Diagnostic results for the surgical specimens from surgical cases.

Supplementary dataset information and Supplementary Figure legend.

Diagnostic features revealed from label-free SRS imaging versus H&E staining of human brain tumors.

The concept of registering SRS imaging with 3D MRI to guide brain tumor delineation intraoperatively.

Neurodegenerative diseases are often accompanied with pathological changes at molecular, cellular, and tissue levels. SRS microscopy, capable of providing intrinsic chemical and spatial resolutions, is in principle suited for detecting the pathological features associated with neural degeneration. In this chapter, two major types of neurodegenerative diseases are studied by SRS. First, motor neuron disease a.k.a. ALS is connected with demyelination, hence the lipid contents and morphological changes of myelin sheath could be imaged by SRS. Second, protein misfolding disease such as Alzheimer’s disease is closely related to the secondary conformational changes of proteins, which leads to the vibrational spectral shifts that could be exploited by SRS to image amyloid plaques in the brain tissues.

Cell mass and chemical composition are important aggregate cellular properties that are especially relevant to physiological processes, such as growth control and tissue homeostasis. Despite their importance, it has been difficult to measure these features quantitatively at the individual cell level in intact tissue. Here, we introduce normalized Raman imaging (NoRI), a stimulated Raman scattering (SRS) microscopy method that provides the local concentrations of protein, lipid, and water from live or fixed tissue samples with high spatial resolution. Using NoRI, we demonstrate that protein, lipid, and water concentrations at the single cell are maintained in a tight range in cells under the same physiological conditions and are altered in different physiological states, such as cell cycle stages, attachment to substrates of different stiffness, or by entering senescence. In animal tissues, protein and lipid concentration varies with cell types, yet an unexpected cell-to-cell heterogeneity was found in cerebellar Purkinje cells. The protein and lipid concentration profile provides means to quantitatively compare disease-related pathology, as demonstrated using models of Alzheimer's disease. This demonstration shows that NoRI is a broadly applicable technique for probing the biological regulation of protein mass, lipid mass, and water mass for studies of cellular and tissue growth, homeostasis, and disease.

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.

Cell growth underlies myriad biological processes including development, regeneration and tumor growth. Diverse extracellular signals such as growth factors, hormones, nutrients, and cell-to-cell contact are transduced by the signaling pathway to regulate growth. Yet it is still an open question how the integration of diverse inputs results in cell growth or maintains cells at the right size in the face of massive protein turnover. Accurate measurement of cell size is critical for probing these questions. State-of-art techniques such as quantitative phase microscopy, suspended microchannel resonator, and novel fluorescence reporter made it possible to measure cellular dry mass in higher precision and throughput than ever. Nevertheless, the existing methods require suspended cells or in vitro cell cultures by which the contextual information of the tissue is lost. While the cell volume can be measured by confocal microscopy, there is no existing method to measure cellular dry mass or protein mass in situ. Here we present Normalized Raman Imaging (NoRI) that fills the technology gap by providing the absolute concentrations of total protein, total lipid, and water of cells and subcellular compartments in situ. NoRI achieves the absolute quantification by combining Stimulated Raman scattering (SRS) microscopy with a computational algorithm that removes the effect of light scattering in thick tissue samples. The protein mass and lipid mass of single cells can be measured by integrating the respective concentrations over the cell volume. NoRI is a label-free technique that can measure live or fixed tissue sections, cultured cells, and small model organisms without staining. Using NoRI we found that cell types and tissue types have characteristic protein and lipid density, which changes with developmental or disease processes. We applied NoRI to study cell growth during the terminal differentiation of epiphyseal chondrocytes.

Background Conventional processing of nerve for histomorphometry is resource-intensive, precluding use in intraoperative assessment of nerve quality during nerve transfer procedures. Stimulated Raman scattering (SRS) microscopy is a label-free technique that enables rapid and high-resolution histology. Methods Segments of healthy murine sciatic nerve, healthy human obturator nerve, and human cross-facial nerve autografts were imaged on a custom SRS microscope. Myelinated axon quantification was performed through segmentation using a random forest machine learning algorithm in commercial software. Results High contrast, high-resolution imaging of nerve morphology was obtained with SRS imaging. Automated myelinated axon quantification from cross-sections of healthy human nerve imaged using SRS was achieved. Conclusions Herein, we demonstrate the use of a label-free technique for rapid imaging of murine and human peripheral nerve cryosections. We illustrate the potential of this technique to inform intraoperative decision-making through rapid automated quantification of myelinated axons using a machine learning algorithm.

Stimulated Raman scattering (SRS) is suitable for combination with superresolution microscopy techniques such as Stimulation Depletion Emission (STED) microscopy to explore nanoscale biological processes. Whereas SRS may be employed for label-free imaging of lipids and proteins, STED microscopy allows for super-resolution of small labelled-vesicles such as exosomes, which play a key role in cell-to-cell communication. The combination of these two imaging techniques allows for more comprehensive study of biological samples under investigation. Herein, we implemented STED microscopy onto an existing custom SRS setup. Herein, the same stoke and pump beam used for SRS were employed for excitation and depletion beam on STED microscopy. The pulse widths of the picosecond lasers were independently adjusted for efficient SRS and STED imaging. The point spread function was engineered to alternate between a conventional Gaussian and Laguerre-Gaussian donut beam for sequential SRS and STED imaging, respectively. The similarity of the techniques facilitated their combination.

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technique. Two-color imaging is often necessary to determine the distribution of chemical species in SRS microscopy. Current multi-color SRS imaging methods involve complicated instrumentation or longer data acquisition time or are limited to transmission imaging. In this Letter, we show that by adding a simple fiber amplifier to a 2 ps laser source and optical-parametric-oscillator-based SRS setup, one can achieve simultaneous two-color or frequency modulation SRS microscopy. The fiber amplifier can generate a wavelength tunable laser of +/- 10 nm around the Stokes laser wavelength at 1031 nm with average power greater than 200 mW. In vivo and ex vivo lipid-protein imaging of mouse brain and skin is demonstrated. To further demonstrate the potential of this technique in high-speed in vivo imaging, white blood cells in a blood stream are imaged in a live mouse. (C) 2017 Optical Society of America

The study of amyotrophic lateral sclerosis (ALS) and potential interventions would be facilitated if motor axon degeneration could be more readily visualized. Here we demonstrate that stimulated Raman scattering (SRS) microscopy could be used to sensitively monitor peripheral nerve degeneration in ALS mouse models and ALS autopsy materials. Three-dimensional imaging of pre-symptomatic SOD1 mouse models and data processing by a correlation-based algorithm revealed that significant degeneration of peripheral nerves could be detected coincidentally with the earliest detectable signs of muscle denervation and preceded physiologically measurable motor function decline. We also found that peripheral degeneration was an early event in FUS as well as C9ORF72 repeat expansion models of ALS, and that serial imaging allowed long-term observation of disease progression and drug effects in living animals. Our study demonstrates that SRS imaging is a sensitive and quantitative means of measuring disease progression, greatly facilitating future studies of disease mechanisms and candidate therapeutics.

Acetylcholine is an important neurotransmitter that relays neural excitation from lower motor neurons to muscles. It also plays significant roles in the central nervous system by modulating neurotransmission. However, there is a lack of tools to directly measure the quantity and distribution of acetylcholine at the subcellular level. In this Communication, we demonstrate for the first time that label-free imaging of acetylcholine is achieved with frequency-modulated spectral-focusing stimulated Raman scattering (FMSF-SRS) microscopy: a technical improvement over traditional SRS microscopy that effectively removes imaging backgrounds. Moreover, we directly quantified the local concentration of acetylcholine at the neuromuscular junction of frog cutaneous pectoris muscle.

Abstract The goal of brain tumor surgery is to maximize tumor removal without injuring critical brain structures. Achieving this goal is challenging as it can be difficult to distinguish tumor from nontumor tissue. While standard histopathology provides information that could assist tumor delineation, it cannot be performed iteratively during surgery as freezing, sectioning, and staining of the tissue require too much time. Stimulated Raman scattering (SRS) microscopy is a powerful label-free chemical imaging technology that enables rapid mapping of lipids and proteins within a fresh specimen. This information can be rendered into pathology-like images. Although this approach has been used to assess the density of glioma cells in murine orthotopic xenografts models and human brain tumors, tissue heterogeneity in clinical brain tumors has not yet been fully evaluated with SRS imaging. Here we profile 41 specimens resected from 12 patients with a range of brain tumors. By evaluating large-scale stimulated Raman imaging data and correlating this data with current clinical gold standard of histopathology for 4,422 fields of view, we capture many essential diagnostic hallmarks for glioma classification. Notably, in fresh tumor samples, we observe additional features, not seen by conventional methods, including extensive lipid droplets within glioma cells, collagen deposition in gliosarcoma, and irregularity and disruption of myelinated fibers in areas infiltrated by oligodendroglioma cells. The data are freely available in a public resource to foster diagnostic training and to permit additional interrogation. Our work establishes the methodology and provides a significant collection of reference images for label-free neurosurgical pathology. Cancer Res; 76(12); 3451–62. ©2016 AACR.

Stimulated Raman Scattering microscopy allows label-free chemical imaging and has enabled exciting applications in biology, material science, and medicine. It provides a major advantage in imaging speed over spontaneous Raman scattering and has improved image contrast and spectral fidelity compared to coherent anti-Stokes Raman. Wider adoption of the technique has, however, been hindered by the need for a costly and environmentally sensitive tunable ultra-fast dual-wavelength source. We present the development of an optimized all-fibre laser system based on the optical synchronization of two picosecond power amplifiers. To circumvent the high-frequency laser noise intrinsic to amplified fibre lasers, we have further developed a high-speed noise cancellation system based on voltage-subtraction autobalanced detection. We demonstrate uncompromised imaging performance of our fibre-laser based stimulated Raman scattering microscope with shot-noise limited sensitivity and an imaging speed up to 1 frame/s.

We investigate picosecond cooperative emission in an ensemble of rubidium atoms coherently excited by 100-fs laser pulses that are three-photon resonant to the 6P-5S transition. The emitted 420-nm light is recorded by a streak camera with 2-ps resolution. The resultant pulse shape shows ringing typical for superradiance in extended sample. The pulse shape is measured as a function of input power and is compared to the solutions of Maxwell-Bloch equations. The observed superradiance, which is in the low-excitation regime, is found to be closely related to optical precursor behavior.

This article demonstrated a new approach for fabrication and sharpening of metal tips of scanning probe microscopes. Experimentally, a metal tip was heated and melted by a focused laser light. The tip was then sharpened by a strong electric field and consolidated as the laser was turned off. With a low-vacuum and a high-voltage source, a 25-µm indium-coated platinum wire was sharpened to a tip with diameters below 50 nm. The minimal tip radius by this method is estimated to be below 1 nm. With this technique, tip sharpening for SPM would be possible. SCANNING 34: 76–79, 2012. © 2011 Wiley Periodicals, Inc.

Recent interest in Sommerfeld-Brillouin optical precursors has brought attention to the possibility of optical precursor observation in bulk matter. We investigate the possible formation of optical precursors in an organic dye solution with a sharp absorption band and anomalous dispersion at a wavelength of approximately 800 nm. We explore this regime experimentally with sub-10-fs pulses with a central wavelength of approximately 800 nm from a Ti : sapphire oscillator. The pulses are passed through a thin layer of the dye solution and characterized by interferometric autocorrelation. The obtained autocorrelation traces are compared with simulations, and we observe important dispersion effects on the shape of the propagated pulses, including precursorlike behavior in their time evolution.

This paper presents the design scheme of nuclear-1E-class isolation amplifiers for safety shutdown system of nuclear power plants.The scheme adopts the switching power supply technology and the analog power supply techniques to design the power module.And then,the design methods for improving the stability and reliability of the isolation amplifiers is detailed from the point view of anti-interference,the device derating and anti-seismic.Performance validation shows that the product has passed all tests of nuclear-1E-class certification program and satisfies the nuclear 1E requirement,and its reliability and various technical indicators have reached or exceeded the similar foreign products.

This article demonstrated a new approach for fabrication and sharpening of metal tips of scanning probe microscopes. Experimentally, a metal tip was heated and melted by a focused laser light. The tip was then sharpened by a strong electric field and consolidated as the laser was turned off. With a low-vacuum and a high-voltage source, a 25-µm indium-coated platinum wire was sharpened to a tip with diameters below 50 nm. The minimal tip radius by this method is estimated to be below 1 nm. With this technique, in situ tip sharpening for SPM would be possible.