Non-invasive detection of regulatory T cells with Raman spectroscopy.

Regulatory T cells (Tregs) are a type of lymphocyte that is key to maintaining immunological self-tolerance, with great potential for therapeutic applications. A long-standing challenge in the study of Tregs is that the only way they can be unambiguously identified is by using invasive intracellular markers. Practically, the purification of live Tregs is often compromised by other cell types since only surrogate surface markers can be used.

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.

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.

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).

Label-free Raman mapping of saturated and unsaturated fatty acid uptake, storage, and return toward baseline levels in macrophages.

Macrophage uptake and metabolism of fatty acids is involved in a large number of important biological pathways including immune activation and regulation of macrophages, as well as pathological conditions including obesity, atherosclerosis, and others lifestyle diseases. There are few methods available to directly probe both the uptake and later redistribution/metabolism of fatty acids within living cells as well as the potential changes induced within the cells themselves. We use Raman imaging and analysis to evaluate the effects of different fatty acids following their uptake in macrophages.

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.

Compressed sensing laser scanning microscopy.

We present a measurement and reconstruction method for laser-scanning microscopy based on compressed sensing, which enables significantly higher frame rates and reduced photobleaching. The image reconstruction accuracy is ensured by including a model of the physical imaging process into the compressed sensing reconstruction procedure. We demonstrate its applicability to unmodified commercial confocal fluorescence microscopy systems and for Raman imaging, showing a potential data reduction of 10-15 times, which directly leads to improvements in acquisition speed, or reduction of photobleaching, without significant loss of spatial resolution.

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.

Cell volume regulation monitored with combined epifluorescence and digital holographic microscopy.

Quantitative phase imaging emerged recently as a valuable tool for cell observation, by enabling label-free imaging through the intrinsic phase-contrast provided by transparent living cells, thus greatly simplifying observation protocols. The quantitative phase signal, unlike the one provided by the widely used phase-contrast microscope, can be related to relevant biological indicators including dry mass, cell volume regulation or transmembrane water movements. Here, we present quantitative phase imaging coupled with live fluorescence, making it possible to follow the phase signal in time to monitor the cell volume regulation, an early indicator of cell viability, along with specific information such as intracellular Ca2+ imaging with Fura-2 ratiometric fluorescence.

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.

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.

A practical inverse-problem approach to digital holographic reconstruction.

In this paper, we propose a new technique for high-quality reconstruction from single digital holographic acquisitions. The unknown complex object field is found as the solution of a nonlinear inverse problem that consists in the minimization of an energy functional. The latter includes total-variation (TV) regularization terms that constrain the spatial amplitude and phase distributions of the reconstructed data.

Local demodulation of holograms using the Riesz transform with application to microscopy.

We propose a Riesz transform approach to the demodulation of digital holograms. The Riesz transform is a higher-dimensional extension of the Hilbert transform and is steerable to a desired orientation. Accurate demodulation of the hologram requires a reliable methodology by which quadrature-phase functions (or simply, quadratures) can be constructed.

Feature-based recognition of surface-enhanced Raman spectra for biological targets.

We propose and compare multiple approaches to automatically process data measured through surface-enhanced Raman scattering (SERS), in the context of intracellular molecule probing. It relies on locally detecting the most relevant spectra to retrieve all data independently through indexing, thus avoiding any pre-filtering which occurs with standard processing methods. We first assess our approach on simulated data of the spectrum of Rhodamine 6G, and then validate high-performing methods on experimental measurements of this compound.

Early cell death detection with digital holographic microscopy.

Background: Digital holography provides a non-invasive measurement of the quantitative phase shifts induced by cells in culture, which can be related to cell volume changes. It has been shown previously that regulation of cell volume, in particular as it relates to ionic homeostasis, is crucially involved in the activation/inactivation of the cell death processes. We thus present here an application of digital holographic microscopy (DHM) dedicated to early and label-free detection of cell death.

Single-shot, simultaneous incoherent and holographic microscopy.

In this work, we demonstrate single-shot, simultaneous recording and subsequent retrieval of one incoherent and two holographic (intensity and phase) images from the same camera frame. Demultiplexing of incoherent and holographic signals in the spatial frequency domain is made possible by carrier frequency modulation and spatial oversampling intrinsic to the off-axis digital holographic configuration. In particular, we show applications to combined fluorescence and digital holographic microscopy, as well as combined bright field and holographic second harmonic generation microscopy.

Determination of transmembrane water fluxes in neurons elicited by glutamate ionotropic receptors and by the cotransporters KCC2 and NKCC1: a digital holographic microscopy study.

Digital holographic microscopy (DHM) is a noninvasive optical imaging technique that provides quantitative phase images of living cells. In a recent study, we showed that the quantitative monitoring of the phase signal by DHM was a simple label-free method to study the effects of glutamate on neuronal optical responses (Pavillon et al., 2010).

Exact complex-wave reconstruction in digital holography.

We address the problem of exact complex-wave reconstruction in digital holography. We show that, by confining the object-wave modulation to one quadrant of the frequency domain, and by maintaining a reference-wave intensity higher than that of the object, one can achieve exact complex-wave reconstruction in the absence of noise. A feature of the proposed technique is that the zero-order artifact, which is commonly encountered in hologram reconstruction, can be completely suppressed in the absence of noise.

Endoscopic low-coherence topography measurement for upper airways and hollow samples.

To evaluate the severity of airway pathologies, quantitative dimensioning of airways is of utmost importance. Endoscopic vision gives a projective image and thus no true scaling information can be directly deduced from it. In this article, an approach based on an interferometric setup, a low-coherence laser source and a standard rigid endoscope is presented, and applied to hollow samples measurements.

Microscopy image resolution improvement by deconvolution of complex fields.

Based on truncated inverse filtering, a theory for deconvolution of complex fields is studied. The validity of the theory is verified by comparing with experimental data from digital holographic microscopy (DHM) using a high-NA system (NA=0.95).

Iterative method for zero-order suppression in off-axis digital holography.

We propose a method to suppress the so-called zero-order term in a hologram, based on an iterative principle. During the hologram acquisition process, the encoded information includes the intensities of the two beams creating the interference pattern, which do not contain information about the recorded complex wavefront, and that can disrupt the reconstructed signal. The proposed method selectively suppresses the zero-order term by employing the information obtained during wavefront reconstruction in an iterative procedure, thus enabling its suppression without any a priori knowledge about the object.