Alison J. Hobro

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.

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.

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.

Distribution of label free cationic polymer-coated gold nanorods in live macrophage cells reveals formation of groups of intracellular SERS signals of probe nanoparticles

Gold nanorods coated with poly(diallydimethylammonium chloride) (PDAC-GNRs) were used to observe the distribution of surface-enhanced Raman signals in live cells and generate distinct groups of surface-enhanced Raman scattering (SERS) spectra in different regions of the cells. Spectra with unique features were clustered into sets of molecules in live murine macrophage cells (Raw 264.7). The distribution of biological substances detected by SERS signals of PDAC-GNRs is also discussed.

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. The resulting particles are pure gold nanocrystals, distributed throughout the laser focus at sizes ranging from 2 to 20u2009nm, and remain in place even after removing the gold solution. We demonstrate the spatial control by scanning a laser beam to write characters in gold inside a cell. Plasmonically enhanced molecular signals could be detected from nanoparticles, allowing their use as nano-chemical probes at targeted locations inside the cell, with intracellular molecular feedback. Such light-based control of the intracellular particle generation reaction also offers avenues for in situ plasmonic device creation in organic targets, and may eventually link optical and electron microscopy.

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

Significance We developed a method enabling the noninvasive study of fine cellular responses that we applied to macrophage activation. The technique is based on a multimodal label-free microscopy system that simultaneously retrieves both morphological and molecular information based on quantitative phase imaging and Raman spectroscopy, respectively. The parameters obtained from these measurements are processed through a machine learning algorithm that makes it possible to reliably assess the macrophage activation state at single-cell level. We found that while each parameter set (morphology and Raman) can detect the activation state, they provide complementary information. Morphology is symptomatic of downstream phenotypes that make the detection dose-dependent, while Raman is indicative of upstream molecular changes that enable the detection of selective inhibition of activation pathways. 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.

Label-Free Raman Imaging

Label-free Raman imaging is a noninvasive spectroscopic method for investigating the nature and distribution of molecular species within a sample. In this chapter, we describe the applications of conventional Raman imaging, as well as the related techniques of coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) imaging for medical, life sciences, and other biological applications.

Raman imaging and analysis: From quantification of cellular dynamics to molecular structure

We develop protocols for Raman microscopy of living cellular changes in response to immunological stimulus and also attempt to quantify molecular structural information through analysis of obtained spectra from purified samples.