Stimulated Raman scattering (SRS) imaging of fresh tissue is an emerging approach to render label-free pathology and diagnosis based on the chemical contrasts of native biomolecules. However, the contrast of cell nuclei is often too weak to perform reliable segmentation and quantification. To advance the development of label-free digital pathology with SRS, we explored three deep learning-based techniques with multicolor and hyperspectral SRS imaging data, including U-Net, Mask R-CNN, and Autoencoder. Our results show that a combination of machine learning and SRS imaging is a promising pathway to transforming the methodology of SRS label-free pathology for real clinical use.
Glioblastoma (GBM) is one of the most aggressive cancer types in nervous system. Due to the limited effectiveness of current treatments, prognosis remains poor for GBM patients. Altered lipid metabolism is a hallmark of GBM. Previous studies show that increased lipid droplets (LDs) could protect cancer cells from chemotherapy and other extracellular stresses. In this study, we apply stimulated Raman scattering (SRS) microscopy to image LDs to monitor uptake of palmitic acid (PA) by U-87 glioma cells. We observe that uptake of free PA rapidly upregulates adipogenesis in glioma cells. We find that glioma cells exhibit distinctive time-dependent and dose-dependent patterns in PA uptake and adipogenesis. Uptake of extra PA significantly decreases the unsaturation degree of LDs. The decreased unsaturation degree and other lipotoxicity effects lead to clear cell death upon PA treatment at a high concentration. The results indicate that inhibition of adipogenesis may have therapeutic effects for GBM utilizing the lipotoxicity effects induced by free fatty acids. This work demonstrates that SRS microscopy for label-free imaging of LDs distribution and their composition is a promising tool for lipid metabolism studies in cancer.
Lymph nodes are an important secondary lymphoid organ in the lymphatic system. It is the major site of B cells, T cells and other white blood cells, leading to the function of identifying and fighting infection. To observe the structure of the lymph node, it is necessary to label it with dyes or fluorescent probes. Multi-color labeling are required to label various cell types and structures, which may be difficult under conditions such as in vivo labeling. Stimulated Raman scattering microscopy (SRS) is a burgeoning label-free biological imaging technique that has been used in many areas, such as tumor detection, pharmacokinetics, and neuroscience. Here, we developed a method of label-free imaging of various cell types and structures in lymph nodes with SRS. Collagenous fibers in the lymph node capsule, subcapsular sinus macrophages, B cells, blood vessels and white blood cells were observed in fresh lymph nodes. Based on the visualization of lymph node structure, we prospected its applications in cancer research, aging research and extracellular matrix research.
Stimulated Raman scattering (SRS) microscopy has been used for rapid label-free imaging of various biomolecules and drugs in living cells and tissues (Science, doi:10.1126/science.aaa8870). Our recent work has demonstrated that lipid and protein mapping of cancer tissue renders pathology-like images, providing essential histopathological information with subcellular resolution of the entire specimen (Cancer Research, doi: 10.1158/0008-5472.CAN-16-027). We have also established the first SRS imaging Atlas of human brain tumors (Harvard Dataverse, doi: (doi:10.7910/DVN/EZW4EK). SRS imaging of tissue could provide invaluable information for cancer diagnosis and surgical guidance in two aspects: rapid surgical pathology and quantitative biomolecular characterization. In this work, we present the use of SRS microscopy for characterization of a few essential biomolecules in breast cancer. Human breast cancer tissue specimens at the tumor core, tumor margin and normal area (5 cm away from the tumor) from surgical cases will be imaged with SRS at multiple Raman shifts, including the peaks for lipid, protein, blood (absorption), collagen, microcalcification (calcium phosphates and calcium oxalate) and carotenoids. Most of these Raman shifts have relatively strong Raman cross sections, which ensures high-quality and fast imaging. This proof-of-principle study is sought to demonstrate the feasibility and potential of SRS imaging for ambient diagnosis and surgical guidance of breast cancer.
Liver steatosis and fibrosis are two prevalence liver diseases and may eventually develop into hepatocellular
carcinoma (HCC) Due to their prevalence and severity, much work has been done to develop efficient
diagnostic methods and therapies. Nonlinear optical microscopy has high sensitivity and chemical specificity
for major biochemical compounds, making it a powerful tool for tissue imaging without staining. In this study,
three nonlinear microscopy imaging modalities are applied to the study of liver diseases in a bile duct ligation
rat modal. CARS shows the distributions of fats or lipids quantitatively across the tissue; SHG visualizes the
collagens; and TPEF reveals the morphology of hepatic cells. The results clearly show the development of
liver steatosis and fibrosis with time, and the hepatic fat and collagen fibrils are quantified. This study
demonstrates the ability of multimodal nonlinear optical microscopy for liver disease diagnosis, and may
provide new insights into the understanding of the mechanisms of steatosis/fibrosis transformations at the
cellular and molecular levels.
We report the implementation of a unique integrated coherent anti-Stokes Raman scattering (CARS), second-harmonic generation (SHG), and two-photon excitation fluorescence (TPEF) microscopy imaging technique developed for label-free monitoring of the progression of liver steatosis and fibrosis generated in a bile duct ligation (BDL) rat model. Among the 21 adult rats used in this study, 18 rats were performed with BDL surgery and sacrificed each week from weeks 1 to 6 (n = 3 per week), respectively; whereas 3 rats as control were sacrificed at week 0. Colocalized imaging of the aggregated hepatic fats, collagen fibrils, and hepatocyte morphologies in liver tissue is realized by using the integrated CARS, SHG, and TPEF technique. The results show that there are significant accumulations of hepatic lipid droplets and collagen fibrils associated with severe hepatocyte necrosis in BDL rat liver as compared to a normal liver tissue. The volume of normal hepatocytes keeps decreasing and the fiber collagen content in BDL rat liver follows a growing trend until week 6; whereas the hepatic fat content reaches a maximum in week 4 and then appears to stop growing in week 6, indicating that liver steatosis and fibrosis induced in a BDL rat liver model may develop at different rates. This work demonstrates that the integrated CARS and multiphoton microscopy imaging technique has the potential to provide an effective means for early diagnosis and detection of liver steatosis and fibrosis without labeling.
KEYWORDS: Microscopy, CARS tomography, Finite-difference time-domain method, Objectives, Signal detection, Molecules, Medical research, Near field optics, Tissue optics, Near field
We report on a unique annular aperture detection scheme in radially and linearly polarized coherent anti-Stokes Raman
scattering (RP- and LP-CARS) microscopy to effectively remove the solvent background for high contrast vibrational
imaging. Our finite-difference time-domain (FDTD) calculations show that the far-field CARS radiation from the
scatterer with size comparable to the excitation wavelength is stronger than that from the solvent at large cone angles
(from 45° to the maximum collection angle of the objective). The annular detection provides about one to two orders
higher contrast for forward detected RP- and LP-CARS microscopy.
Liver steatosis/fibrosis represents the major conditions and symptoms for many liver diseases. Nonlinear optical
microscopy has emerged as a powerful tool for label-free tissue imaging with high sensitivity and chemical specificity for
several typical biochemical compounds. Three nonlinear microscopy imaging modalities are implemented on the sectioned
tissues from diseased livers induced by high fat diet (HFD). Coherent anti-Stokes Raman scattering (CARS) imaging
visualizes and quantifies the lipid droplets accumulated in the liver, Second harmonic generation (SHG) is used to map the
distribution of aggregated collagen fibers, and two-photon excitation fluorescence (TPEF) reveals the morphology of
hepatic cells based on the autofluorescence signals from NADH and flavins within the hepatocytes. Our results
demonstrate that obesity induces liver steatosis in the beginning stage, which may progress into liver fibrosis with high
risk. There is a certain correlation between liver steatosis and fibrosis. This study may provide new insights into the
understanding of the mechanisms of steatosis/fibrosis transformations at the cellular and molecular levels.
Liver fibrosis is the excessive accumulation of extracellular matrix proteins such as collagens, which may result in
cirrhosis, liver failure, and portal hypertension. In this study, we apply a multimodal nonlinear optical microscopy
platform developed to investigate the fibrotic liver diseases in rat models established by performing bile duct ligation
(BDL) surgery. The three nonlinear microscopy imaging modalities are implemented on the same sectioned tissues of
diseased model sequentially: i.e., second harmonic generation (SHG) imaging quantifies the contents of the collagens,
the two-photon excitation fluorescence (TPEF) imaging reveals the morphology of hepatic cells, while coherent
anti-Stokes Raman scattering (CARS) imaging maps the distributions of fats or lipids quantitatively across the tissue.
Our imaging results show that during the development of liver fibrosis (collagens) in BDL model, fatty liver disease also
occurs. The aggregated concentrations of collagen and fat constituents in liver fibrosis model show a certain
correlationship between each other.
We report on a unique annular aperture detection scheme in radially polarized coherent anti-Stokes Raman scattering
(RP-CARS) microscopy to effectively remove the solvent background for high contrast vibrational imaging. Our
finite-difference time-domain (FDTD) calculations show that the far-field RP-CARS radiation from the scatterer with
size comparable to the excitation wavelength is stronger than that from the solvent at large cone angles (around 45° to
150°). The annular detection provides about one order higher contrast for both forward and backward detected RP-CARS
microscopy.
We report a novel interferometric-detection method for conventional polarization coherent anti-Stokes
Raman scattering (CARS) imaging with both high vibrational contrast and high signal strength. We
demonstrate this technique by imaging 10-μm polystyrene beads immersed in water.
Conventional polarization coherent anti-Stokes Raman scattering (P-CARS) microscopy always suffers
from serious signal attenuation in imaging cells and tissue. We develop a novel heterodyne-detection
method for P-CARS imaging with an ability of high imaging vibrational contrast and signal strength. We
demonstrate this method by imaging 4.69-μm polystyrene beads immersed in water.
According to the incident angle domain, the design of an ultraviolet omnidirectional reflector deposited on substrate of quartz glass is briefly presented. The spectral wavelength region of the design is from 328.95nm to 352.11nm and the relative bandwidth is 6.80% in theory. An experiment has been done to verify the design by using HfO2 and SiO2. The experimental result shows that the band gap corresponds to wavelength band from 331.2nm to 350.4nm and the relative bandwidth is 5.63%.
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