Combining coherent Raman scattering microscopy and deuterium labeling provides insight into metabolism in live cancer cells during cancer development and progression. The dynamic data is modeled with Michaelis-Menten-kinetics to quantify lipid synthesis and utilization in cancer cells. Changes in lipid levels are found to originate from de novo lipid synthesis using glucose as a source. In this work, we isolate fatty acid synthesis/consumption rates and elucidated effects of altered lipid metabolism in T47D breast cancer cells in response to estradiol stimulation and etomoxir treatment, dynamic processes that cannot be easily observed without the application of appropriate models.
Epithelial breast cells, which are involved in the production of milk fats, display altered lipid metabolism when they grow more malignant. At the same time, breast cancer cells show a higher degree of glycolysis, a mechanism to meet an increased demand for biomass as cells proliferate. Somewhat counterintuitively, breast cancer cells exhibit lower amounts of stored lipid, while glycolysis rates are up. We have investigated the kinetics of glycolysis, lipid synthesis, and lipid consumption rates in healthy and diseased epithelial breast cells. Using stimulated Raman scattering (SRS) microscopy of deuterated precursors, the cellular chemistry can be followed in space and time, revealing a clear link between glycolytic rate and the kinetics of lipid metabolism.
Two-photon excited fluorescence (TPEF) imaging of the cellular cofactors nicotinamide adenine dinucleotide and oxidized flavin adenine dinucleotide is widely used to measure cellular metabolism, both in normal and pathological cells and tissues. When dual-wavelength excitation is used, ratiometric TPEF imaging of the intrinsic cofactor fluorescence provides a metabolic index of cells—the “optical redox ratio” (ORR). With increased interest in understanding and controlling cellular metabolism in cancer, there is a need to evaluate the performance of ORR in malignant cells. We compare TPEF metabolic imaging with seahorse flux analysis of cellular oxygen consumption in two different breast cancer cell lines (MCF-7 and MDA-MB-231). We monitor metabolic index in living cells under both normal culture conditions and, for MCF-7, in response to cell respiration inhibitors and uncouplers. We observe a significant correlation between the TPEF-derived ORR and the flux analyzer measurements (R=0.7901, p<0.001). Our results confirm that the ORR is a valid dynamic index of cell metabolism under a range of oxygen consumption conditions relevant for cancer imaging.
We report on a direct comparison between Ti:Sapphire and Yb fiber lasers for depth-resolved label-free multimodal imaging of human skin. We found that the penetration depth achieved with the Yb laser was 80% greater than for the Ti:Sapphire. Third harmonic generation (THG) imaging with Yb laser excitation provides additional information about skin structure. Our results indicate the potential of fiber-based laser systems for moving into clinical use.
Clinical examination crucially relies on the ability to quickly examine large tissue areas and rapidly zoom in to regions of interest. Skin lesions often show irregularity in color and appearance in general, especially when they start to progress towards malignancy. Large field of view (FOV) and automatic translation of the imaging area are critical in the assessment of the entire lesion. Imaging of limited FOVs of the lesion can easily result in false negative diagnosis. We present a multiphoton microscope based on two-photon excited fluorescence and second-harmonic generation that images FOVs of about 0.8 mm2 (without stitching adjacent FOVs) at speeds of 10 frames/second (800 x 800 pixels) with lateral and axial resolutions of 0.5 μm and 2.5 μm, respectively. The main novelty of this instrument is the design of the scan head, which includes a fast galvanometric scanner, relay optics, a beam expander and a high NA objective lens. We optimized the system based on the Olympus 25x, 1.05NA water immersion lens, that features a long working distance of 1 mm. Proper tailoring of the beam expander, which consists of the scan and tube lens elements, enables scaling of the FOV. The design criteria include a flat wavefront of the beam, minimum field curvature, and suppressed spherical aberrations. All aberrations in focus are below the Marechal criterion of 0.07λ rms for diffraction-limited performance. We demonstrate the practical utility of this microscope by ex-vivo imaging of wide FOVs in normal human skin.
Advancing the practical utility of nonlinear optical microscopy requires continued improvement in imaging depth and contrast. We evaluated second‐harmonic generation (SHG) and third‐harmonic generation images from ex vivo human skin and showed that a sub‐40 fs, 1060‐nm Yb‐fiber laser can enhance SHG penetration depth by up to 80% compared to a >100 fs, 800 nm Ti:sapphire source. These results demonstrate the potential of fiber‐based laser systems to address a key performance limitation related to nonlinear optical microscopy (NLOM) technology while providing a low‐barrier‐to‐access alternative to Ti:sapphire sources that could help accelerate the movement of NLOM into clinical practice.
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