Imaging vs Nonimaging Raman Spectroscopy for High-Throughput Single-Cell Phenotyping.

Raman spectroscopy can provide nonbiased single-cell analysis based on the endogenous ensemble of biomolecules, with alterations in cellular content indicative of cell state and disease. The measurements themselves can be performed in a variety of modes: generally, full imaging takes the most time but can provide the most information. By reducing the imaging resolution and generating the most characteristic single-cell Raman spectrum in the shortest time, we optimize the utility of the Raman measurement for cell phenotyping. Here, we establish methods to compare these different measurement approaches and assess what, if any, undesired effects occur in the cell. Assuming that laser-induced damage should be apparent as a change in molecular spectra across sequential measurements, and by defining the information content as the Raman-based separability of two cell lines, we thereby establish a parameter range for optimum measurement sensitivity and single-cell throughput in single-cell Raman spectroscopic analysis. While the work here uses 532 nm irradiation, the same approach can be generalized to Raman analysis at other wavelengths.

Saturated-excitation image scanning microscopy.

Image scanning microscopy (ISM) overcomes the trade-off between spatial resolution and signal volume in confocal microscopy by rearranging the signal distribution on a two-dimensional detector array to achieve a spatial resolution close to the theoretical limit achievable by infinitesimal pinhole detection without sacrificing the detected signal intensity. In this paper, we improved the spatial resolution of ISM in three dimensions by exploiting saturated excitation (SAX) of fluorescence. We theoretically investigated the imaging properties of ISM, when the fluorescence signals are nonlinearly induced by SAX, and show combined SAX-ISM fluorescence imaging to demonstrate the improvement of the spatial resolution in three dimensions. In addition, we confirmed that the SNR of SAX-ISM imaging of fluorescent beads and biological samples, which is one of the challenges in conventional SAX microscopy, was improved.

Hyperspectral two-photon excitation microscopy using visible wavelength.

We demonstrate hyperspectral imaging by visible-wavelength two-photon excitation microscopy using line illumination and slit-confocal detection. A femtosecond pulsed laser light at 530 nm was used for the simultaneous excitation of fluorescent proteins with different emission wavelengths. The use of line illumination enabled efficient detection of hyperspectral images and achieved simultaneous detection of three fluorescence spectra in the observation of living HeLa cells with an exposure time of 1 ms per line, which is equivalent to about 2 µs per pixel in point scanning, with 160 data points per spectrum. On combining linear spectral unmixing techniques, localization of fluorescent probes in the cells was achieved. A theoretical investigation of the imaging property revealed high-depth discrimination property attained through the combination of nonlinear excitation and slit detection.

Heparin induces neutrophil elastase-dependent vital and lytic NET formation.

Heparin is used extensively as an anticoagulant in a broad range of diseases and procedures; however, its biological effects are not limited to coagulation and remain incompletely understood. Heparin usage can lead to the life-threatening complication known as heparin-induced thrombocytopenia (HIT), caused by the development of antibodies against heparin/PF4 complexes. Here, we demonstrate the ability of heparin to induce neutrophil extracellular traps (NETs). NETs occurred with cell lysis and death, but live neutrophils releasing extracellular DNA strands, known as vital NETs, also occurred abundantly. Formation of NETs was time and dose dependent, and required reactive oxygen species and neutrophil elastase. Other compounds related to heparin such as low molecular weight heparin, fondaparinux and heparan sulfate either failed to induce NETs, or did so to a much lesser extent. Our findings suggest the ability of heparin to directly induce NET formation should be considered in the context of heparin treatment and HIT pathogenesis.

Deriving accurate molecular indicators of protein synthesis through Raman-based sparse classification.

Raman spectroscopy has the ability to retrieve molecular information from live biological samples non-invasively through optical means. Coupled with machine learning, it is possible to use this large amount of information to create models that can predict the state of new samples. We study here linear models, whose separation coefficients can be used to interpret which bands are contributing to the discrimination, and compare the performance of principal component analysis coupled with linear discriminant analysis (PCA/LDA), with regularized logistic regression (Lasso). By applying these methods to single-cell measurements for the detection of macrophage activation, we found that PCA/LDA yields poorer performance in classification compared to Lasso, and underestimates the required sample size to reach stable models. Direct use of Lasso (without PCA) also yields more stable models, and provides sparse separation vectors that directly contain the Raman bands most relevant to classification. To further evaluate these sparse vectors, we apply Lasso to a well-defined case where protein synthesis is inhibited, and show that the separating features are consistent with RNA accumulation and protein levels depletion. Surprisingly, when features are selected purely in terms of their classification power (Lasso), they consist mostly of side bands, while typical strong Raman peaks are not present in the discrimination vector. We propose that this occurs because large Raman bands are representative of a wide variety of intracellular molecules and are therefore less suited for accurate classification.

Detecting nitrile-containing small molecules by infrared photothermal microscopy.

The use of infrared (IR) photothermal microscopy (IR-PTM) is emerging for imaging chemical substances in various samples. In this research, we demonstrated the use of a nitrile group as a vibrational tag to image target molecules in the low water-background region. We performed IR photothermal imaging of trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP) in cells and confirmed the high spatial resolution by photothermal detection using visible light as a probe beam. We imaged FCCP-treated HeLa cells and confirmed that the photothermal signal was indeed produced from the vibrational tag in lipid droplets. We also compared the results with nitrile imaging by stimulated Raman scattering (SRS) microscopy. From both the calculated and experimental results, IR-PTM demonstrated a signal-to-noise ratio (SNR) several tens of times better than that of SRS microscopy on the basis of the same power input.

Noninvasive detection of macrophage activation with single-cell resolution through machine learning.

We present a method enabling the noninvasive study of minute cellular changes in response to stimuli, based on the acquisition of multiple parameters through label-free microscopy. The retrieved parameters are related to different attributes of the cell. Morphological variables are extracted from quantitative phase microscopy and autofluorescence images, while molecular indicators are retrieved via Raman spectroscopy. We show that these independent parameters can be used to build a multivariate statistical model based on logistic regression, which we apply to the detection at the single-cell level of macrophage activation induced by lipopolysaccharide (LPS) exposure and compare their respective performance in assessing the individual cellular state. The models generated from either morphology or Raman can reliably and independently detect the activation state of macrophage cells, which is validated by comparison with their cytokine secretion and intracellular expression of molecules related to the immune response. The independent models agree on the degree of activation, showing that the features provide insight into the cellular response heterogeneity. We found that morphological indicators are linked to the phenotype, which is mostly related to downstream effects, making the results obtained with these variables dose-dependent. On the other hand, Raman indicators are representative of upstream intracellular molecular changes related to specific activation pathways. By partially inhibiting the LPS-induced activation using progesterone, we could identify several subpopulations, showing the ability of our approach to identify the effect of LPS activation, specific inhibition of LPS, and also the effect of progesterone alone on macrophage cells.

Saturated two-photon excitation fluorescence microscopy with core-ring illumination.

We demonstrated resolution improvement in two-photon excitation microscopy by combining saturated excitation (SAX) of fluorescence and pupil manipulation. We theoretically estimated the resolution improvement and the sidelobe effect in the point spread function with various pupil designs and found that the combination of SAX and core-ring illumination can effectively enhance the spatial resolution in 3D and suppress sidelobe artifacts. The experimental demonstration shows that the proposed technique is effective for observation with a depth of 100 µm in a tissue phantom and can be applied to 3D observations of tissue samples with higher spatial resolution than conventional two-photon excitation microscopy.

Raman spectroscopy as a tool for label-free lymphocyte cell line discrimination.

Unactivated lymphocytes are morphologically identical and biochemically relatively similar, making them difficult to distinguish from one another with conventional light microscopy. Here, we use Raman spectroscopy to provide biochemical information on the composition of different lymphocyte cell lines. As could be expected, the biochemical differences measured with Raman spectroscopy between lymphocyte cell lines are small, but in combination with partial least squares discriminant analysis it is possible not only to distinguish between T- and B-cells, but also between individual T-cell and B-cell lines.

Label-free Raman imaging of the macrophage response to the malaria pigment hemozoin.

Hemozoin, the 'malaria pigment', is engulfed by phagocytic cells, such as macrophages, during malaria infection. This biocrystalline substance is difficult to degrade and often accumulates in phagocytes. The macrophage response to hemozoin relates to the severity of the disease and the potential for malaria-related disease complications. In this study we have used Raman spectroscopy as a label-free method to investigate the biochemical changes occurring in macrophages during the first few hours of hemozoin uptake. We found a number of distinct spectral groups, spectrally or spatially related to the presence of the hemozoin inside the cell. Intracellular hemozoin was spectrally identical to extracellular hemozoin, regardless of the location in the cell. A small proportion of hemozoin was found to be associated with lipid-based components, consistent with the uptake of hemozoin into vesicles such as phagosomes and lysosomes. The spatial distribution of the hemozoin was observed to be inhomogeneous, and its presence largely excluded that of proteins and lipids, demonstrating that cells were not able to break down the biocrystals on the time scales studied here. These results show that Raman imaging can be used to answer some of the open questions regarding the role of hemozoin in the immune response. How different combinations of hemozoin and other molecules are treated by macrophages, whether hemozoin can be broken down by the cell, and more importantly, which co-factors or products are involved in the subsequent cell reaction are the expected issues to be elucidated by this technique.

3D SERS (surface enhanced Raman scattering) imaging of intracellular pathways.

We demonstrate dynamic surface-enhanced Raman scattering (SERS) imaging with 3 dimensional mapping in a living cell by combining a confocal Raman spectrometer and dual-focus dark-field microscopy. Gold nanoparticles acting as a probe traveling through the living cell, are tracked by dual-focus dark-field imaging, with position feedback to the confocal Raman spectrometer in order to record evolving SERS signals from the same gold nanoparticle moving through the cell. Simultaneous detection of motion and the SERS spectrum enables the visualization of the SERS pathways. Observation of how the SERS spectra change in space and time during biological functions or by changes of the molecular environment provides exciting opportunities for understanding the molecular basis of the cell dynamics.

Saturated excitation microscopy with optimized excitation modulation.

Saturated excitation (SAX) microscopy utilizes the nonlinear relation between fluorescence emission and excitation under saturated excitation to improve the spatial resolution of confocal microscopy. In this study, we theoretically and experimentally investigate the saturation of fluorescence excitation under modulated excitation to optimize the excitation conditions for SAX microscopy. Calculation of the relationships between fluorescence and excitation intensity with different modulation frequencies reveals that the lifetime of the triplet state of the fluorescent probe strongly affects the strength of the demodulated fluorescence signals. We also find that photobleaching shows little dependence on the modulation frequency. These investigations allow us to determine the optimum excitation conditions, that is, the conditions providing sufficient fluorescence saturation without strong photobleaching. For a sample stained with ATTO Rho6G phalloidin, we estimate the optimal excitation conditions, which are produced with 50 kHz excitation modulation and a 50 µsec pixel dwell time, and successfully perform three-dimensional imaging with sub-diffraction resolution.

Cell optical density and molecular composition revealed by simultaneous multimodal label-free imaging.

We show how Raman imaging can be combined with independent but simultaneous phase measurements of unlabeled cells, with the resulting data providing information on how the light is retarded and/or scattered by molecules in the cell. We then show, for the first time to our knowledge, how the chemistry of the cell highlighted in the Raman information is related to the cell quantitative phase information revealed in digital holographic microscopy by quantifying how the two sets of spatial information are correlated. The results show that such a multimodal implementation is highly useful for the convenience of having video rate imaging of the cell during the entire Raman measurement time, allowing us to observe how the cell changes during Raman acquisition. More importantly, it also shows that the two sets of label-free data, which result from different scattering mechanisms, are complementary and can be used to interpret the composition and dynamics of the cell, where each mode supplies label-free information not available from the other mode.

Raman spectroscopic analysis of malaria disease progression via blood and plasma samples.

In this study Raman spectroscopy has been used to monitor the changes in erythrocytes and plasma during Plasmodium infection in mice, following malaria disease progression over the course of 7 days. The Raman spectra of both samples are dominated by the spectra of hemoglobin and hemozoin, due to their resonant enhancement. In plasma samples, due to the inherently low heme background, heme-based changes in the Raman spectra could be detected in the very early stages of infection, as little as one day after Plasmodium infection, where parasitemia levels were low, on the order of 0.2%, and typically difficult to detect by existing methods. Further principal component analysis also indicates concurrent erythrocyte membrane changes at around day 4, where parasitemia levels reached 3%. These results show that plasma analysis has significant potential for early, quantitative and automated detection of malaria, and to quantify heme levels in serum which modulate malarial effects on the immune system.

Deconstructing RNA: optical measurement of composition and structure.

RNA molecules are involved in many pathways within the cell and their sequence composition, structure, conformational transitions and interactions with other molecules are all important factors in determining RNA function. Here we present a method for systematically and quantitatively determining characteristics of RNA using Raman spectroscopy. This method can be used to assess the composition and structure of a given RNA molecule, including ribose-phosphate sugar-pucker conformation, face-to-face base stacking and hydrogen bonding interactions. Three RNA molecules with different sequence and structural features (the exon splicing silencer 3 from HIV-1, an RNA aptamer against Runt-related transcription factor, and the SARS coronaviral stem loop 2) are presented as examples where the structure is crucial to the function of the RNA. We carry out piecewise analysis of the RNA spectra and show that using a nucleotide spectra library helps to unlock the entire ensemble of vibrational information. This analysis demonstrates the extent to which RNA characteristics can be elucidated, using purely optical methods.

Non-invasive monitoring of T cell differentiation through Raman spectroscopy.

The monitoring of dynamic cellular behaviors remains a technical challenge for most established techniques used nowadays for single-cell analysis, as most of them are either destructive, or rely on labels that can affect the long-term functions of cells. We employ here label-free optical techniques to non-invasively monitor the changes that occur in murine naive T cells upon activation and subsequent differentiation into effector cells. Based on spontaneous Raman single-cell spectra, we develop statistical models that allow the detection of activation, and employ non-linear projection methods to delineate the changes occurring over a several day period spanning early differentiation. We show that these label-free results have very high correlation with known surface markers of activation and differentiation, while also providing spectral models that allow the identification of the underlying molecular species that are representative of the biological process under study.

Dynamic SERS imaging of cellular transport pathways with endocytosed gold nanoparticles.

Dynamic SERS imaging inside a living cell is demonstrated with the use of a gold nanoparticle, which travels through the intracellular space to probe local molecular information over time. Simultaneous tracking of particle motion and SERS spectroscopy allows us to detect intracellular molecules at 65 nm spatial resolution and 50 ms temporal resolution, providing molecular maps of organelle transport and lisosomal accumulation. Multiplex spectral and trajectory imaging will enable imaging of specific dynamic biological functions such as membrane protein diffusion, nuclear entry, and rearrangement of cellular cytoskeleton.

Spectral focusing in picosecond pulsed stimulated Raman scattering microscopy.

We introduce spectral focusing of picosecond laser pulses in stimulated Raman scattering (SRS) microscopy to improve spectral resolution, reduce nonlinear background signals, and decrease nonlinear photodamage. We produce a pair of 14 ps pump and Stokes laser pulses by spectral focusing of a 2 ps laser and achieve a spectral resolution of 2 cm-1. Due to instantaneous narrow-band excitation, we find that the chirped 14 ps laser pulses can be used to improve the signal-to-background ratio in SRS microscopy of various samples such as polymer particles and small molecules in HeLa cells. The lower peak powers produced by chirped picosecond laser pulses also reduce nonlinear photodamage, allowing long-term SRS imaging of living cells with higher SNR.

Bessel-beam illumination Raman microscopy.

We demonstrate the use of Bessel beams for side illumination slit-scanning Raman imaging for label-free and hyperspectral analysis of cell spheroids. The background elimination by the side illumination and the aberration-resistant Bessel beam drastically improves the image contrast in Raman observation, allowing label-free investigation of intracellular molecules in thick biological samples. Live cell spheroids were observed to confirm the improvement in image contrast and background reduction with Bessel illumination compared to conventional epi-line illumination.

Cellular Adhesion Is a Controlling Factor in Neutrophil Extracellular Trap Formation Induced by Anti-Neutrophil Cytoplasmic Antibodies.

Anti-neutrophil cytoplasmic Ab (ANCA)-associated vasculitis (AAV) is a life-threatening condition characterized by improper activation of neutrophils and the release of neutrophil extracellular traps (NETs) in small vessels. This study aimed to explain the role of NETs in AAV pathogenesis by investigating a link between adhesion and NET release using human neutrophils. We leveraged an imaging flow cytometry-based assay and three-dimensional culture to demonstrate that neutrophil adhesion is essential for ANCA-induced NET formation. We confirmed this requirement for cell adhesion using standard microscopy on ultra-low attachment hydrogel surfaces and demonstrate that this depends on the focal adhesion kinase pathway as determined using inhibitors for multiple targets in this process. ANCA increased expression of ß2 integrins on neutrophils, and we confirmed that these integrins were required for NET formation using blocking Abs. Finally, inhibitors for oxidative burst prevented NET formation, and this oxidative burst was mediated by the focal adhesion pathway. Overall, our findings reveal a central role for neutrophil attachment in NET formation in response to ANCAs, helping to explain the restricted localization pattern of vessel damage, and suggesting that targeting neutrophil adhesion factors may be beneficial in preventing pathological damage from NETs during AAV.