RESUMO
Nonlinear optical microscopy allows for rapid high-resolution microscopy with image contrast generated from the intrinsic properties of the sample. Established modalities, such as multiphoton excited fluorescence and second/third-harmonic generation, can be combined with other nonlinear techniques, such as coherent Raman spectroscopy, which typically allow chemical imaging of a single resonant vibrational mode of a sample. Here, we utilize a single ultrafast laser source to obtain broadband coherent Raman spectra on a microscope, together with other nonlinear microscopy approaches on the same instrument. We demonstrate that the coherent Raman modality allows broadband measurement (>1000 cm-1), with high spectral resolution (<5 cm-1), with a rapid spectral acquisition rate (3-12 kHz). This enables Raman hyperspectral imaging of kilo-pixel images at >11 frames per second.
RESUMO
The spectral resolution of broadband Fourier-transform coherent anti-Stokes Raman spectroscopy is limited by the maximum optical path length difference that can be scanned within a short time in an interferometer. However, alternatives to the Fourier transform exist which can bypass this limitation with certain assumptions. We apply one such approach to broadband coherent Raman spectroscopy using interferometers with a short delay line (low Fourier spectral resolution) and large delay line (high Fourier spectral resolution). With this method, we demonstrate that broadband coherent Raman spectroscopy of closely spaced vibrational bands is possible using a short delay line interferometer with comparable spectral resolution to the longer delay line instrument. We discuss how this approach may be particularly useful for more complex Raman spectra, such as those measured from biological samples.
RESUMO
Cellular plasma membrane deformability and stability is important in a range of biological processes. Changes in local curvature of the membrane affect the lateral movement of lipids, affecting the biophysical properties of the membrane. An integrated holographic optical tweezers and Raman microscope was used to investigate the effect of curvature gradients induced by optically stretching individual giant unilamellar vesicles (GUVs) on lipid packing and lateral segregation of cholesterol in the bilayer. The spatially resolved Raman analysis enabled detection of induced phase separation and changes in lipid ordering in individual GUVs. Using deuterated cholesterol, the changes in lipid ordering and phase separation were linked to lateral sorting of cholesterol in the stretched GUVs. Stretching the GUVs in the range of elongation factors 1-1.3 led to an overall decrease in cholesterol concentration at the edges compared to the center of stretched GUVs. The Raman spectroscopy results were consistent with a model of the bilayer accounting for cholesterol sorting in both bilayer leaflets, with a compositional asymmetry of 0.63 ± 0.04 in favor of the outer leaflet. The results demonstrate the potential of the integrated holographic optical tweezers-Raman technique to induce deformations to individual lipid vesicles and to simultaneously provide quantitative and spatially resolved molecular information. Future studies can extend to include more realistic models of cell membranes and potentially live cells.
Assuntos
Bicamadas Lipídicas/química , Análise Espectral Raman , Lipossomas Unilamelares/química , Colesterol/química , Pinças ÓpticasRESUMO
We present a new approach for combining holographic optical tweezers with confocal Raman spectroscopy. Multiple laser foci, generated using a liquid-crystal spatial light modulator, are individually used for both optical trapping and excitation of spontaneous Raman spectroscopy from trapped objects. Raman scattering from each laser focus is spatially filtered using reflective apertures on a digital micro-mirror device, which can be reconfigured with flexible patterns at video rate. We discuss operation of the instrument, and performance and viability considerations for biological measurements. We then demonstrate the capability of the instrument for fast, flexible, and interactive manipulation with molecular measurement of interacting live cell systems.
Assuntos
Bactérias/citologia , Células Dendríticas/citologia , Holografia/instrumentação , Pinças Ópticas , Análise Espectral Raman/instrumentação , Linfócitos T/citologia , Desenho de Equipamento , LuzRESUMO
We investigated the feasibility of using spatially offset Raman spectroscopy (SORS) for nondestructive characterization of bone tissue engineering scaffolds. The deep regions of these scaffolds, or scaffolds implanted subcutaneously in live animals, are typically difficult to measure by confocal Raman spectroscopy techniques because of the limited depth penetration of light caused by the high level of light scattering. Layered samples consisting of bioactive glass foams (IEIC16), three-dimensional (3D)-printed biodegradable poly(lactic-co-glycolic acid) scaffolds (PLGA), and hydroxyapatite powder (HA) were used to mimic nondestructive detection of biomineralization for intact real-size 3D tissue engineering constructs. SORS spectra were measured with a new SORS instrument using a digital micromirror device (DMD) to allow software selection of the spatial offsets. The results show that HA can be reliably detected at depths of 0-2.3 mm, which corresponds to the maximum accessible spatial offset of the current instrument. The intensity ratio of Raman bands associated with the scaffolds and HA with the spatial offset depended on the depth at which HA was located. Furthermore, we show the feasibility for in vivo monitoring mineralization of scaffold implanted subcutaneously by demonstrating the ability to measure transcutaneously Raman signals of the scaffolds and HA (fresh chicken skin used as a top layer). The ability to measure spectral depth profiles at high speed (5 s acquisition time) and the ease of implementation make SORS a promising approach for noninvasive characterization of cell/tissue development in vitro, and for long-term in vivo monitoring the mineralization in 3D scaffolds subcutaneously implanted in small animals.
Assuntos
Osso e Ossos/química , Durapatita/análise , Engenharia Tecidual , Materiais Biocompatíveis/análise , Ácido Láctico/química , Tamanho da Partícula , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Análise Espectral RamanRESUMO
Spectral depth-profiling of optically turbid samples is of high interest to a broad range of applications. We present a method for measuring spatially-offset Raman spectroscopy (SORS) over a range of length scales by incorporating a digital micro-mirror device (DMD) into a sample-conjugate plane in the detection optical path. The DMD can be arbitrarily programmed to collect/reject light at spatial positions in the 2D sample-conjugate plane, allowing spatially offset Raman measurements. We demonstrate several detection geometries, including annular and simultaneous multi-offset modalities, for both macro- and micro-SORS measurements, all on the same instrument. Compared to other SORS modalities, DMD-based SORS provides more flexibility with only minimal additional experimental complexity for subsurface Raman collection.
RESUMO
Raman micro-spectroscopy (RMS) is a non-invasive technique for imaging live cells in vitro. However, obtaining quantitative molecular information from Raman spectra is difficult because the intensity of a Raman band is proportional to the number of molecules in the sampled volume, which depends on the local molecular concentration and the thickness of the cell. In order to understand these effects, we combined RMS with atomic force microscopy (AFM), a technique that can measure accurately the thickness profile of the cells. Solution-based calibration models for RNA and albumin were developed to create quantitative maps of RNA and proteins in individual fixed cells. The maps were built by applying the solution-based calibration models, based on partial least squares fitting (PLS), on raster-scan Raman maps, after accounting for the local cell height obtained from the AFM. We found that concentrations of RNA in the cytoplasm of mouse neuroprogenitor stem cells (NSCs) were as high as 25 ± 6 mg ml(-1), while proteins were distributed more uniformly and reached concentrations as high as â¼50 ± 12 mg ml(-1). The combined AFM-Raman datasets from fixed cells were also used to investigate potential improvements for normalization of Raman spectral maps. For all Raman maps of fixed cells (n = 10), we found a linear relationship between the scores corresponding to the first component (PC1) and the cell height profile obtained by AFM. We used PC1 scores to reconstruct the relative height profiles of independent cells (n = 10), and obtained correlation coefficients with AFM maps higher than 0.99. Using this normalization method, qualitative maps of RNA and protein were used to obtain concentrations for live NSCs. While this study demonstrates the potential of using AFM and RMS for measuring concentration maps for individual NSCs in vitro, further studies are required to establish the robustness of the normalization method based on principal component analysis when comparing Raman spectra of cells with large morphological differences.
Assuntos
Microscopia de Força Atômica , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Análise Espectral Raman , Animais , Análise dos Mínimos Quadrados , Camundongos , Células-Tronco Neurais/química , Análise de Componente Principal , Proteínas/análise , RNA/análiseRESUMO
We report a new approach in tip-enhanced Raman spectroscopy (TERS) in which TERS-active tips with enhancement factors of â¼10(-5)× can be rapidly (1-3 min) produced in situ by laser-induced synthesis of silver nanoparticles at the tip apex. The technique minimizes the risks of tip contamination and damage during handling and provides in situ feedback control, which allows the prediction of the tip performance. We show that TERS tips produced by this technique enable the measurement of spatially resolved TERS spectra of self-assembled peptide nanotubes with a spatial resolution of â¼20 nm.
Assuntos
Lasers , Nanopartículas Metálicas/química , Nanotecnologia/métodos , Nanotubos de Peptídeos/química , Prata/química , Análise Espectral Raman/métodos , Dipeptídeos , Microscopia de Força Atômica , Fenilalanina/análogos & derivados , Fenilalanina/químicaRESUMO
Various toxic compounds disrupt bacterial physiology. While bacteria harbor defense mechanisms to mitigate the toxicity, these mechanisms are often coupled to the physiological state of the cells and become ineffective when the physiology is severely disrupted. Here, we characterized such feedback by exposing Escherichia coli to protonophores. Protonophores dissipate the proton motive force (PMF), a fundamental force that drives physiological functions. We found that E. coli cells responded to protonophores heterogeneously, resulting in bimodal distributions of cell growth, substrate transport, and motility. Furthermore, we showed that this heterogeneous response required active efflux systems. The analysis of underlying interactions indicated the heterogeneous response results from efflux-mediated positive feedback between PMF and protonophores' action. Our studies have broad implications for bacterial adaptation to stress, including antibiotics. IMPORTANCE An electrochemical proton gradient across the cytoplasmic membrane, alternatively known as proton motive force, energizes vital cellular processes in bacteria, including ATP synthesis, nutrient uptake, and cell division. Therefore, a wide range of organisms produce the agents that collapse the proton motive force, protonophores, to gain a competitive advantage. Studies have shown that protonophores have significant effects on microbial competition, host-pathogen interaction, and antibiotic action and resistance. Furthermore, protonophores are extensively used in various laboratory studies to perturb bacterial physiology. Here, we have characterized cell growth, substrate transport, and motility of Escherichia coli cells exposed to protonophores. Our findings demonstrate heterogeneous effects of protonophores on cell physiology and the underlying mechanism.
Assuntos
Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Ionóforos de Próton/farmacologia , Força Próton-Motriz , Transporte Biológico , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genéticaRESUMO
Understanding and quantifying the temporal acquisition of host cell molecules by intracellular pathogens is fundamentally important in biology. In this study, a recently developed holographic optical trapping (HOT)-based Raman microspectroscopy (RMS) instrument is applied to detect, characterize and monitor in real time the molecular trafficking of a specific molecular species (isotope-labeled phenylalanine (L-Phe(D8)) at the single cell level. This approach enables simultaneous measurement of the chemical composition of human cerebrovascular endothelial cells and the protozoan parasite Toxoplasma gondii in isolation at the very start of the infection process. Using a model to decouple measurement contributions from host and pathogen sampling in the excitation volume, the data indicate that manipulating parasites with HOT coupled with RMS chemical readout was an effective method for measurement of L-Phe(D8) transfer from host cells to parasites in real-time, from the moment the parasite enters the host cell.
Assuntos
Interações Hospedeiro-Parasita , Toxoplasma , Células Endoteliais , Humanos , Pinças ÓpticasRESUMO
Advances in consumer display screen technologies have historically been adapted by researchers across the fields of optics as they can be used as electronically controlled spatial light modulators (SLMs) for a variety of uses. The performance characteristics of such SLM devices based on liquid crystal (LC) and digital micromirror device (DMD) technologies, in particular, has developed to the point where they are compatible with increasingly sensitive instrumental applications, for example, Raman spectroscopy. Spatial light modulators provide additional flexibility, from modulation of the laser excitation (including multiple laser foci patterns), manipulation of microscopic samples (optical trapping), or selection of sampling volume (adaptive optics or spatially offset Raman spectroscopy), to modulation in the spectral domain for high-resolution spectral filtering or multiplexed/compressive fast detection. Here, we introduce the benefits of different SLM devices as a part of Raman instrumentation and provide a variety of recent example applications which have benefited from their incorporation into a Raman system.
RESUMO
Using phantom samples, we investigated the feasibility of spatially-offset Raman spectroscopy (SORS) as a tool for monitoring non-invasively the mineralization of bone tissue engineering scaffold in-vivo. The phantom samples consisted of 3D-printed scaffolds of poly-caprolactone (PCL) and hydroxyapatite (HA) blends, with varying concentrations of HA, to mimic the mineralisation process. The scaffolds were covered by a 4 mm layer of skin to simulate the real in-vivo measurement conditions. At a concentration of HA approximately 1/3 that of bone (~0.6 g/cm3), the characteristic Raman band of HA (960 cm-1) was detectable when the PCL:HA layer was located at 4 mm depth within the scaffold (i.e. 8 mm below the skin surface). For the layers of the PCL:HA immediately under the skin (i.e. top of the scaffold), the detection limit of HA was 0.18 g/cm3, which is approximately one order of magnitude lower than that of bone. Similar results were also found for the phantoms simulating uniform and inward gradual mineralisation of the scaffold, indicating the suitability of SORS to detect early stages of mineralisation. Nevertheless, the results also show that the contribution of the materials surrounding the scaffold can be significant and methods for subtraction need to be investigated in the future. In conclusion, these results indicate that spatially-offset Raman spectroscopy is a promising technique for in-vivo longitudinal monitoring scaffold mineralization and bone re-growth.
RESUMO
While Raman hyperspectral imaging has been widely used for label-free mapping of biomolecules in cells, these measurements require the cells to be cultured on weakly Raman scattering substrates. However, many applications in biological sciences and engineering require the cells to be cultured on polymer substrates that often generate large Raman scattering signals. Here, we discuss the theoretical limits of the signal-to-noise ratio in the Raman spectra of cells in the presence of polymer signals and how optical aberrations may affect these measurements. We show that Raman spectra of cells cultured on polymer substrates can be obtained using automatic subtraction of the polymer signals and demonstrate the capabilities of these methods in two important applications: tissue engineering and in vitro toxicology screening of drugs. Apart from their scientific and technological importance, these applications are examples of the two most common measurement configurations: (1) cells cultured on an optically thick polymer substrate measured using an immersion/dipping objective; and (2) cells cultured on a transparent polymer substrate and measured using an inverted optical microscope. In these examples, we show that Raman hyperspectral data sets with sufficient quality can be successfully acquired to map the distribution of common biomolecules in cells, such as nucleic acids, proteins, and lipids, as well as detecting the early stages of apoptosis. We also discuss strategies for further improvements that could expand the application of Raman hyperspectral imaging on polymer substrates even further in biomedical sciences and engineering.
Assuntos
Técnicas de Cultura de Células/métodos , Análise de Célula Única/métodos , Análise Espectral Raman/métodos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Técnicas de Cultura de Células/instrumentação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Células MCF-7 , Polímeros/química , Polímeros/farmacologia , Razão Sinal-Ruído , Análise de Célula Única/instrumentação , Análise Espectral Raman/instrumentaçãoRESUMO
We describe a multifocal Raman micro-spectroscopy detection method based on a digital micromirror device, which allows for simultaneous "power-sharing" acquisition of Raman spectra from ad hoc sampling points. As the locations of the points can be rapidly updated in real-time via software control of a liquid-crystal spatial light modulator (LC-SLM), this technique is compatible with automated adaptive- and selective-sampling Raman spectroscopy techniques, the latter of which has previously been demonstrated for fast diagnosis of skin cancer tissue resections. We describe the performance of this instrument and show examples of multiplexed measurements on a range of test samples. Following this, we show the feasibility of reducing measurement time for power-shared multifocal Raman measurements combined with confocal auto-fluorescence imaging to provide guided diagnosis of tumours in human skin samples.