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.

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.

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.

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.

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.

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.

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.

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.

A pilot study of brain injury in police officers: A source of mental health problems?

WHAT IS KNOWN ON THE SUBJECT?: Traumatic brain injury (TBI) has been linked to poor outcomes in terms of mental health, specifically, PTSD, depression and alcohol abuse. A lack of research evidence exists relevant to exploring the presence and implications of TBI in the police in the UK and globally, despite the elevated risk of physical and emotional trauma specific to policing. WHAT DOES THE PAPER ADD TO EXISTING KNOWLEDGE?: The rate of traumatic brain injury is highly prevalent in a small sample of police officers. Traumatic brain injury is a major source of post-concussion symptoms (physical, cognitive and emotional deficits) in police officers, which, in general, are associated with greater mental health difficulties and drinking alcohol to cope. WHAT ARE THE IMPLICATIONS FOR PRACTICE?: Traditional mental health treatments should be supplemented with elements of concussion care to address any cognitive, emotional and physical issues due to head injury. Interventions should be made more accessible to those suffering from a mild brain injury. This can be done through regular reminders of appointments, pictograms and by providing a concrete follow-up. ABSTRACT: Introduction Police officers have a high risk of injury through assaults, road traffic incidents and attending domestic calls, with many officers developing post-traumatic stress disorder (PTSD) as a consequence. Traumatic brain injury (TBI) is a common injury in populations involved in conflict and has been extensively linked to mental health difficulties. However, current research has not explored the frequency and sequelae of TBI in police populations, despite the elevated risk of physical and emotional trauma specific to policing. Aim To explore self-reported TBI, PTSD, post-concussion symptoms, depression and drinking to cope in a small sample of UK police, to determine the frequency of these conditions and their relationships. Method Measures of TBI, mental health, and drinking alcohol to cope were administered to 54 police officers from a Midshire Police Constabulary. Results Mild TBI with loss of consciousness was reported by 38.9% of the sample. TBI was associated with increased post-concussion symptoms (PCS). PCS were associated with greater severity of PTSD, depression and drinking to cope. Discussion Exploring TBI in the police could identify a major factor contributing towards ongoing mental health difficulties in a population where, based on previous research, the implications of TBI should not be overlooked, highlighting the need for further research in this area. Implications for Practice This research spans to identify the importance of routine assessment and increasing awareness within mental health services. Mental health treatments should be made amenable to a population with potential memory, planning and impulse control deficits. Further work in mental health services is needed to understand the level of ongoing issues that are due to post-concussion symptoms and those that are due to other mental health difficulties, such as PTSD, thereby educating patients on the association between TBI and emotional difficulties. A graduated return-to-work plan should be developed to enable a safe transition back to work, whilst managing any ongoing symptoms.

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.

Immune cell type, cell activation, and single cell heterogeneity revealed by label-free optical methods.

Measurement techniques that allow the global analysis of cellular responses while retaining single-cell sensitivity are increasingly needed in order to understand complex and dynamic biological processes. In this context, compromises between sensitivity, degree of multiplexing, throughput, and invasiveness are often unavoidable. We present here a noninvasive optical approach that can retrieve quantitative biomarkers of both morphological and molecular phenotypes of individual cells, based on a combination of quantitative phase imaging and Raman spectroscopy measurements. We then develop generalized statistical tools to assess the influence of both controlled (cell sub-populations, immune stimulation) and uncontrolled (culturing conditions, animal variations, etc.) experimental parameters on the label-free biomarkers. These indicators can detect different macrophage cell sub-populations originating from different progenitors as well as their activation state, and how these changes are related to specific differences in morphology and molecular content. The molecular indicators also display further sensitivity that allow identification of other experimental conditions, such as differences between cells originating from different animals, allowing the detection of outlier behaviour from given cell sub-populations.

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.

Structured line illumination Raman microscopy.

In the last couple of decades, the spatial resolution in optical microscopy has increased to unprecedented levels by exploiting the fluorescence properties of the probe. At about the same time, Raman imaging techniques have emerged as a way to image inherent chemical information in a sample without using fluorescent probes. However, in many applications, the achievable resolution is limited to about half the wavelength of excitation light. Here we report the use of structured illumination to increase the spatial resolution of label-free spontaneous Raman microscopy, generating highly detailed spatial contrast from the ensemble of molecular information in the sample. Using structured line illumination in slit-scanning Raman microscopy, we demonstrate a marked improvement in spatial resolution and show the applicability to a range of samples, including both biological and inorganic chemical component mapping. This technique is expected to contribute towards greater understanding of chemical component distributions in organic and inorganic materials.

Visible-wavelength two-photon excitation microscopy for fluorescent protein imaging.

The simultaneous observation of multiple fluorescent proteins (FPs) by optical microscopy is revealing mechanisms by which proteins and organelles control a variety of cellular functions. Here we show the use of visible-light based two-photon excitation for simultaneously imaging multiple FPs. We demonstrated that multiple fluorescent targets can be concurrently excited by the absorption of two photons from the visible wavelength range and can be applied in multicolor fluorescence imaging. The technique also allows simultaneous single-photon excitation to offer simultaneous excitation of FPs across the entire range of visible wavelengths from a single excitation source. The calculation of point spread functions shows that the visible-wavelength two-photon excitation provides the fundamental improvement of spatial resolution compared to conventional confocal microscopy.

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.

Maximizing throughput in label-free microspectroscopy with hybrid Raman imaging.

Raman spectroscopy is an optical method providing sample molecular composition, which can be analyzed (by point measurements) or spatially mapped by Raman imaging. These provide different information, signal-to-noise ratios, and require different acquisition times. Here, we quantitatively assess Raman spectral features and compare the two measurement methods by multivariate analysis. We also propose a hybrid method: scanning the beam through the sample but optically binning the signal at one location on the detector. This approach generates significantly more useful spectral signals in terms of peak visibility and statistical information. Additionally, by combination with a complementary imaging mode such as quantitative phase microscopy, hybrid imaging allows high throughput and robust spectral analysis while retaining sample spatial information. We demonstrate the improved ability to discriminate between cell lines when using hybrid scanning compared to typical point mode measurements, by quantitatively evaluating spectra taken from two macrophage-like cell lines. Hybrid scanning also provides better classification capability than the full Raman imaging mode, while providing higher signal-to-noise signals with shorter acquisition times. This hybrid imaging approach is suited for various applications including cytometry, cancer versus noncancer detection, and label-free discrimination of cell types or tissues.

Dual-polarization Raman spectral imaging to extract overlapping molecular fingerprints of living cells.

Raman spectral imaging is gaining more and more attention in biological studies because of its label-free characteristic. However, the discrimination of overlapping chemical contrasts has been a major challenge. In this study, we introduce an optical method to simultaneously obtain two orthogonally polarized Raman images from a single scan of the sample. We demonstrate how this technique can improve the quality and quantity of the hyperspectral Raman dataset and how the technique is expected to further extend the horizons of Raman spectral imaging in biological studies by providing more detailed chemical information. The dual-polarization Raman images of a HeLa cell.