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.

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.

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.

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.

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.