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Broadband coherent anti-Stokes Raman scattering (BCARS) is capable of producing high-quality Raman spectra spanning broad bandwidths, 400-4000 cm-1, with millisecond acquisition times. Raw BCARS spectra, however, are a coherent combination of vibrationally resonant (Raman) and non-resonant (electronic) components that may challenge or degrade chemical analyses. Recently, we demonstrated a deep convolutional autoencoder network, trained on pairs of simulated BCARS-Raman datasets, which could retrieve the Raman signal with high quality under ideal conditions. In this work, we present a new computational system that incorporates experimental measurements of the laser system spectral and temporal properties, combined with simulated susceptibilities. Thus, the neural network learns the mapping between the susceptibility and the measured response for a specific BCARS system. The network is tested on simulated and measured experimental results taken with our BCARS system.
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Removing distortions in coherent anti-Stokes Raman scattering (CARS) spectra due to interference with the nonresonant background (NRB) is vital for quantitative analysis. Popular computational approaches, the Kramers-Kronig relation and the maximum entropy method, have demonstrated success but may generate significant errors due to peaks that extend in any part beyond the recording window. In this work, we present a learned matrix approach to the discrete Hilbert transform that is easy to implement, fast, and dramatically improves accuracy of Raman retrieval using the Kramers-Kronig approach.
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It has been proposed that entangled two-photon absorption (E2PA) can be observed with up to 1010 lower photon flux than its classical counterpart, therefore enabling ultralow-power two-photon fluorescence microscopy. However, there is a significant controversy regarding the magnitude of this quantum enhancement in excitation efficiency. We investigated the fluorescence signals from Rhodamine 6G and LDS798 excited with a CW laser or an entangled photon pair source at â¼1060 nm. We observed a signal that originates from hot-band absorption (HBA), which is one-photon absorption from thermally populated vibrational levels of the ground electronic state. This mechanism, which has not been previously discussed in the context of E2PA, produces a signal with a linear power dependence, as would be expected for E2PA. For the typical conditions under which E2PA measurements are performed, contributions from the HBA process could lead to a several orders of magnitude overestimate of the quantum advantage.
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We aim to develop a quantitative viability method that distinguishes individual quiescent from dead cells and is measured in time (ns) as a referenceable, comparable quantity. We demonstrate that fluorescence lifetime imaging of an anionic, fluorescent membrane voltage probe fulfills these requirements for Streptococcus mutans. A random forest machine-learning model assesses whether individual S. mutans can be correctly classified into their original populations: stationary phase (quiescent), heat killed and inactivated via chemical fixation. We compare the results to intensity using three models: lifetime variables (τ1 , τ2 and p1 ), phasor variables (G, S) or all five variables, with the five variable models having the most accurate classification. This initial work affirms the potential for using fluorescence lifetime of a membrane voltage probe as a viability marker for quiescent bacteria, and future efforts on other bacterial species and fluorophores will help refine this approach.
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Corantes Fluorescentes , Imagem Óptica , Bactérias , Aprendizado de Máquina , Microscopia de FluorescênciaRESUMO
We demonstrate the preservation of the time-energy entanglement of near-IR photons through thick biological media (≤1.55 mm) and tissue (≤ 235 µm) at room temperature. Using a Franson-type interferometer, we demonstrate interferometric contrast of over 0.9 in skim milk, 2% milk, and chicken tissue. This work supports the many proposed opportunities for nonclassical light in biological imaging and analyses from sub-shot noise measurements to entanglement-enhanced fluorescence imaging, clearly indicating that the entanglement characteristics of photons can be maintained even after propagation through thick, turbid biological samples.
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Surface-textured polymer nanocomposite (PNC) films are utilized in many device applications, and therefore understanding the relaxation behavior of such films is important. By extending an in situ wrinkle relaxation method, we observed that the thermal stability of wrinkled PNC films, both above and below the glass transition temperature (Tg), is proportional to a film's nanoparticle (polymer grafted and bare) concentration, with a slope that changes sign at a compensation temperature (Tcomp) that is determined to be in the vicinity of the film's Tg. This provides unambiguous confirmation of entropy-enthalpy compensation (EEC) as a general feature of PNC films, implying that the stability of PNC films changes from being enhanced to becoming diminished by simply passing through this characteristic temperature, a phenomenon having evident practical ramifications. We suggest EEC will also arise in films where residual stresses are associated with the film fabrication process, which is relevant to nanotech device applications.
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We present a new collection of processing techniques, collectively "factorized Kramers-Kronig and error correction" (fKK-EC), for (a) Raman signal extraction, (b) denoising, and (c) phase- and scale-error correction in coherent anti-Stokes Raman scattering (CARS) hyperspectral imaging and spectroscopy. These new methods are orders-of-magnitude faster than conventional methods and are capable of real-time performance, owing to the unique core concept: performing all processing on a small basis vector set and using matrix/vector multiplication afterwards for direct and fast transformation of the entire dataset. Experimentally, we demonstrate that a 703026 spectra image of chicken cartilage can be processed in 70 s (≈ 0.1 ms / spectrum), which is ≈ 70 times faster than with the conventional workflow (≈7.0 ms / spectrum). Additionally, we discuss how this method may be used for machine learning (ML) by re-using the transformed basis vector sets with new data. Using this ML paradigm, the same tissue image was processed (post-training) in ≈ 33 s, which is a speed-up of ≈ 150 times when compared with the conventional workflow.
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Caenorhabditis elegans serves as a model for understanding adiposity and its connections to aging. Current methodologies do not distinguish between fats serving the energy needs of the parent, akin to mammalian adiposity, from those that are distributed to the progeny, making it difficult to accurately interpret the physiological implications of fat content changes induced by external perturbations. Using spectroscopic coherent Raman imaging, we determine the protein content, chemical profiles and dynamics of lipid particles in live animals. We find fat particles in the adult intestine to be diverse, with most destined for the developing progeny. In contrast, the skin-like epidermis contains fats that are the least heterogeneous, the least dynamic and have high triglyceride content. These attributes are most consistent with stored somatic energy reservoirs. These results challenge the prevailing practice of assessing C. elegans adiposity by measurements that are dominated by the intestinal fat content.
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Caenorhabditis elegans/fisiologia , Lipídeos/química , Análise Espectral Raman/métodos , Animais , Metabolismo dos Lipídeos/fisiologiaRESUMO
Histopathology plays a central role in diagnosis of many diseases including solid cancers. Efforts are underway to transform this subjective art to an objective and quantitative science. Coherent Raman imaging (CRI), a label-free imaging modality with sub-cellular spatial resolution and molecule-specific contrast possesses characteristics which could support the qualitative-to-quantitative transition of histopathology. In this work we briefly survey major themes related to modernization of histopathology, review applications of CRI to histopathology and, finally, discuss potential roles for CRI in the transformation of histopathology that is already underway.
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Coherent anti-Stokes Raman scattering (CARS) microspectroscopy has demonstrated significant potential for biological and materials imaging. To date, however, the primary mechanism of disseminating CARS spectroscopic information is through pseudocolor imagery, which explicitly neglects a vast majority of the hyperspectral data. Furthermore, current paradigms in CARS spectral processing do not lend themselves to quantitative sample-to-sample comparability. The primary limitation stems from the need to accurately measure the so-called nonresonant background (NRB) that is used to extract the chemically-sensitive Raman information from the raw spectra. Measurement of the NRB on a pixel-by-pixel basis is a nontrivial task; thus, reference NRB from glass or water are typically utilized, resulting in error between the actual and estimated amplitude and phase. In this manuscript, we present a new methodology for extracting the Raman spectral features that significantly suppresses these errors through phase detrending and scaling. Classic methods of error-correction, such as baseline detrending, are demonstrated to be inaccurate and to simply mask the underlying errors. The theoretical justification is presented by re-developing the theory of phase retrieval via the Kramers-Kronig relation, and we demonstrate that these results are also applicable to maximum entropy method-based phase retrieval. This new error-correction approach is experimentally applied to glycerol spectra and tissue images, demonstrating marked consistency between spectra obtained using different NRB estimates, and between spectra obtained on different instruments. Additionally, in order to facilitate implementation of these approaches, we have made many of the tools described herein available free for download.
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Coherent Raman imaging requires high-peak power laser pulses to maximize the nonlinear multiphoton signal generation, but accompanying photo-induced sample damage often poses a challenge to microscopic imaging studies. We demonstrate that beam scanning by a 3.5-kHz resonant mirror in a broadband coherent anti-Stokes Raman scattering (BCARS) imaging system can reduce photo-induced damage without compromising signal intensity. Additionally, beam scanning enables slit acquisition, in which spectra from a thin line of sample illumination are acquired in parallel during a single charge-coupled device exposure. Reflective mirrors are employed in the beam-scanning assembly to minimize chromatic aberration and temporal dispersion. The combined approach of beam scanning and slit acquisition is compared with the sample-scanning mode in terms of spatial resolution, photo-induced damage, and imaging speed at the maximum laser power below the sample-damage threshold. We show that the beam-scanning BCARS imaging method can reduce photodamage probability in biological cells and tissues, enabling faster imaging speed by using a higher excitation laser power than could be achieved without beam scanning.
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Imagem Óptica/métodos , Análise Espectral Raman , Células 3T3 , Animais , Camundongos , Poliestirenos , Fatores de TempoRESUMO
Many scaffold systems have evolved for tissue engineering and in vitro tissue models to provide a 3D (three-dimensional) microenvironment that enables cells to behave more physiologically. We hypothesized that cells would adopt morphologies with more 3D character during culture in scaffolds as compared to planar substrates. Cell shape and function are tightly linked and effects of scaffold niche properties on cell shape and dimensionality are important for directing cell function. Herein, primary human bone marrow stromal cells (hBMSCs) were cultured in 6 different scaffolds and on a planar control substrate. hBMSCs were imaged using 3D confocal microscopy, and 3D image analyses were used to assess hBMSC shape and dimensionality. A characteristic gyration tensor ellipsoid was calculated for hBMSCs in the different scaffolds which enabled hBMSC dimensionality to be classified based on shape. A "Dimensionality Matrix" was developed that showed that hBMSC shape and dimensionality were influenced by scaffold properties, and that scaffolds could drive hBMSCs into 1D, 2D or 3D shapes. In addition, the hBMSC Z-Depth was measured to determine if hBMSCs became less flat during culture in scaffolds. Z-Depth results showed that all 6 scaffolds caused an increase in cell Z-Depth compared to the 2D planar substrate. These results demonstrate that hBMSCs take on morphologies with greater 3D character in scaffolds than on a planar substrate and that scaffold properties can be adjusted to modify cell dimensionality. In addition, biomaterialists can use this measurement approach to assess and compare scaffold design modifications as they strive to create optimal cell niches that provide a 3D microenvironment.
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Forma Celular , Células-Tronco/citologia , Alicerces Teciduais/química , Humanos , Imageamento Tridimensional , Células-Tronco Mesenquimais/citologiaRESUMO
An imaging platform based on broadband coherent anti-Stokes Raman scattering (BCARS) has been developed which provides an advantageous combination of speed, sensitivity and spectral breadth. The system utilizes a configuration of laser sources that probes the entire biologically-relevant Raman window (500 cm-1 to 3500 cm-1) with high resolution (< 10 cm-1). It strongly and efficiently stimulates Raman transitions within the typically weak "fingerprint" region using intrapulse 3-colour excitation, and utilizes the nonresonant background (NRB) to heterodyne amplify weak Raman signals. We demonstrate high-speed chemical imaging in two- and three-dimensional views of healthy murine liver and pancreas tissues and interfaces between xenograft brain tumours and the surrounding healthy brain matter.
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We compare a coherent Raman imaging modality, broadband coherent anti-Stokes Raman scattering (BCARS) microscopy, with spontaneous Raman microscopy for quantitative and qualitative assessment of multicomponent pharmaceuticals. Indomethacin was used as a model active pharmaceutical ingredient (API) and was analyzed in a tabulated solid dosage form, embedded within commonly used excipients. In comparison with wide-field spontaneous Raman chemical imaging, BCARS acquired images 10× faster, at higher spatiochemical resolution and with spectra of much higher SNR, eliminating the need for multivariate methods to identify chemical components. The significant increase in spatiochemical resolution allowed identification of an unanticipated API phase that was missed by the spontaneous wide-field method and bulk Raman spectroscopy. We confirmed the presence of the unanticipated API phase using confocal spontaneous Raman, which provided spatiochemical resolution similar to BCARS but at 100× slower acquisition times.
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Formas de Dosagem , Microscopia/métodos , Preparações Farmacêuticas/análise , Análise Espectral Raman/métodos , Preparações Farmacêuticas/química , Difração de Raios X/métodosRESUMO
We present the first demonstration, to our knowledge, of a label-free flow cytometer for the analysis of biological specimens using multiplex coherent anti-Stokes Raman scattering (MCARS) and elastic scatter measurements. The MCARS system probes the Raman vibrational energy levels and the elastic scatter provides morphological information. We demonstrate these capabilities by probing a culture of Saccharomyces cerevisiae at 100 spectra/s and constructing a background-free Raman reconstruction using a Kramers-Kronig relation. A theoretical analysis shows that this system could operate at speeds of 10 kHz with appropriate hardware; thus facilitating integration into commercial flow cytometers or use as a high-speed, stand-alone device.
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Citometria de Fluxo/métodos , Análise Espectral Raman , Citometria de Fluxo/instrumentação , Processamento de Imagem Assistida por Computador , Saccharomyces cerevisiae/citologiaRESUMO
Flow cytometry is an ever-advancing high-throughput multivariate analysis tool that natively provides size and morphological information. To obtain molecular information, however, typically requires the addition of fluorophores, which are limited by spectral overlap, nonspecific binding, available conjugation chemistries, and cellular toxicity. A complementary or alternative, label-free approach to molecular information is through multiplex coherent anti-Stokes Raman scattering (MCARS), which is a coherent, nonlinear optical method that provides a wealth of molecular information by probing the Raman energies within a molecule. In this work, we demonstrate the unique capability of our MCARS flow cytometer to distinguish flowing particles and discuss system performance capabilities and possibilities.