Raman microscopy of cryofixed biological specimens for high-resolution and high-sensitivity chemical imaging.

Raman microscopy is an emerging molecular imaging technology, yet its signal-to-noise ratio (SNR) in measurements of biological specimens is severely limited because of the small cross section of Raman scattering. Here, we present Raman imaging techniques of cryofixed specimens to overcome SNR limitations by enabling long exposure of specimens under highly stabilized low-temperature conditions. The observation of frozen specimens in a cryostat at a constant low temperature immediately after rapid freezing enabled the improvement of SNR and enhanced the spatial and spectral resolution.

Correlative Quantitative Raman Chemical Imaging and MCR-ALS in Mouse NASH Model Reveals Direct Relationships between Diet and Resultant Liver Pathology.

Raman imaging has the capability to provide unlabeled, spatially aware analysis of chemical components, with no assumptions. Several lifestyle diseases such as nonalcoholic steatohepatitis (NASH) can appear in the liver as changes in the nature, abundance, and distribution of lipids, proteins, and other biomolecules and are detectable by Raman imaging. In order to identify which of these liver-associated changes occur as a direct result of the diet and which are secondary effects, we developed correlative imaging and analysis of diet and liver samples.

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.

Single-cell Raman microscopy with machine learning highlights distinct biochemical features of neutrophil extracellular traps and necrosis.

The defining biology that distinguishes neutrophil extracellular traps (NETs) from other forms of cell death is unresolved, and techniques which unambiguously identify NETs remain elusive. Raman scattering measurement provides a holistic overview of cell molecular composition based on characteristic bond vibrations in components such as lipids and proteins. We collected Raman spectra from NETs and freeze/thaw necrotic cells using a custom built high-throughput platform which is able to rapidly measure spectra from single cells.

High-throughput line-illumination Raman microscopy with multislit detection.

Raman microscopy is an emerging tool for molecular imaging and analysis of living samples. Use of Raman microscopy in life sciences is, however, still limited because of its slow measurement speed for spectral imaging and analysis. We developed a multiline-illumination Raman microscope to achieve ultrafast Raman spectral imaging.

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.

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.

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. 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.

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.

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).

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.

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.

Quantitative Drug Dynamics Visualized by Alkyne-Tagged Plasmonic-Enhanced Raman Microscopy.

Visualizing live-cell uptake of small-molecule drugs is paramount for drug development and pharmaceutical sciences. Bioorthogonal imaging with click chemistry has made significant contributions to the field, visualizing small molecules in cells. Furthermore, recent developments in Raman microscopy, including stimulated Raman scattering (SRS) microscopy, have realized direct visualization of alkyne-tagged small-molecule drugs in live cells.

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).

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.

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.

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.

Deep-UV biological imaging by lanthanide ion molecular protection.

Deep-UV (DUV) light is a sensitive probe for biological molecules such as nucleobases and aromatic amino acids due to specific absorption. However, the use of DUV light for imaging is limited because DUV can destroy or denature target molecules in a sample. Here we show that trivalent ions in the lanthanide group can suppress molecular photodegradation under DUV exposure, enabling a high signal-to-noise ratio and repetitive DUV imaging of nucleobases in cells.

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.

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.

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.

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.

Laser-targeted photofabrication of gold nanoparticles inside cells.

Nanoparticle manipulation is of increasing interest, since they can report single molecule-level measurements of the cellular environment. Until now, however, intracellular nanoparticle locations have been essentially uncontrollable. Here we show that by infusing a gold ion solution, focused laser light-induced photoreduction allows in situ fabrication of gold nanoparticles at precise locations.