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Optimized signal-to-noise ratio with shot noise limited detection in Stimulated Raman Scattering microscopy
摘要： We describe our set-up for Stimulated Raman Scattering (SRS) microscopy with shot noise limited detection for a broad window of biologically relevant laser powers. This set-up is used to demonstrate that the highest signal-to-noise ratio (SNR) in SRS with shot noise limited detection is achieved with a time-averaged laser power ratio of 1:2 of the unmodulated and modulated beam. In SRS, two different coloured laser beams are incident on a sample. If the energy difference between them matches a molecular vibration of a molecule, energy can be transferred from one beam to the other. By applying amplitude modulation to one of the beams, the modulation transfer to the other beam can be measured. The efficiency of this process is a direct measure for the number of molecules of interest in the focal volume. Combined with laser scanning microscopy, this technique allows for fast and sensitive imaging with sub-micrometre resolution. Recent technological advances have resulted in an improvement of the sensitivity of SRS applications, but few show shot noise limited detection. The dominant noise source in this SRS microscope is the shot noise of the unmodulated, detected beam. Under the assumption that photodamage is linear with the total laser power, the optimal SNR shifts away from equal beam powers, where the most signal is generated, to a 1:2 power ratio. Under these conditions the SNR is maximized and the total laser power that could induce photodamage is minimized. Compared to using a 1:1 laser power ratio, we show improved image quality and a signal-to-noise ratio improvement of 8 % in polystyrene beads and C. Elegans worms. Including a non-linear damage mechanism in the analysis, we find that the optimal power ratio converges to a 1:1 ratio with increasing order of the non-linear damage mechanism.
关键词： shot noise limited detection,signal-to-noise ratio,Nonlinear Raman microscopy
更新于2025-11-21 11:08:12
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Vibrational spectroscopic imaging and live cell video microscopy for studying differentiation of primary human alveolar epithelial cells
摘要： Alveolar type II(ATII) cells in the peripheral human lung spontaneously differentiate towards alveolar type I(ATI) cells, thus enabling air-blood barrier formation. Here, linear Raman and coherent anti-Stokes Raman scattering (CARS) microscopy are applied to study cell differentiation of freshly isolated ATII cells. The Raman spectra can successfully be correlated to gradual morphological and molecular changes during cell differentiation. Alveolar surfactant rich vesicles in ATII cells are identified based on phospholipid vibrations, while ATI-like cells are characterized by the absence of vesicular structures. Complementary, CARS microscopy allows for 3D visualization of lipid vesicles within ATII cells and their secretion, while hyperspectral CARS enables the distinction between cellular proteins and lipids according to their vibrational signatures. This study paves the path for further label-free investigations of lung cells and the role of the pulmonary surfactant, thus also providing a basis for rational development of future lung therapeutics.
关键词： confocal laser scanning microscopy,confocal Raman microscopy,pneumocyte type II differentiation,CARS microscopy,cell imaging
更新于2025-09-23 15:23:52
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Cross striation in human permanent and deciduous enamel measured with confocal Raman microscopy
摘要： Dental enamel is the most mineralized tissue in the human body and has a very complex organization. The aim of the present study was to evaluate chemical composition and orientation of crystals in human enamel cross striation using confocal Raman microscope. Slices of teeth were prepared and scanned using a confocal Raman microscope. Cross striation spacing was calculated according to the variation of PO4 3? peak intensity. The measurements showed a characteristic length between two cross striations of 2.70 (SD 0.43) μm for permanent teeth and 1.75 (SD 0.37) for deciduous teeth. Ratios between OH and PO4 3? peaks, for 0 and 90 degrees angulation, are, respectively, 9.18 (SD 0.80) and 44.74 (SD 3.12) for decidual teeth, and 11.72 (SD 1.46) and 22.12 (SD 3.15) for adult teeth. This confirmed that the hydroxyapatite crystal is well oriented along the enamel prism, as reported in the literature, but with a significant increase in deciduous teeth. K‐mean cluster binary images were calculated to compare the ratio of the centroid peaks of the cross striation zone and inter cross striation material. The regions corresponding to inner prism, included cross striations, are more mineralized than the outer part of enamel prism. In the prism, the Pearson correlation test was performed based on the intensity of hydroxyapatite characteristic peaks (ν1 PO4 3?, ν1 CO3 2?, and OH). The variations were well correlated: These results indicated that cross striations corresponded to the variation of hydroxyapatite concentration and not to a variation of crystals orientation.
关键词： Raman microscopy,cross striation,enamel prism,K‐mean cluster analysis,decidual teeth
更新于2025-09-23 15:23:52
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Bone quality assessment of osteogenic cell cultures by Raman microscopy
摘要： The use of autologous stem/progenitor cells represents a promising approach to the repair of craniofacial bone defects. The calvarium is recognized as a viable source of stem/progenitor cells that can be transplanted in vitro to form bone. However, it is unclear if bone formed in cell culture is similar in quality to that found in native bone. In this study, the quality of bone mineral formed in osteogenic cell cultures were compared against calvarial bone from postnatal mice. Given the spectroscopic resemblance that exists between cell and collagen spectra, the feasibility of extracting information on cell activity and bone matrix quality were also examined. Stem/progenitor cells isolated from fetal mouse calvaria were cultured onto fused‐quartz slides under osteogenic differentiation conditions for 28 days. At specific time intervals, slides were removed and analyzed by Raman microscopy and mineral staining techniques. We show that bone formed in culture at Day 28 resembled calvarial bone from 1‐day‐old postnatal mice with comparable mineralization, mineral crystallinity, and collagen crosslinks ratios. In contrast, bone formed at Day 28 contained a lower degree of ordered collagen fibrils compared with 1‐day‐old postnatal bone. Taken together, bone formed in osteogenic cell culture exhibited progressive matrix maturation and mineralization but could not fully replicate the high degree of collagen fibril order found in native bone.
关键词： Raman microscopy,osteogenic differentiation,tissue engineering,stem cells,bone quality
更新于2025-09-23 15:21:01
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Nanoplastic Analysis by On-line Coupling of Raman Microscopy and Field-Flow Fractionation Enabled by Optical Tweezers
摘要： Nanoplastic pollution is of emerging environmental concern, but current analytical approaches are facing limitations in this size range. However, the coupling of nanoparticle separation with chemical characterization bears potential to close this gap. Here, we realize the hyphenation of particle separation / characterization (field-flow fractionation (FFF), UV and multi angle light scattering (MALS)) with subsequent chemical identification by on-line Raman microspectroscopy (RM). The problem of low Raman scattering was overcome by trapping particles with 2D optical tweezers. This setup enabled RM to identify particles of different materials (polymers and inorganic) in the size range from 200 nm to 5 μm, with concentrations in the order of 1 mg/L (109 particles L-1). The hyphenation was realized for asymmetric flow FFF (AF4) and centrifugal FFF (CF3), which separate particles based on different properties. This technique shows potential for application in nanoplastic analysis, as well as many other fields of nanomaterials.
关键词： Nanoplastic,Particle Separation,Optical Tweezers,Raman Microscopy,Field-Flow Fractionation
更新于2025-09-23 15:19:57
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An Expandable Mechanopharmaceutical Device (3): a Versatile Raman Spectral Cytometry Approach to Study the Drug Cargo Capacity of Individual Macrophages
摘要： Purpose To improve cytometric phenotyping abilities and better understand cell populations with high interindividual variability, a novel Raman-based microanalysis was developed to characterize macrophages on the basis of chemical composition, specifically to measure and characterize intracellular drug distribution and phase separation in relation to endogenous cellular biomolecules. Methods The microanalysis was developed for the commercially-available WiTec alpha300R confocal Raman microscope. Alveolar macrophages were isolated and incubated in the presence of pharmaceutical compounds nilotinib, chloroquine, or etravirine. A Raman data processing algorithm was specifically developed to acquire the Raman signals emitted from single-cells and calculate the signal contributions from each of the major molecular components present in cell samples. Results Our methodology enabled analysis of the most abundant biochemicals present in typical eukaryotic cells and clearly identified Bfoamy^ lipid-laden macrophages throughout cell populations, indicating feasibility for cellular lipid content analysis in the context of different diseases. Single-cell imaging revealed differences in intracellular distribution behavior for each drug; nilotinib underwent phase separation and self-aggregation while chloroquine and etravirine accumulated primarily via lipid partitioning. Conclusions This methodology establishes a versatile cytometric analysis of drug cargo loading in macrophages requiring small numbers of cells with foreseeable applications in toxicology, disease pathology, and drug discovery.
关键词： lipid-laden foamy macrophages,intracellular drug bioaccumulation,single-cell chemical imaging,confocal Raman microscopy,pulmonary alveolar macrophages
更新于2025-09-23 15:19:57
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Nanoscale characterization of photonic metasurface made of lens-like SiGe Mie-resonators formed on Si (100) substrate
摘要： Submicrometer-sized high-index Mie resonators attract significant interest in photonic applications due to their capabilities to manipulate light. 2-dimensional metamaterials or metasurfaces consisting of arrays of such resonators on a device surface can be used in the flat optics, sensors, and other applications. Here, we report on the comprehensive nanoscale characterization and optical properties of nearly regular SiGe Mie resonator arrays on a Si surface fabricated using a simple and low-cost method. We achieved control on the surface morphology by depositing Ge on the Si(100) surface at elevated temperatures 890–960 °C and obtained arrays of submicrometer/micrometer low-Ge-content SiGe lenslike islands via dewetting when the Ge content was >4%. At the lower Ge content, we observed the formation of a continuous SiGe film via wetting. We used Raman microscopy not only for the Ge content and stress control but also for studying photonic properties of the islands and their coupling with the Si substrate. In contrast to the elastic light scattering, we clearly distinguished visible light Raman signals from the islands themselves and from the substrate areas under the islands enhanced compared to the signal from the open substrate. Calculation of the light electric field distribution in the islands and the substrate demonstrate how the islands trap the light and forward it into the high-index substrate. This explains the island-induced reflection suppression and Si substrate Raman enhancement, which we observe experimentally. Such an SiGe-island array is a promising metasurface for the improvement of Si photosensors and solar-energy device performance.
关键词： SiGe,metasurfaces,photonic properties,Mie resonators,Raman microscopy
更新于2025-09-16 10:30:52
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Three‐stage kinetics of laser‐induced LiFePO <sub/>4</sub> decomposition
摘要： This paper reports about new insight into a problem of a laser–matter interaction during Raman probing of lithium iron phosphate (LiFePO4), discusses phase transformation kinetics in powder samples, and provides methodological recommendations. LiFePO4 is the second most popular positive electrode material in the global lithium battery industry, but the use of Raman spectroscopy for its structural characterization is hampered by the laser‐induced degradation. The statistical/big‐data approach to Raman spectra measurements is utilized to revise the problem and suggest a simple model of laser‐induced phase transformations in powder LiFePO4. The results are proposed to be used for better understanding of the physics and chemistry of other processes taking place in electrochemical cells, namely, lithiation/delithiation and thermal stability/safety studies. Simple recommendations for nondestructive LiFePO4 characterization by Raman spectroscopy are formulated.
关键词： lithium iron phosphate,nondamaging characterization,statistical Raman spectroscopy,lithium batteries,statistical Raman microscopy
更新于2025-09-12 10:27:22
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Functionalized graphene transistor for ultrasensitive detection of carbon quantum dots
摘要： Ubiquitous carbon nanomaterials have great potential for emerging technologies, but they also pose a threat to human health at the end of their lifecycle, especially when they are introduced into waste or ground waters. Graphene field-effect transistors (GFET) and real time in situ confocal Raman microscopy (CRM) were employed to detect a submonolayer of carbon quantum dots (CQDs) in water. An effective GFET channel was formed on exfoliated and chemical vapor deposited (CVD) graphene. The adsorption of CQDs was monitored by measuring conductance changes in GFETs. The graphene channel was functionalized with (3-aminopropyl) triethoxysilane (APTES), which allowed for easy observation of a shift in the charge neutrality point (CNP) when the graphene channel was exposed to CQDs. The affinity of the CQD’s carboxyl terminal groups to the aminofunctionalized channel enabled a highly sensitive CQD detection based on changes in the GFET conductivity. The adsorption of the CQDs induced a positive shift of the CNP with a limit of detection at concentrations of 239 ppm and 62 ppm for the exfoliated and CVD graphene, respectively. A supporting study of graphene functionalization and CQD adsorption was performed by real time in situ CRM, ellipsometry, and ex situ X-ray photoelectron spectroscopy.
关键词： graphene field-effect transistors,carbon quantum dots,confocal Raman microscopy,chemical vapor deposited graphene,aminofunctionalized channel
更新于2025-09-12 10:27:22
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Reference Module in Materials Science and Materials Engineering || Tip-Enhanced Raman Microscopy: Instrumentation, Techniques and Applications in Practice
摘要： The idea of optical microscopy is to use a set of lenses to magnify the object to see. Scientists changed the idea of this conventional optical microscopy since the scanning probe microscope (SPM) came out in 1980s. Inouye and Kawata proposed to use a sharp metal probe to create an optical image [1]. They scanned the metal tip on a sample surface while recording the optical near-field signals scattered by the tip [1,2]. The spatial resolution is not determined by the numerical aperture (N.A.) of the objective lens but by the tip radius that is typically a few tens of nanometers. The use of a metal tip provides not only high spatial resolution but also an enhancement of optical signals as a result of excitation of surface plasmons at the sharp metal tip. With the tip-enhancement effect, it becomes possible to detect even a very weak optical signal scattered from an extremely tiny volume. One such process is Raman scattering. The application of tip-enhancement to Raman spectroscopy was reported in the year of 2000 by three independent groups led by Kawata, Zenobi, and Anderson, respectively [3–6]. Using TERS, distribution of molecules [7–11] and electronic [12] and chemical properties within nanoscale materials [13–18] have been successfully visualized through Raman signatures with a nanoscale spatial resolution. There are a number of review articles for TERS microscopy and spectroscopy available in literatures [14,19–22]. During the past 20 years, the development of TERS has been mostly driven by research labs having research-grade apparatus operated by skillful researchers. The situation has been changing in the recent years, some companies have started to sell commercial products of TERS microscopes in the market. However, in order to realize TERS microscope as an analytical tool for the routine use of research and industry, challenges in TERS probes, reproducibility issues remain such as stable implementation of atomic force microscopes (AFMs), and peripheral operational techniques.
关键词： Raman spectroscopy,nanoscale imaging,plasmonic enhancement,Tip-Enhanced Raman Microscopy,TERS
更新于2025-09-10 09:29:36