Chemical Fingerprint Imaging with Broadband Coherent Anti-Stokes Raman Scattering Microscopy.

Plants are inherently complex systems dynamically interacting at different size scale levels. Spontaneous Raman microscopy links the molecular with the cellular structural level; however, as Raman scattering is a low-probability phenomenon, pixel dwell times for biological applications are not compatible with high-resolution imaging. Due to absorption and autofluorescence interferences, Raman methods are often restricted to pigment-poor regions in plant samples.

Solvent Exclusion Effect on Infrared Absorption Spectroscopy.

Absorption spectroscopy of a solution containing an analyte solute typically uses the transmission through a solvent as a reference based on the assumption that the solvent does not absorb light. However, when a solvent absorbs light, the resulting absorbance becomes lower and often negative because of the reduced number of solvent molecules due to the space taken by solute molecules. The solvent exclusion (SE) effect is problematic for accurate quantitation and analysis across various absorption spectroscopies, especially near the broad, strong water absorption peaks.

A Liquid-Core Fiber Platform for Classical and Entangled Two-Photon Absorption Measurements.

We introduce a toluene-filled fiber platform for two-photon absorption measurements. By confining both the light and molecular sample inside the 5 μm hollow core of the fiber, we increase the distance over which the nonlinear light-matter interaction occurs. With only a 7.

Removing non-resonant background from broadband CARS using a physics-informed neural network.

Broadband coherent anti-Stokes Raman scattering (BCARS) is capable of producing high-quality Raman spectra spanning broad bandwidths, 400-4000 cm, 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.

Raman signal extraction from CARS spectra using a learned-matrix representation of the discrete Hilbert transform.

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.

Hot-Band Absorption Can Mimic Entangled Two-Photon Absorption.

It has been proposed that entangled two-photon absorption (E2PA) can be observed with up to 10 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 ∼1060 nm.

Toward absolute viability measurements for bacteria.

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.

Witnessing the survival of time-energy entanglement through biological tissue and scattering media.

We demonstrate the preservation of the time-energy entanglement of near-IR photons through thick biological media (≤1.55 mm) and tissue (≤ 235 m) at room temperature. Using a Franson-type interferometer, we demonstrate interferometric contrast of over 0.

Observation of General Entropy-Enthalpy Compensation Effect in the Relaxation of Wrinkled Polymer Nanocomposite Films.

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 (), is proportional to a film's nanoparticle (polymer grafted and bare) concentration, with a slope that changes sign at a compensation temperature () that is determined to be in the vicinity of the film's . 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.

Real-time and high-throughput Raman signal extraction and processing in CARS hyperspectral imaging.

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.

Spectroscopic coherent Raman imaging of Caenorhabditis elegans reveals lipid particle diversity.

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.

Histological coherent Raman imaging: a prognostic review.

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.

Quantitative, Comparable Coherent Anti-Stokes Raman Scattering (CARS) Spectroscopy: Correcting Errors in Phase Retrieval.

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.

Beam scanning for rapid coherent Raman hyperspectral imaging.

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.

High-Speed Coherent Raman Fingerprint Imaging of Biological Tissues.

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 to 3500 cm) with high resolution (< 10 cm). 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.

Measuring stem cell dimensionality in tissue scaffolds.

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.

Multicomponent chemical imaging of pharmaceutical solid dosage forms with broadband CARS microscopy.

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.

Label-free flow cytometry using multiplex coherent anti-Stokes Raman scattering (MCARS) for the analysis of biological specimens.

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.

Multiplex coherent anti-Stokes Raman scattering (MCARS) for chemically sensitive, label-free flow cytometry.

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.