Label-Free Nanometer-Resolution Imaging of Biological Architectures through Surface Enhanced Raman Scattering (original) (raw)
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Nature Photonics, 2011
Label-free microscopy that has chemical contrast and high acquisition speeds up to video rates has recently been made possible using stimulated Raman scattering (SRS) microscopy. SRS imaging offers high sensitivity, but the spectral specificity of the original narrowband implementation is limited, making it difficult to distinguish chemical species with overlapping Raman bands. Here, we present a highly specific imaging method that allows mapping of a particular chemical species in the presence of interfering species, based on tailored multiplex excitation of its vibrational spectrum. This is implemented by spectral modulation of a broadband pump beam at a high frequency (>1 MHz), allowing detection of the SRS signal of the narrowband Stokes beam with high sensitivity. Using the scheme, we demonstrate quantification of cholesterol in the presence of lipids, and real-time three-dimensional spectral imaging of protein, stearic acid and oleic acid in live Caenorhabditis elegans.
Super-resolution stimulated Raman scattering (SRS) microscopy
Unlike traditionally-mapped Raman imaging, stimulated Raman scattering (SRS) imaging achieved the capability of imaging metabolic dynamics and a greatly improved signal-noise-ratio. However, its spatial resolution is still limited by the numerical aperture or scattering cross-section. To achieve super-resolved SRS imaging, we developed a new deconvolution algorithm-Adam optimization-based Pointillism Deconvolution (A-PoD)-for SRS imaging, and demonstrated a spatial resolution of 52 nm on polystyrene beads. By applying A-PoD to spatially correlated multi-photon uorescence (MPF) imaging and deuterium oxide (D 2 O)-probed SRS (DO-SRS) imaging data from diverse samples, we compared nanoscopic distributions of proteins and lipids in cells and subcellular organelles. We successfully differentiated newly synthesized lipids in lipid droplets using A-PoD coupled with DO-SRS. The A-PoDenhanced DO-SRS imaging method was also applied to reveal the metabolic change in brain samples from Drosophila on different diets. This new approach allows us to quantitatively measure the nanoscopic co-localization of biomolecules and metabolic dynamics in organelles. We expect that the A-PoD algorithm will have a wide range of applications, from nano-scale measurements of biomolecules to processing astronomical images.
Plasmon-enhanced stimulated Raman scattering microscopy with single-molecule detection sensitivity
Nature Communications
Stimulated Raman scattering (SRS) microscopy allows for high-speed label-free chemical imaging of biomedical systems. The imaging sensitivity of SRS microscopy is limited to ~10 mM for endogenous biomolecules. Electronic pre-resonant SRS allows detection of sub-micromolar chromophores. However, label-free SRS detection of single biomolecules having extremely small Raman cross-sections (~10−30 cm2 sr−1) remains unreachable. Here, we demonstrate plasmon-enhanced stimulated Raman scattering (PESRS) microscopy with single-molecule detection sensitivity. Incorporating pico-Joule laser excitation, background subtraction, and a denoising algorithm, we obtain robust single-pixel SRS spectra exhibiting single-molecule events, verified by using two isotopologues of adenine and further confirmed by digital blinking and bleaching in the temporal domain. To demonstrate the capability of PESRS for biological applications, we utilize PESRS to map adenine released from bacteria due to starvation st...
Super-resolved Raman microscopy using random structured light illumination: Concept and feasibility
The Journal of Chemical Physics, 2021
In this article, we report the use of randomly structured light illumination for chemical imaging of molecular distribution based on Raman microscopy with improved image resolution. Random structured basis images generated from temporal and spectral characteristics of the measured Raman signatures were superposed to perform structured illumination microscopy (SIM) with the blind-SIM algorithm. For experimental validation, Raman signatures corresponding to Rhodamine 6G (R6G) in the waveband of 730-760 nm and Raman shift in the range of 1096-1634 cm −1 were extracted and reconstructed to build images of R6G. The results confirm improved image resolution using the concept and hints at super-resolution by almost twice better than the diffraction-limit.
Raman microscopy is a vibrational imaging technology that can detect molecular chemical bond vibrational signals. Since this signal is originated from almost every vibrational mode of molecules with different vibrational energy levels, it provides spatiotemporal distribution of various molecules in living organisms without the need for any labeling. The limitations of low signal strength in Raman microscopy have been effectively addressed by incorporating a stimulated emission process, leading to the development of stimulated Raman scattering (SRS) microscopy. Furthermore, the issue of low spatial resolution has been resolved through the application of computational techniques, specifically image deconvolution. In this article, we present a comprehensive guide to super-resolution SRS microscopy using an Adam-based pointillism deconvolution (A-PoD) algorithm, complemented by a user-friendly graphical user interface (GUI). We delve into the crucial parameters and conditions necessary for achieving super-resolved images through SRS imaging. Additionally, we provide a step-by-step walkthrough of the preprocessing steps and the use of GUI-supported A-PoD. This complete package offers a user-friendly platform for super-resolution SRS microscopy, enhancing the versatility and applicability of this advanced microscopy technique to reveal nanoscopic multimolecular nature.
Super-resolution vibrational imaging using expansion stimulated Raman scattering microscopy
2021
Stimulated Raman scattering (SRS) microscopy is an emerging technology that provides high chemical specificity for endogenous biomolecules and can circumvent common constraints of fluorescence microscopy including limited capabilities to probe small biomolecules and difficulty resolving many colors simultaneously due to spectral overlap. However, the resolution of SRS microscopy remains governed by the diffraction limit. To overcome this, we describe a new technique called Molecule Anchorable Gel-enabled Nanoscale Imaging of Fluorescence and stImulatEd Raman Scattering microscopy (MAGNIFIERS), that integrates SRS microscopy with expansion microscopy (ExM). ExM is a powerful strategy providing significant improvement in imaging resolution by physical magnification of hydrogel-embedded preserved biological specimens. MAGNIFIERS offers chemical-specific nanoscale imaging with sub-50 nm resolution and has scalable multiplexity when combined with multiplex Raman probes and fluorescent la...
Chinese Optics Letters, 2020
The recent development of stimulated Raman scattering (SRS) microscopy allows for highly sensitive biological imaging with molecular vibrational contrast, opening up a variety of applications including label-free imaging, metabolic imaging, and super-multiplex imaging. This paper introduces the principle of SRS microscopy and the methods of multicolor SRS imaging and describes an overview of biomedical applications.
Super-resolution vibrational microscopy by stimulated Raman excited fluorescence
Light: Science & Applications, 2021
Inspired by the revolutionary impact of super-resolution fluorescence microscopy, super-resolution Raman imaging has been long pursued because of its much higher chemical specificity than the fluorescence counterpart. However, vibrational contrasts are intrinsically less sensitive compared with fluorescence, resulting in only mild resolution enhancement beyond the diffraction limit even with strong laser excitation power. As such, it is still a great challenge to achieve biocompatible super-resolution vibrational imaging in the optical far-field. In 2019 Stimulated Raman Excited Fluorescence (SREF) was discovered as an ultrasensitive vibrational spectroscopy that combines the high chemical specificity of Raman scattering and the superb sensitivity of fluorescence detection. Herein we developed a novel super-resolution vibrational imaging method by harnessing SREF as the contrast mechanism. We first identified the undesired role of anti-Stokes fluorescence background in preventing di...
Volumetric chemical imaging by clearing-enhanced stimulated Raman scattering microscopy
PNAS, 2019
Three-dimensional visualization of tissue structures using optical microscopy facilitates the understanding of biological functions. However, optical microscopy is limited in tissue penetration due to severe light scattering. Recently, a series of tissue-clearing techniques have emerged to allow significant depth-extension for fluorescence imaging. Inspired by these advances, we develop a volumetric chemical imaging technique that couples Raman-tailored tissue-clearing with stimulated Raman scattering (SRS) microscopy. Compared with the standard SRS, the clearing-enhanced SRS achieves greater than 10-times depth increase. Based on the extracted spatial distribution of proteins and lipids, our method reveals intricate 3D organizations of tumor spheroids, mouse brain tissues, and tumor xenografts. We further develop volumetric phasor analysis of multispectral SRS images for chemically specific clustering and segmentation in 3D. Moreover, going beyond the conventional label-free paradigm, we demonstrate metabolic volu-metric chemical imaging, which allows us to simultaneously map out metabolic activities of protein and lipid synthesis in glioblas-toma. Together, these results support volumetric chemical imaging as a valuable tool for elucidating comprehensive 3D structures, compositions, and functions in diverse biological contexts, complementing the prevailing volumetric fluorescence microscopy. volumetric imaging | stimulated Raman scattering | tissue clearing | metabolic imaging | cancer metabolism T hree-dimensional structures of biological tissues are closely associated with their functions in both health and disease. Representative examples range from neuroscience and cancer biology, to developmental biology: 3D distributions of neurites determine neuronal wiring of the brain (1-6); 3D interactions between tumor cells and their microenvironment influence tumor growth, invasion, and metastasis (7, 8); and 3D organ morphogenesis reveals key mechanisms of embryonic development (9, 10). Therefore, the ability to visualize structures and functions of tissues in 3D is crucial for enhancing fundamental understanding across biomedical disciplines. Toward this goal, light microscopy presents an appealing tool to noninvasively probe biological processes with subcellular resolution. In particular, fluorescence microscopy is the method of choice for bio-imaging, offering high sensitivity, molecular specificity, and biocompatibility (11-13). However, its imaging depth is limited to superficial layers of tissues due to inevitable light scattering originating from heterogeneous refractive indices (RIs) within tissues. Typically, fluorescence imaging depth in tissues is limited to ∼500 μm (1 mm in rare cases), achieved by two-photon excited fluorescence microscopy (14). A number of advanced techniques have been developed to extend this depth limit, including adaptive optics, longer excitation wavelengths, and higher-order nonlinear excitation (15-19). Unfortunately, these techniques usually require complicated instrumentations or procedures, and the achievable depth extensions are often within a factor of two or three. Recently, a series of tissue-clearing-based fluorescence micros-copies have emerged and demonstrated remarkable results toward volumetric visualization of whole organs (20, 21). Compared with instrumentation-based approaches, these sample-centered methods adopt a fundamentally different strategy by reducing light scattering with active homogenization of tissue RIs (20, 21). Enabled by tissue clearing, volumetric fluorescence imaging provides a window to peer deep into a variety of tissues (e.g., brain, lung, bones) of different organisms in both physiology and pathology (e.g., Alz-heimer's disease, cancer) (7, 8, 22-25). Complementary to fluorescence microscopy in many aspects, stimulated Raman scattering (SRS) microscopy allows for imaging of chemical bonds in biological samples with subcellular resolution. By harnessing quantum amplification via stimulated emission, SRS mi-croscopy offers bond-selective vibrational specificity with high sensitivity (down to micromolars), fast acquisition (up to video-rate), and general biocompatibility, making it a powerful technique for bio-medicine (26-29). Compared with X-ray tomography (30), magnetic resonance imaging (31), and positron emission tomography (32), SRS offers higher spatial resolution and unique bond-selective specificity. However, the typical imaging depth of SRS has been limited to ∼100 μm inside highly scattering tissues, such as the brain, because of the scattering loss of laser power and overwhelming background (29). In less scattering tissues, the imaging depth is still limited to 300-500 μm. Therefore, extending SRS microscopy to deep volumetric imaging (millimeter range) would provide holistic chemical information in a host of environments relevant to biomedicine. Inspired by the success of tissue clearing, we devised an SRS-based volumetric chemical imaging method to generate chemical-specific 3D maps deep into tissues. We note that many of tissue-clearing Significance Cells form structures and perform functions through intricate 3D tissue organizations. However, due to tissue scattering, coherent Raman microscopy-a powerful method complementary to fluorescence imaging-suffers from limited imaging depth in tissues. Here, we develop a volumetric chemical imaging method with greater than 10-fold depth increase. We formulate a Raman-tailored tissue-clearing recipe and combine it with advanced Raman microscopies. Equipped with the toolbox of volumetric chemical imaging and analyses, we elucidate complex 3D structures, chemical compositions, and metabolic dynamics in diverse tissues including lipid synthesis throughout tumor spheroids, 3D networks of axons, vascula-tures, and cell bodies in brain regions, as well as heterogeneous tumor structures and tumor metabolism.