Author response for "Bioorthogonal chemical imaging of solid lipid nanoparticles with minimal labeling by stimulated Raman scattering microscopy"
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Bioorthogonal Chemistry
Substantial enhancement in Raman back scattering is obtained when the solution investigated is placed in the core of a hollow core photonic crystal fiber. The origin of this enhancement for liquid core waveguides is discussed. The enhancement is then used to study the Raman modes of several types of nanoparticles the Raman spectroscopy of colloidal nanoparticles in dilute solution. By employing this technique, four different stages of the synthesis ZnO nanoparticles were studied with record low pump power levels. The concentration of ZnO in the system was <1% by weight of the total mass. Also the different synthesis stages could be differentiated, and the differences between these stages could be identified through the Raman modes obtained. The concentration of nanoparticles in solution could be obtained with sensitivity for concentrations in the millimolar range. Raman modes of other nanoparticles such as CdTe have also been examined.
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Raman spectroscopy can identify cancerous from healthy tissue, with a chemical analysis from the measurement of vibrational bond frequencies. However, to detect small tumors a form of Raman imaging is required. Such imaging—by acquiring a Raman spectrum at each imaging pixel—can detect tumors but is rather slow. Multiphoton versions of Raman—anti-Stokes Raman scattering (CARS) microscopy and stimulated Raman scattering (SRS) microscopy—offer similar accuracies in identifying cancerous tissue and tumor margins but with a far higher speed, which is beneficial for diagnosis of small tumors in tissue. SRS microscopy can also be used to image extrinsic molecules in living cells, such as anti-cancer drugs at typical concentrations.
Raman microscope
Raman microspectroscopy
Chemical Imaging
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Since the first introduction of Raman microscope in 1973, optical and laser technology has made a tremendous step forward. However, despite of the obvious advantages of being a very informative and nondestructive method of studying biological samples, spontaneous Raman scattering suffers from a series of limitations such as a fluorescent background and a low signal level. Nonlinear Raman spectroscopy and, in particular, spectroscopy of coherent anti-Stokes Raman scattering (CARS) can resolve most of the problems associated with conventional Raman spectroscopy. In this report, the most critical issues of the CARS microspectroscopy setup design are reviewed and several exciting potential applications of the broadband CARS microspectroscopy, where the CARS microscopy has an advantage with respect to Raman microscopy, are outlined.
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Raman microspectroscopy
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Raman spectroscopy has been an attractive tool for scientists because of its capability of label-free analysis of materials. Raman spectra reflect molecular or lattice vibration in a sample and provide rich information about the sample compositions and their environments. However, due to the small cross-section of Raman scattering, it has been difficult to utilize Raman scattering for imaging biological samples under physiological conditions. We have developed Raman imaging techniques that utilize the advantages of spontaneous Raman scattering. The simple optical process of Raman scattering allows us to perform spatial multiplexing of signal detection, which has been enabled by the recent development of high-power lasers and 2D sensors with large pixel numbers. We utilized this advantage to realize high-speed Raman imaging by illuminating a sample by a line-shaped focus. The parallel detection of hundreds of Raman spectra from the illumination line drastically shortened the image acquisition time. We applied the line-illumination Raman imaging technique to observe molecular dynamics in cellular events, such as apoptosis, cell division, and cell differentiation. The use of laser light at 532 nm for excitation allows us to monitor mitochondrial dysfunction in the subcellular scale via the resonant Raman effect on heme proteins. We also proposed and demonstrated the use of alkyne as a tiny tag for imaging small molecules, which enabled the observation of molecules too small to be labeled by fluorescent probes.
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Raman spectroscopy has been widely utilized to analyze various type of material. However the low scattering cross-section of Raman scattering prevents the application in microscopic imaging. In our research, we have developed a Raman microscope system with improved imaging speed, where using line-shaped illumination is used to detect Raman spectra simultaneously at multiple points in a sample. This improvement allowed us to utilize Raman imaging in biological research such as label-free hemeprotein imaging, diagnosis of cell differentiations.
Biomolecule
Raman microscope
Chemical Imaging
Molecular biophysics
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We study the dependence of surface-enhanced Raman scattering from metallic arrays on geometry by angle-resolved Raman spectroscopy and confocal microscopy. It is found that both Raman profile and intensity are strongly dependent on hole size.
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Raman spectroscopy has been widely used for material analysis because of its capability of detecting molecular vibrations. However, microscopic imaging of live cells has not been well utilized for biological studies since a long exposure time is required for measuring a Raman spectrum due to the low efficiency of Raman scattering.
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Raman spectroscopy has enabled researchers to map the specific chemical makeup of surfaces, solutions, and even cells. However, the inherent insensitivity of the technique makes it difficult to use and statistically complicated. When Raman active molecules are near gold or silver nanoparticles, the Raman intensity is significantly amplified. This phenomenon is referred to as surface‐enhanced Raman spectroscopy (SERS). The extent of SERS enhancement is due to a variety of factors such as nanoparticle size, shape, material, and configuration. The choice of Raman reporters and protective coatings will also influence SERS enhancement. This review provides an introduction to how these factors influence signal enhancement and how to optimize them during synthesis of SERS nanoparticles.
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Raman microscope
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Raman microscopes are currently used in various fields of research because they allow for label-free sample investigation. Moreover, the inherently low scattering cross section of Raman spectroscopy, as well as its diffraction-limited lateral resolution, has been overcome by new Raman microscopy techniques. Nonlinear methods such as coherent anti-Stokes Raman spectroscopy and stimulated Raman spectroscopy reduce measurement times and improve z resolution, allowing for three-dimensional spectroscopic imaging of biological samples. Moreover, tip-enhanced Raman spectroscopy, a near-field optical technique that combines scanning-probe microscopy with the enhancement offered by surface-enhanced Raman scattering, enables Raman spectroscopic imaging far below the optical diffraction limit. We cover the theoretical and technical aspects of Raman microscopy and related new imaging techniques and review some very recent applications in graphene research and cell biology.
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