Benchtop IR Imaging of Live Cells: Monitoring the Total Mass of Biomolecules in Single Cells.

Absolute quantity imaging of biomolecules on a single cell level is critical for measurement assurance in biosciences and bioindustries. While infrared (IR) transmission microscopy is a powerful label-free imaging modality capable of chemical quantification, its applicability to hydrated biological samples remains challenging due to the strong IR absorption by water. Traditional IR imaging of hydrated cells relies on powerful light sources, such as synchrotrons, to mitigate the light absorption by water. However, we overcome this challenge by applying a solvent absorption compensation (SAC) technique to a home-built benchtop IR microscope based on an external-cavity quantum cascade laser. SAC-IR microscopy adjusts the incident light using a pair of polarizers to precompensate the IR absorption by water while retaining the full dynamic range. Integrating the IR absorbance over a cell yields the total mass of biomolecules per cell. We monitor the total mass of the biomolecules of live fibroblast cells over 12 h, demonstrating promise for advancing our understanding of the biomolecular processes occurring in live cells on the single-cell level.

Quantum Cascade Laser Infrared Spectroscopy for Glycan Analysis of Glycoprotein Solutions.

Glycans are oligosaccharides attached to proteins or lipids and affect their functions, such as drug efficacy, structural contribution, metabolism, immunogenicity, and molecular recognition. Conventional glycosylation analysis has relied on destructive, slow, system-sensitive methods, including enzymatic reactions, chromatography, fluorescence labeling, and mass spectrometry. Herein, we propose quantum cascade laser (QCL) infrared (IR) spectroscopy as a rapid, nondestructive method to quantify glycans and their monosaccharide composition. Previously, we demonstrated high-sensitivity IR spectroscopy of protein solution using solvent absorption compensation (SAC) and double-beam modulation (DBM) techniques. However, the SAC-DBM approach suffered a limited frequency scanning range (<400 cm-1) due to the light dispersion by acousto-optic modulators (AOMs). Here, we implemented a mirror-based double-pass AOM in the SAC-DBM scheme and successfully extended the frequency range to (970 to 1840 cm-1), which encompasses the vibrational fingerprint of biomolecules. The extended frequency range allowed the simultaneous observation of monosaccharide ring bands (1000 to 1200 cm-1) and protein amide bands (1500 to 1700 cm-1). We compared the IR spectra of six glycoproteins and two nonglycosylated proteins with the results from intact mass spectrometry. The IR absorbance ratios of the ring band to the amide band of glycoproteins in solutions showed a linear correlation with the ratios of glycan to protein backbone masses. Furthermore, a multivariate analysis produced monosaccharide compositions consistent with the reported database for the glycoproteins, and the monosaccharide compositions were used to improve the predictability of the glycan-protein mass ratio from the IR-absorbance ratio. This nondestructive, high-sensitivity QCL-IR spectroscopy could be used as a standard method to monitor batch-to-batch comparability during drug manufacturing and quantify the glycosylation and monosaccharide composition of new glycoproteins and other glycosylated biosystems.

Single-detector double-beam modulation for high-sensitivity infrared spectroscopy.

Balanced detection based on double beams is widely used to reduce common-mode noises, such as laser intensity fluctuation and irregular wavelength scanning, in absorption spectroscopy. However, employing an additional detector can increase the total system noise due to added non-negligible thermal noise of the detector, particularly with mid-infrared (IR) detectors. Herein, we demonstrate a new optical method based on double-beam modulation (DBM) that uses a single-element detector but keeps the advantage of double-beam balanced detection. The sample and reference path beams were modulated out-of-phase with each other at a high frequency, and their average and difference signals were measured by two lock-in amplifiers and converted into absorbance. DBM was coupled with our previously reported solvent absorption compensation (SAC) method to eliminate the IR absorption contribution of water in aqueous solutions. The DBM-SAC method enabled us to acquire IR absorption spectra of bovine serum albumin solutions down to 0.02 mg/mL. We investigated the noise characteristics of DBM measurements when the wavelength was either fixed or scanned. The results demonstrate that DBM can lower the limit of detection by ten times compared to the non-modulation method.

Effects of Salts and Surface Charge on the Biophysical Stability of a Low pI Monoclonal Antibody.

The impact of five representative Hofmeister salts (NaCl, KCl, MgCl2, Na2SO4, and NaSCN) on the thermal stability and aggregation kinetics of a slightly acidic monoclonal antibody (mAb) were investigated under different pH conditions. The thermal stability of the mAb was assessed by measuring the lowest unfolding transition temperature, Tm, with differential scanning fluorimetry. MgCl2 and NaSCN significantly decreased Tm at all three charged states of the mAb, but to the greatest extent when the mAb surface charge was net positive. Non-native aggregation kinetics was monitored by measuring Rayleigh light scattering. When the mAb surface charge was net positive or net neutral, the nucleation rate increased non-monotonically with MgCl2 and NaSCN but decreased monotonically with NaCl, KCl, and Na2SO4. By contrast, when the mAb surface was negatively charged, there were only minor changes in the nucleation rate with all salts tested. Furthermore, there was less structural perturbation and slower aggregation rates when the mAb was net negatively charged than when it was net neutrally or positively charged. The observed salt effects on thermal unfolding are consistent with ion-specific mechanisms dominated by short-range amide backbone binding. On the other hand, the salt effects on nucleation rates appear to be influenced by both amide backbone binding and long-range electrostatic binding of ions to charged amino acid side chains.

3D Orientation Imaging of Polymer Chains with Polarization-Controlled Coherent Raman Microscopy.

Despite the ubiquity of three-dimensional (3D) anisotropic materials, their 3D molecular alignment cannot be measured using conventional two-dimensional (2D) polarization imaging. Here, we present images of the 3D angles of molecular orientations with submicrometer spatial resolution acquired through polarization-controlled coherent anti-Stokes Raman scattering microscopy. The hyperspectral Raman data of a polyethylene (PE) film were converted into images, showing the polymer chains' 3D angles and order parameters. The 3D orientation images of PE chains in ring-banded spherulites show that the azimuthal angles of the chains are perpendicular to the crystal growth direction, while the out-of-plane angles display limited-range oscillations synchronous with ring banding. The prevailing crystal growth model of fully twisting lamellae is inconsistent with the observed restricted oscillations of the out-of-plane direction, which are unobservable through conventional 2D projected imaging. This high-resolution, label-free, quantitative imaging of 3D molecular orientation can become a standard measurement tool for the microscopic structures of complex synthetic and biological materials.

Imaging 3D molecular orientation by orthogonal-pair polarization IR microscopy.

Anisotropic molecular alignment occurs ubiquitously and often heterogeneously in three dimensions (3D). However, conventional imaging approaches based on polarization can map only molecular orientation projected onto the 2D polarization plane. Here, an algorithm converts conventional polarization-controlled infrared (IR) hyperspectral data into images of the 3D angles of molecular orientations. The polarization-analysis algorithm processes a pair of orthogonal IR transition-dipole modes concurrently; in contrast, conventional approaches consider individual IR modes separately. The orthogonal-pair polarization IR (OPPIR) method, introduced theoretically but never demonstrated experimentally, was used to map the 3D orientation angles and the order parameter of the local orientational distribution of polymer chains in a poly(ε-caprolactone) film. The OPPIR results show that polymer chains in the semicrystalline film are aligned azimuthally perpendicular to the radial direction of a spherulite and axially tilted from the film normal direction. This newly available information on the local alignments in continuously distributed molecules helps to understand the molecular-level structure of highly anisotropic and spatially heterogeneous materials.

Compensation of Strong Water Absorption in Infrared Spectroscopy Reveals the Secondary Structure of Proteins in Dilute Solutions.

Infrared (IR) absorption spectroscopy is a powerful tool that can quantify complex biomolecules and their structural conformations. However, conventional approaches to protein analysis in aqueous solutions have been significantly challenged because the strong IR absorption of water overwhelms the limited dynamic range of the detection system and thus allows only a very short path length and a limited concentration sensitivity. Here, we demonstrate a solvent absorption compensation (SAC) approach that can improve the concentration sensitivity and extend the available path length by distinguishing the analyte signal over the full dynamic range at each wavelength. Absorption spectra without any postprocessing show good linearity from 100 to 0.1 mg/mL protein concentration, allowing a >100 times enhanced signal-to-noise ratio in the amide I band compared to the non-SAC results. We apply this method to in situ investigate the isothermal kinetics of insulin fibrillation at two clinical concentrations at 74 °C for 18 h. Simultaneous monitoring of both reactants (native forms) and products (fibrils) allows quantitative discussion of the detailed fibrillation mechanisms, which are not accessible with other single modality measurements. This simple optical technique can be applied to other absorption spectroscopies of analytes in strongly absorbing solvents, allowing for enhanced sensitivity without changing the detection system.

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 (≈ 0.1 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.

Theory of birefringence correction for polarization-controlled CARS.

Polarization-controlled coherent Raman spectroscopy is used as a high-throughput method to characterize the anisotropic nature of a molecular system, such as the molecular orientation distribution. However, optical birefringence originating from the molecular anisotropy can cause the observed Raman spectrum to be significantly distorted, making it extremely challenging to obtain quantitative information from polarization Raman measurements. Here, the birefringence effect on the signal intensity and the spectral shape of a polarization-controlled coherent anti-Stokes Raman scattering (CARS) is theoretically described using a uniaxially symmetrical model system. Due to the complexity, the effect of phase delay in the incident lights is not considered but only that of the generated CARS signal is considered. A new analytical method is presented to eliminate the birefringence contribution from polarization-controlled CARS data by analyzing polarization intensity profiles and retrieving the resonant Raman susceptibility spectra. This method is tested with two sets of polarization-controlled CARS data simulated with various combinations of symmetries of multiple underlying Raman modes. The analysis result clearly demonstrates that the effect of birefringence can be corrected for polarization-controlled CARS data and the symmetry tensor elements of all underlying Raman modes can be quantitatively characterized.

Position- and Polarization-Specific Waveguiding of Multi-Emissions in Single ZnO Nanorods.

We examine multiphoton-produced optical signals waveguided through single ZnO nanorods (NRs) using a newly developed, scanning offset-emission hyperspectral microscopy (SOHM) technique. Specifically, we concurrently analyze waveguiding behaviors of sum-frequency generation (SFG), deep-trap emissions (DTE), and coherent anti-Stokes Raman scattering (CARS) occurring in individual ZnO NRs. SOHM acquires spectrally-indexed and spatially-resolved intensity maps/spectra of waveguided light intensity while excitation/emission collection positions and light polarization are scanned. Hence, the powerful measurement capabilities of SOHM enable quantitative analyses of the different ZnO NR waveguiding behaviors specific to the multiphoton-generated emissions as a function of measurement position, light-matter interaction geometry, and the optical origin of the guided signal. We subsequently reveal the distinct waveguiding behaviors of single ZnO NRs pertaining to the SFG-, DTE-, and CARS-originated signals and discuss particularly attractive ZnO NR properties in CARS waveguiding.

Concurrent polarization IR analysis to determine the 3D angles and the order parameter for molecular orientation imaging.

A non-tomographic analysis method is proposed to determine the 3D angles and the order parameter of molecular orientation using polarization-dependent infrared (IR) spectroscopy. Conventional polarization-based imaging approaches provide only 2D-projected orientational information of single vibrational modes. The newly proposed method concurrently analyses polarization angle-dependent absorptance of two non-parallel transition dipole moments. The relative phase angle and the maximum-to-minimum ratios observed from the two polarization profiles are used to calculate the 3D angles of the mean molecular orientation and the order parameter of the orientational distribution. Usage of those relative observables as intermediate input parameters makes the analysis results robust against variations in concentration, thickness, absorption peak, and absorption cross-section, which can occur in typical imaging conditions. This analysis is based on a single-step, non-iterative calculation that does not require any analytical model function of an orientational distribution function. This concurrent polarization analysis method is demonstrated using two simulation data examples, followed by associated error propagation analysis and discussion on the effect of absorption strength. Application of this robust spectral analysis method to polarization IR microscopy will provide a full molecular orientation image without tilting that tomographies require.

Stoichiometric analysis of competing intermolecular hydrogen bonds using infrared spectroscopy.

We quantitatively analyze multiple hydrogen bonds in mixtures of two monomers: urethane dimethacrylate (UDMA) and triethylene glycol-divinylbenzyl ether (TEG-DVBE). The carbonyl stretching band in infrared (IR) absorption spectra is deconvoluted into free and hydrogen-bonded carbonyl groups. The amounts of the sub-components are determined for 21 mixture compositions and initially analyzed using a simple stoichiometric model (based on one dominant hydrogen acceptor group per monomer species) for the equilibrium state of hydrogen bond formation. However, our in-depth stoichiometric analysis suggests that at least two UDMA acceptor groups (carbonyl and alkoxy oxygens) and one TEG-DVBE acceptor group (ether oxygen) contribute to intermolecular hydrogen bonding interactions. This finding is further supported by a quantitative analysis of the hydrogen bonding effect on the N-H stretching band. Moreover, the equilibrium constants of these hydrogen bond formations confirm that the interassociation between UDMA and TEG-DVBE is non-negligible in comparison to the UDMA selfassociations. Such quantitative information on intermolecular interactions provides insight into the effect of hydrogen bonding on the copolymerization kinetics of these monomer mixtures.

Effects of Sparassis crispa in Medical Therapeutics: A Systematic Review and Meta-Analysis of Randomized Controlled Trials.

In this study, we investigated the therapeutic potential and medical applications of Sparassis crispa (S. crispa) by conducting a systematic review of the existing literature and performing a meta-analysis. The original efficacy treatment of the mushroom extract is considered primarily and searched in electronic databases. A total of 623 articles were assessed, 33 randomized controlled experiments were included after the manual screening, and some papers, review articles, or editorials that did not contain data were excluded. A comparative standard means difference (SMD) and a funnel plot between control and S. crispa groups were used as parameters to demonstrate the beneficial effects of S. crispa for diabetes and cancer treatment, as well as anti-inflammatory, anti-fungal and antioxidant activities. The meta-analysis was carried out using Review Manager 5.1 software. Although for therapeutic diabetes there was heterogeneity in the subgroup analysis (I² = 91.9%), the overall results showed statistically significant SMDs in major symptoms that decreased serum insulin levels (SMD = 1.92, 95% CI (1.10, 2.75), I² = 0%), wound rates (SMD = 3.55 (2.56, 4.54), I² = 40%) and contributions to an increase in nutrient intake content (SMD = 0.32 (-0.15, 0.78), I² = 0%). Simultaneously, the study confirmed the utility of S. crispa treatment in terms of not only anti-cancer activity (reduction of tumor activity and survival of cancer cells I² = 42 and 34%, respectively) but also anti-inflammatory, anti-fungal and antioxidant activities (I² = 50, 44, and 10%, respectively). Our findings suggest that S. crispa extracts are useful for prevention and treatment of human diseases and might be the best candidates for future medicines.

Analytical and Numerical Characterization of Autocorrelation and Perturbation-Correlation Moving-Window Methods.

Moving-window (MW) approaches to two-dimensional correlation spectroscopy (2D-COS) make it possible to characterize spectral changes occurring in a narrow range of perturbation variable (e.g., time, temperature, and concentration). Despite the wide range of application, the physical meanings of MW correlation intensities have been only qualitatively associated with the direction and curvature of spectral intensity change with regard to a perturbation variable. Here are full and simplified analytical expressions of autocorrelation moving-window (ACMW) and synchronous and asynchronous perturbation-correlation moving-window ( s-PCMW and as-PCMW) intensities. When the window is set sufficiently narrower than the bandwidth of spectral change, the square root of ACMW intensity and s-PCMW intensity becomes proportional to the first order derivative, and as-PCMW intensity becomes proportional to the negative of the second order derivative. This paper demonstrates that both ACMW and PCMW profiles can be significantly altered by non-uniform perturbation spacing. It is also found that intensity noise can cause ACMW to display a false offset drift. This analytical and numerical characterization of the two MW correlation intensities elucidates their physical meanings and ascertains the analysis conditions for reliable interpretation.

Least Squares Moving-Window Spectral Analysis.

Least squares regression is proposed as a moving-windows method for analysis of a series of spectra acquired as a function of external perturbation. The least squares moving-window (LSMW) method can be considered an extended form of the Savitzky-Golay differentiation for nonuniform perturbation spacing. LSMW is characterized in terms of moving-window size, perturbation spacing type, and intensity noise. Simulation results from LSMW are compared with results from other numerical differentiation methods, such as single-interval differentiation, autocorrelation moving-window, and perturbation correlation moving-window methods. It is demonstrated that this simple LSMW method can be useful for quantitative analysis of nonuniformly spaced spectral data with high frequency noise.

Prunus mume leaf extract lowers blood glucose level in diabetic mice.

Context Diabetes is a common metabolic disease with long-term complications. Prunus mume Sieb. et Zucc. (Rosaceae) fruits have shown to ameliorate glucose intolerance. However, the antidiabetic effects of P. mume leaves have not been investigated. Objective This study evaluated the effects of P. mume leaf 70% ethanol extract (PMLE) on alleviating diabetes in vivo and in vitro. Materials and methods PMLE was fractionated into n-hexane, dichloromethane (CH2Cl2), ethyl acetate (EtOAc), n-butanol (BuOH) and water. Polyphenol and flavonoid contents in PMLE fractions were determined using Folin-Ciocalteu reagent and the aluminium chloride colorimetric method, respectively. We evaluated α-glucosidase inhibition using a microplate reader at 400 nm. Adipocyte differentiation by lipid accumulation was measured using Nile Red staining. Male imprinting control region (ICR) mice were injected with streptozotocin (STZ, 100 mg/kg, i.p.). High-fat diets were provided for three weeks prior to PMLE treatments to induce type 2 diabetes. PMLE (0, 5, 25 or 50 mg/kg) was administrated for four weeks with high-fat diets. Results The EtOAc fraction of PMLE inhibited α-glucosidase activity (IC50 = 68.2 µg/mL) and contained 883.5 ± 14.9 mg/g of polyphenols and 820.1 ± 7.7 mg/g of flavonoids. The 50 mg/kg PMLE supplement reduced 40% of blood glucose level compared to obese/diabetes mice. Obese/diabetic mice treated with 50 mg/kg PMLE showed a lower level of triacylglycerol (320.7 ± 20.73 mg/dL) compared to obese/diabetes mice (494.9 ± 14.80 mg/dL). Conclusion The data demonstrate that P. mume leaves exert antidiabetic effects that may be attributable to high concentrations of polyphenols and flavonoids.

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. 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.

Phase-Specific Raman Analysis of n-Alkane Melting by Moving-Window Two-Dimensional Correlation Spectroscopy.

We use moving-window two-dimensional correlation spectroscopy (MW-2DCOS) for phase-specific Raman analysis of the n-alkane (C21H44) during melting from the crystalline solid phase to the intermediate rotator phase and to the amorphous molten phase. In MW-2DCOS, individual peak-to-peak correlation analysis within a small subset of spectra provides both temperature-resolved and spectrally disentangled Raman assignments conducive to understanding phase-specific molecular interactions and chain configurations. We demonstrate that autocorrelation MW-2DCOS can determine the phase transition temperatures with a higher resolving power than commonly-used analysis methods including individual peak intensity analysis or principal component analysis. Besides the enhanced temperature resolving power, we demonstrate that asynchronous 2DCOS near the orthorhombic-to-rotator transition temperature can spectrally resolve the two overlapping peaks embedded in the Raman CH2 twisting band in the orthorhombic phase, which had been only predicted but not observed due to thermal broadening near the melting temperature.

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. 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.

Determination of 3D molecular orientation by concurrent polarization analysis of multiple Raman modes in broadband CARS spectroscopy.

A theoretical description is presented about a new analysis method to determine three-dimensional (3D) molecular orientation by concurrently analyzing multiple Raman polarization profiles. Conventional approaches to polarization Raman spectroscopy are based on single peaks, and their 2D-projected polarization profiles are limited in providing 3D orientational information. Our new method analyzes multiple Raman profiles acquired by a single polarization scanning measurement of broadband coherent anti-Stokes Raman scattering (BCARS). Because the analysis uses only dimensionless quantities, such as intensity ratios and phase difference between multiple profiles, the results are not affected by sample concentration and the system response function. We describe how to determine the 3D molecular orientation with the dimensionless observables by using two simplified model cases. In addition, we discuss the effect of orientational broadening on the polarization profiles in the two model cases. We find that in the presence of broadening we can still determine the mean 3D orientation angles and, furthermore, the degree of orientational broadening.