our findings show that adjustable-dispersion glass blocks allow the user to effortlessly modify their optical system to suit their imaging requirements. These blocks can be used to significantly simplify and miniaturize experimental configurations utilizing spectral focusing.

Exploring the Use of Raman Spectroscopy and Covariate-Adjusted Multivariate Analysis for the Detection of Irradiated Blood.

Biological dosimetry is a key technique for retrospective radiation dosimetry that provides individual estimates of absorbed dose of ionizing radiation, applicable for use in a large scale radiological/nuclear event. Current techniques for biodosimetry are labor intensive and time consuming and not high through-put. In this proof-of-concept study, we developed a new approach for detecting irradiated blood based on Raman spectroscopy of blood combined with multivariate analysis. Peripheral blood samples from 8 healthy male and female, anonymous donors, were exposed to either 5 Gy X ray radiation or unirradiated (0 Gy). At 3 h postirradiation, the blood was immediately frozen at -80°C. Raman spectra were measured from thawed blood using a portable spectrometer system. Data were preprocessed and analyzed using principal component analysis (PCA) to observe trends in the data, and by using partial least squares-discriminant analysis (PLS-DA) to build a model to discriminate between Raman spectra of control (0 Gy) and irradiated (5 Gy) blood. We found strong evidence of inter-donor variability in the form of donor-wise clustering of PCA scores corresponding to the control Raman spectra, in addition to the poor separation of PLS-DA scores associated with Raman intensities of 0 Gy vs. 5 Gy spectra. However, after adjustment for donor covariates using a linear mixed-effects model, we obtained a better separation between control and irradiated blood using PLS-DA. Evaluation of the coefficients of the PLS-DA loading vectors indicated radiation-induced changes in proteins, lipids and hemoglobin to be major contributors for this discrimination. This pilot study demonstrates the potential of application of Raman spectroscopy to support biodosimetry of blood and blood components.

Multispectral label-free Raman spectroscopy can detect ovarian and endometrial cancer with high accuracy.

Up to 70% of ovarian cancer patients are diagnosed with advanced-stage disease and the degree of cytoreduction is an important survival prognostic factor. The aim of this study was to evaluate if Raman spectroscopy could detect cancer from different organs within the abdominopelvic region, including the ovaries. A Raman spectroscopy probe was used to interrogate specimens from a cohort of nine patients undergoing cytoreductive surgery, including four ovarian cancer patients and three patients with endometrial cancer. A feature-selection algorithm was developed to determine which spectral bands contributed to cancer detection and a machine-learning model was trained. The model could detect cancer using only eight spectral bands. The receiver-operating-characteristic curve had an area-under-the-curve of 0.96, corresponding to an accuracy, a sensitivity and a specificity of 90%, 93% and 88%, respectively. These results provide evidence multispectral Raman spectroscopy could be developed to detect ovarian cancer intraoperatively.

Label-free two-photon imaging of mitochondrial activity in murine macrophages stimulated with bacterial and viral ligands.

Mitochondria are the metabolic hub of the cell, playing a central role in regulating immune responses. Dysfunction of mitochondrial reprogramming can occur during bacterial and viral infections compromising hosts' immune signaling. Comparative evaluation of these alterations in response to bacterial and viral ligands can provide insights into a cell's ability to mount pathogen-specific responses. In this study, we used two-photon excitation fluorescence (TPEF) imaging to quantify reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) and flavin adenine dinucleotide (FAD) levels in the cell and to calculate the optical redox ratio (ORR), an indicator of mitochondrial dysfunction. Analyses were performed on RAW264.7 cells and murine bone marrow derived macrophages (BMM) stimulated with bacterial (LPS) and viral (Poly(I:C)) ligands. Responses were cell type dependent, with primary cells having significantly higher levels of FAD and higher oxygen consumption rates suggesting BMM may be more dependent on mitochondrial metabolism. Our findings also suggest that FAD-TPEF intensity may be a better predictor of mitochondrial activity and localization since it demonstrates unique mitochondrial clustering patterns in LPS vs. Poly(I:C) stimulated macrophages. Collectively, we demonstrate that TPEF imaging is a powerful label-free approach for quantifying changes in mitochondrial function and organization in macrophages following bacterial and viral stimuli.

High spatial resolution dosimetry with uncertainty analysis using Raman micro-spectroscopy readout of radiochromic films.

PURPOSE: The purpose of this work is to develop a new approach for high spatial resolution dosimetry based on Raman micro-spectroscopy scanning of radiochromic film (RCF). The goal is to generate dose calibration curves over an extended dose range from 0 to 50 Gy and with improved sensitivity to low (<2 Gy) doses, in addition to evaluating the uncertainties in dose estimation associated with the calibration curves. METHODS: Samples of RCF (EBT3) were irradiated at a broad dose range of 0.03-50 Gy using an Elekta Synergy clinical linear accelerator. Raman spectra were acquired with a custom-built Raman micro-spectroscopy setup involving a 500 mW, multimode 785 nm laser focused to a lateral spot diameter of 30 µm on the RCF. The depth of focus of 34 µm enabled the concurrent collection of Raman spectra from the RCF active layer and the polyester laminate. The preprocessed Raman spectra were normalized to the intensity of the 1614 cm-1 Raman peak from the polyester laminate that was unaltered by radiation. The mean intensities and the corresponding standard deviation of the active layer Raman peaks at 696, 1445, and 2060 cm-1 were determined for the 150 × 100 µm2 scan area per dose value. This was used to generate three calibration curves that enabled the conversion of the measured Raman intensity to dose values. The experimental, fitting, and total dose uncertainty was determined across the entire dose range for the dosimetry system of Raman micro-spectroscopy and RCF. RESULTS: In contrast to previous work that investigated the Raman response of RCFs using different methods, high resolution in the dose response of the RCF, even down to 0.03 Gy, was obtained in this study. The dynamic range of the calibration curves based on all three Raman peaks in the RCF extended up to 50 Gy with no saturation. At a spatial resolution of 30 × 30 µm2 , the total uncertainty in estimating dose in the 0.5-50 Gy dose range was [6-9]% for all three Raman calibration curves. This consisted of the experimental uncertainty of [5-8]%, and the fitting uncertainty of [2.5-4.5]%. The main contribution to the experimental uncertainty was determined to be from the scan area inhomogeneity which can be readily reduced in future experiments. The fitting uncertainty could be reduced by performing Raman measurements on RCF samples at further intermediate dose values in the high and low dose range. CONCLUSIONS: The high spatial resolution experimental dosimetry technique based on Raman micro-spectroscopy and RCF presented here, could become potentially useful for applications in microdosimetry to produce meaningful dose estimates in cellular targets, as well as for applications based on small field dosimetry that involve high dose gradients.

Measurement of cartilage sub-component distributions through the surface by Raman spectroscopy-based multivariate analysis.

Articular cartilage posesses unique material properties due to a complex depth-dependent composition of sub-components. Raman spectroscopy has proven valuable in quantifying this composition through cartilage cross-sections. However, cross-sectioning requires tissue destruction and is not practical in situ. In this work, Raman spectroscopy-based multivariate curve resolution (MCR) was employed in porcine cartilage samples (n = 12) to measure collagen, glycosaminoglycan, and water distributions through the surface for the first time; these were compared against cross-section standards. Through the surface Raman measurements proved reliable in predicting composition distribution up to a depth of approximately 0.5 mm. A fructose-based optical clearing agent (OCA) was also used in an attempt to further improve depth of resolution of this measurement method. However, it did not; mainly due to a high-spectral overlap with the Raman spectra of main cartilage sub-components. This measurement technique potentially could be used in situ, to better understand the etiology of joint diseases such as osteoarthritis (OA).

Coherent anti-Stokes Raman scattering imaging using silicon photomultipliers.

Silicon photomultipliers (SiPMs) are an emerging solid-state alternative to photomultiplier tubes (PMTs) for low light detection, with similar gain but lower cost and lower operating voltage. We demonstrate coherent anti-Stokes Raman scattering (CARS) imaging in a side-by-side comparison of an uncooled SiPM with an uncooled multialkali PMT as well as a state-of-the-art cooled GaAsP PMT. We determine the optimum reverse-bias voltage for acquiring the best signal-to-noise ratio (SNR) for CARS imaging of lipids at ${2850}\;{{\rm cm}^{ - 1}}$2850cm-1. We find that despite the higher dark counts, the SNR of CARS images acquired with the uncooled SiPM biased at an optimum voltage is better than that of the multialkali PMT and close to that of the cooled GaAsP PMT (${\sim}{1.5}$∼1.5 and ${\sim}{0.8}$∼0.8 times, respectively). This is due to the higher gain and lower excess noise factor related to the pulse height variability in the SiPM.

Metformin Abrogates Age-Associated Ovarian Fibrosis.

PURPOSE: The ovarian cancer risk factors of age and ovulation are curious because ovarian cancer incidence increases in postmenopausal women, long after ovulations have ceased. To determine how age and ovulation underlie ovarian cancer risk, we assessed the effects of these risk factors on the ovarian microenvironment. EXPERIMENTAL DESIGN: Aged C57/lcrfa mice (0-33 months old) were generated to assess the aged ovarian microenvironment. To expand our findings into human aging, we assembled a cohort of normal human ovaries (n = 18, 21-71 years old). To validate our findings, an independent cohort of normal human ovaries was assembled (n = 9, 41-82 years old). RESULTS: We first validated the presence of age-associated murine ovarian fibrosis. Using interdisciplinary methodologies, we provide novel evidence that ovarian fibrosis also develops in human postmenopausal ovaries across two independent cohorts (n = 27). Fibrotic ovaries have an increased CD206+:CD68+ cell ratio, CD8+ T-cell infiltration, and profibrotic DPP4+αSMA+ fibroblasts. Metformin use was associated with attenuated CD8+ T-cell infiltration and reduced CD206+:CD68+ cell ratio. CONCLUSIONS: These data support a novel hypothesis that unifies the primary nonhereditary ovarian cancer risk factors through the development of ovarian fibrosis and the formation of a premetastatic niche, and suggests a potential use for metformin in ovarian cancer prophylaxis.See related commentary by Madariaga et al., p. 523.

Development of a flow cell based Raman spectroscopy technique to overcome photodegradation in human blood.

Raman spectroscopy of blood offers significant potential for label-free diagnostics of disease. However, current techniques are limited by the use of low laser power to avoid photodegradation of blood; this translates to a low signal to noise ratio in the Raman spectra. We developed a novel flow cell based Raman spectroscopy technique that provides reproducible Raman spectra with a high signal to noise ratio and low data acquisition time while ensuring a short dwell time in the laser spot to avoid photodamage in blood lysates. We show that our novel setup is capable of detecting minute changes in blood lysate spectral features from natural aging. Moreover, we demonstrate that by rigorously controlling the experimental conditions, the aging effect due to natural oxidation does not confound the Raman spectral measurements and that blood treated with hydrogen peroxide to induce oxidative stress can be discriminated from normal blood with a high accuracy of greater than 90% demonstrating potential for use in a clinical setting.

Automated classification of multiphoton microscopy images of ovarian tissue using deep learning.

Histopathological image analysis of stained tissue slides is routinely used in tumor detection and classification. However, diagnosis requires a highly trained pathologist and can thus be time-consuming, labor-intensive, and potentially risk bias. Here, we demonstrate a potential complementary approach for diagnosis. We show that multiphoton microscopy images from unstained, reproductive tissues can be robustly classified using deep learning techniques. We fine-train four pretrained convolutional neural networks using over 200 murine tissue images based on combined second-harmonic generation and two-photon excitation fluorescence contrast, to classify the tissues either as healthy or associated with high-grade serous carcinoma with over 95% sensitivity and 97% specificity. Our approach shows promise for applications involving automated disease diagnosis. It could also be readily applied to other tissues, diseases, and related classification problems.

Oxidative and nitrative stress-related changes in human lens epithelial cells following exposure to X-rays.

PURPOSE: There is limited understanding of the mechanistic effects of ionizing radiation (IR) exposure in cataract formation. In this study, we explored the effects of IR on reactive oxygen/nitrogen species (ROS and RNS) generation in human lens epithelial (HLE) cells as an early key event to long-term damage. MATERIALS AND METHODS: HLE cell-line was exposed to X-rays at varied doses (0-5 Gy) and dose-rates. Cell lysates and supernatants were collected 20 h post-exposure and analysed for viability, cell cycling and metabolites of ROS (p, m-, o-, tyrosines, 3-chlorotyrosine (cl-tyrosine), 8-hydroxy deoxyguanosine, (8-OH-dG) and RNS (3-nitrotyrosine). RESULTS AND CONCLUSIONS: HLE cell-line exhibited a bi-phasic response in terms of cell viability, ROS and RNS profiles. At doses <0.5 Gy, ROS and RNS levels were lower than control and at higher doses (>0.5 Gy) a steady increase was observed in each metabolite. This response was observed irrespective of dose-rate. Among the associations tested, cl, p, m-tyrosine and 3-nitrotyrosine revealed changes (p < .05) at 5 Gy compared exclusively to 0.05 and 0.01 Gy. In addition, dose-rate related differences were observed. Overall, the data suggests that ROS and RNS are key events in radiation induced damage and this response is dependent on the dose and dose-rate of IR exposure.

Raman micro-spectroscopy analysis of human lens epithelial cells exposed to a low-dose-range of ionizing radiation.

Recent findings in populations exposed to ionizing radiation (IR) indicate dose-related lens opacification occurs at much lower doses (<2 Gy) than indicated in radiation protection guidelines. As a result, research efforts are now being directed towards identifying early predictors of lens degeneration resulting in cataractogenesis. In this study, Raman micro-spectroscopy was used to investigate the effects of varying doses of radiation, ranging from 0.01 Gy to 5 Gy, on human lens epithelial (HLE) cells which were chemically fixed 24 h post-irradiation. Raman spectra were acquired from the nucleus and cytoplasm of the HLE cells. Spectra were collected from points in a 3 × 3 grid pattern and then averaged. The raw spectra were preprocessed and principal component analysis followed by linear discriminant analysis was used to discriminate between dose and control for 0.25, 0.5, 2, and 5 Gy. Using leave-one-out cross-validation accuracies of greater than 74% were attained for each dose/control combination. The ultra-low doses 0.01 and 0.05 Gy were included in an analysis of band intensities for Raman bands found to be significant in the linear discrimination, and an induced repair model survival curve was fit to a band-difference-ratio plot of this data, suggesting HLE cells undergo a nonlinear response to low-doses of IR. A survival curve was also fit to clonogenic assay data done on the irradiated HLE cells, showing a similar nonlinear response.

Raman micro-spectroscopy applied to treatment resistant and sensitive human ovarian cancer cells.

Despite the many advances intended to enhance the response to treatment, the survival rate of patients with ovarian cancer has only marginally improved in the past few decades. One major cause for this, is the lack of diagnostics for platinum-resistant disease. The goal of this study was to determine whether Raman micro-spectroscopy in conjunction with multivariate statistical analysis could discriminate between chemically fixed cisplatin-resistant (A2780cp) and cisplatin-sensitive (A2780s) human ovarian carcinoma cells. Raman spectra collected from individual cells were pre-processed and subsequently analyzed with Principal Component Analysis - Linear Discriminant Analysis (PCA-LDA). Statistically significant differences (P <  0.0001) were observed between the Raman spectra of A2780s and A2780cp cells. A diagnostic accuracy of 82% was obtained using the PCA-LDA classifier model for the discrimination between the A2780s and A2780cp cells. The loading plot analysis suggests that relative increases in proteins and glutathione in the cisplatin-resistant cells compared to the cisplatin-sensitive cells are most likely the major source of discrimination between the two types of cells. These results support the potential application of Raman spectroscopy in the identification of chemo-resistant tumors prior to treatment.

Determining optimum operating conditions of the polarization-maintaining fiber with two far-lying zero dispersion wavelengths for CARS microscopy.

Single femtosecond laser-based coherent anti-Stokes Raman scattering (CARS) microscopy, using a photonic crystal fiber (PCF) pumped in the near-IR to generate a supercontinuum for the Stokes source, is rapidly being adopted as a cost-effective approach. A PCF with two closely-lying zero dispersion wavelengths is a popular choice for the Stokes source, but it is often limited to imaging lipids. A polarization-maintaining PCF with two far-lying zero dispersion wavelengths offers important advantages for polarization CARS microscopy, and for CARS imaging in the fingerprint region. This PCF fiber, though commercially available, has limited use for CARS microscopy in the C-H bond region. The main problem is that the supercontinuum from this fiber is typically noisier than that from a standard PCF with two closely-lying zero dispersion wavelengths. To overcome this, we determined the optimum operating conditions for generating a low-noise supercontinuum out of a PCF with two far-lying zero dispersion wavelengths, in terms of the input parameters of the excitation pulse. We measured the relative intensity noise (RIN) of the Stokes and the corresponding CARS signal as a function of the input laser parameters in this fiber. We showed that the results of CARS imaging using this alternate fiber are comparable to those achieved using the standard fiber, for input laser pulse conditions of low average power, narrow pulse width with slightly positive chirp, and polarization direction parallel to the slow axis of the selected fiber.

Enhancing optical field intensities in Gaussian-profile fiber Bragg gratings.

Gaussian profile fiber Bragg gratings exhibit narrow-bandwidth transmission peaks with significant group delay at the edge of their photonic bandgap. We demonstrate group delays ranging from 0.2 to 5.6 ns from a 1.2 cm structure. Simulations suggest such a device would be capable of enhancing the field intensity of incoming light by a factor of 800. Enhancement is confirmed by photothermally induced bistability of these peaks even at sub-milliwatt input powers with as much as a four-fold difference in the magnitude of their responses. The strong field intensities of these modes could significantly enhance desired nonlinear optical responses in fiber, provided the impact of absorption is addressed.

Portable, miniaturized, fibre delivered, multimodal CARS exoscope.

We demonstrate for the first time, a portable multimodal coherent anti-Stokes Raman scattering microscope (exoscope) for minimally invasive in-vivo imaging of tissues. This device is based around a micro-electromechanical system scanning mirror and miniaturized optics with light delivery accomplished by a photonic crystal fibre. A single Ti:sapphire femtosecond pulsed laser is used as the light source to produce CARS, two photon excitation fluorescence and second harmonic generation images. The high resolution and distortion-free images obtained from various resolution and bio-samples, particularly in backward direction (epi) successfully demonstrate proof of concept, and pave the path towards future non or minimally-invasive in vivo imaging.

Miniaturized multimodal CARS microscope based on MEMS scanning and a single laser source.

We demonstrate a novel miniaturized multimodal coherent anti-Stokes Raman scattering (CARS) microscope based on microelectromechanical systems (MEMS) scanning mirrors and custom miniature optics. A single Ti:sapphire femtosecond pulsed laser is used as the light source to produce the CARS, two photon excitation fluorescence (TPEF) and second harmonic generation (SHG) images using this miniaturized microscope. The high resolution and distortion-free images obtained from various samples such as a USAF target, fluorescent and polystyrene microspheres and biological tissue successfully demonstrate proof of concept, and pave the path towards future integration of parts into a handheld multimodal CARS probe for non- or minimally-invasive in vivo imaging.

Coherent anti-Stokes Raman scattering microscopy using photonic crystal fiber with two closely lying zero dispersion wavelengths.

We demonstrate coherent anti-Stokes Raman scattering (CARS) microscopy of lipid-rich structures using a single unamplified femtosecond Ti:sapphire laser and a photonic crystal fiber (PCF) with two closely lying zero dispersion wavelengths (ZDW) for the Stokes source. The primary enabling factor for the fast data acquisition (84 micros per pixel) in the proof-of-principle CARS images, is the low noise supercontinuum (SC) generated in this type of PCF, in contrast to SC generated in a PCF with one ZDW. The dependence of the Stokes pulse on average input power, pump wavelength, pulse duration and polarization is experimentally characterized. We show that it is possible to control the spectral shape of the SC by tuning the pump wavelength of the input pulse and the consequence for CARS microscopy is discussed.