ultra-high-information-content-chemical-imaging-with-broadband

Ultra-high Information-content Chemical Imaging with Broadband Coherent Anti-Stokes Raman and Two-photon Fluorescence Lifetime Microscopy

Raman fingerprint spectroscopy and fluorescence lifetime imaging are emerging tools for studying metabolic profiles of biological specimens. While Raman ...

Georgia Institute of Technology

Potential energy landscape (PEL) concepts have been useful in conceptualizing the effects of intermolecular interactions on dynamic and thermodynamic properties of liquids and glasses. "Basins", or regions of reduced potential energy associated with locally preferred molecular packing are important PEL features. The molecular configurations at the bottom of these basins are referred to as inherent structures (ISs). Experimental methods for directly characterizing PEL features such as these are rare, largely relegating PEL concepts to theory and simulation studies, and impeding their exploration in real systems. Recently, we showed that quasielastic neutron scattering (QENS) data from propylene carbonate (PC) exhibit signatures of picosecond timescale motion that are consistent with intrabasin motion and interbasin transitions [Cicerone et al., J. Chem. Phys., 2017, 146, 054502]. Here we present optically-heterodyne-detected optical Kerr effect (OHD-OKE) spectroscopy studies on PC. The data exhibit signatures of motion within and transitions between basins that agree quantitatively with and extend the QENS results. We show that the librational component of the OKE response corresponds to intrabasin dynamics, and the enigmatic intermediate OKE response corresponds to interbasin transition events. The OKE data extend the measurement range of these parameters and reveal their utility in characterizing PEL features of real systems.

The polarizability response of a glass-forming liquid reveals intrabasin motion and interbasin transitions on a potential energy landscape.

Caenorhabditis elegans serves as a model for understanding adiposity and its connections to aging. Current methodologies do not distinguish between fats serving the energy needs of the parent, akin to mammalian adiposity, from those that are distributed to the progeny, making it difficult to accurately interpret the physiological implications of fat content changes induced by external perturbations. Using spectroscopic coherent Raman imaging, we determine the protein content, chemical profiles and dynamics of lipid particles in live animals. We find fat particles in the adult intestine to be diverse, with most destined for the developing progeny. In contrast, the skin-like epidermis contains fats that are the least heterogeneous, the least dynamic and have high triglyceride content. These attributes are most consistent with stored somatic energy reservoirs. These results challenge the prevailing practice of assessing C. elegans adiposity by measurements that are dominated by the intestinal fat content.

Spectroscopic coherent Raman imaging of Caenorhabditis elegans reveals lipid particle diversity.

We describe a simple approach to dispersion-free optical delay line design that provides very low aberration over an extended delay range. In this approach, we minimize aberrations by directing non-axial beam displacements along a line of symmetry built into the apparatus. We show improved performance and significant reduction of wavefront aberrations by comparing simulation and experimental results with a similar delay line that lacks this line of symmetry. The new design facilitates transform-limited recovery of spectral resolution in Fourier transform coherent anti-Stokes Raman scattering, and accordingly, we demonstrate 3.5 spectral resolution with a 10 ps delay scan range.

Low-aberration high-speed-compatible optical delay line.

We demonstrate the preservation of the time-energy entanglement of near-IR photons through thick biological media (≤1.55 mm) and tissue (≤ 235 m) at room temperature. Using a Franson-type interferometer, we demonstrate interferometric contrast of over 0.9 in skim milk, 2% milk, and chicken tissue. This work supports the many proposed opportunities for nonclassical light in biological imaging and analyses from sub-shot noise measurements to entanglement-enhanced fluorescence imaging, clearly indicating that the entanglement characteristics of photons can be maintained even after propagation through thick, turbid biological samples.

Witnessing the survival of time-energy entanglement through biological tissue and scattering media.

While Raman spectroscopy can provide label-free discrimination between highly similar biological species, the discrimination is often marginal, and optimal use of spectral information is imperative. Here, we compare two machine learning models, an artificial neural network and a support vector machine, for discriminating between Raman spectra of 11 bacterial mutants of Escherichia coli MDS42. While we find that both models discriminate the 11 bacterial strains with similarly high accuracy, sensitivity and specificity, it is clear that the models form different class boundaries. By extracting strain-specific (and function-specific) spectral features utilized by the models, we find that both models utilize a small subset of high intensity peaks while separate subsets of lower intensity peaks are utilized by only one method or the other. This analysis highlights the need for methods to use the complete spectral information more effectively, beginning with a better understanding of the distinct information gained from each model.

Discriminating cell line specific features of antibiotic-resistant strains of Escherichia coli from Raman spectra via machine learning analysis.

Structural excitations that enable interbasin (IB) barrier crossings on a potential energy landscape are thought to play a facilitating role in the relaxation of liquids. Here, we show that the population of these excitations exhibits the same density scaling observed for α relaxation in propylene carbonate, even though they are heavily influenced by intramolecular modes. We also find that IB crossing modes exhibit a Grüneisen parameter (γ) that is approximately equivalent to the density scaling parameter γ. These observations suggest that the well-documented relationship between γ and γ may be a direct result of the pressure dependence of the frequency of unstable (relaxation) modes associated with IB motion.

Excitations follow (or lead?) density scaling in propylene carbonate.

Fat metabolism is an important modifier of aging and longevity in . Given the anatomy and hermaphroditic nature of , a major challenge is to distinguish fats that serve the energetic needs of the parent from those that are allocated to the progeny. Broadband coherent anti-Stokes Raman scattering (BCARS) microscopy has revealed that the composition and dynamics of lipid particles are heterogeneous both within and between different tissues of this organism. Using BCARS, we have previously succeeded in distinguishing lipid-rich particles that serve as energetic reservoirs of the parent from those that are destined for the progeny. While BCARS microscopy produces high-resolution images with very high information content, it is not yet a widely available platform. Here we report a new approach combining the lipophilic vital dye Nile Red and two-photon fluorescence lifetime imaging microscopy (2p-FLIM) for the  discrimination of lipid particle sub-types. While it is widely accepted that Nile Red staining yields unreliable results for detecting lipid structures in live  due to strong interference of autofluorescence and non-specific staining signals, our results show that simple FLIM phasor analysis can effectively separate those signals and is capable of differentiating the non-polar lipid-dominant (lipid-storage), polar lipid-dominant (yolk lipoprotein) particles, and the intermediates that have been observed using BCARS microscopy. An advantage of this approach is that images can be acquired using common, commercially available 2p-FLIM systems within about 10% of the time required to generate a BCARS image. Our work provides a novel, broadly accessible approach for analyzing lipid-containing structures in a complex, live whole organism context.

Identifying lipid particle sub-types in live  with two-photon fluorescence lifetime imaging.

Raman spectroscopy has long been known to provide sufficient information to discriminate distinct cell phenotypes. Underlying this discriminating capability is that Raman spectra provide an overall readout of the metabolic profiles that change with transcriptomic activity. Robustly associating Raman spectral changes with the regulation of specific signaling pathways may be possible, but the spectral signals of interest may be weak and vary somewhat among individuals. Establishing a Raman-to-transcriptome mapping will thus require tightly controlled and easily manipulated biological systems and high-throughput spectral acquisition. We attempt to meet these requirements using broadband coherent anti-Stokes Raman scattering (BCARS) microscopy to spatio-spectrally map the  hermaphrodite gonad  at subcellular resolution. The  hermaphrodite gonad is an ideal model system with a sequential, continuous process of highly regulated spatiotemporal cellular events. We demonstrate that the BCARS spatio-spectral signatures correlate with gene expression profiles in the gonad, evincing that BCARS has potential as a spatially resolved omics surrogate.

Toward Gene-Correlated Spatially Resolved Metabolomics with Fingerprint Coherent Raman Imaging.

Correction to "Toward Gene-Correlated Spatially Resolved Metabolomics with Fingerprint Coherent Raman Imaging".

The increasing global demand for food, coupled with concerns about the environmental impact of synthetic fertilizers, underscores the urgency of developing sustainable agricultural practices. Nitrogen-fixing bacteria, known as diazotrophs, offer a potential solution by converting atmospheric nitrogen into bioavailable forms, reducing the reliance on synthetic fertilizers. However, a deeper understanding of their interactions with plants and other microbes is needed. In this study, we introduce a recently developed label-free 3D quantitative phase imaging technology called dynamic quantitative oblique back-illumination microscopy (DqOBM) to assess the dynamic activity of diazotrophs  and . Our experiments involved three different diazotrophs (, , and ) cultured on media with amendments of carbon and nitrogen sources. Over five days, we observed increased dynamic activity in nutrient-amended media. These results suggest that the observed bacterial dynamics correlate with their metabolic activity. Furthermore, we applied qOBM to visualize bacterial activity within the root cap and elongation zone of  primary roots. This allowed us to identify distinct areas of microbial infiltration in plant roots without the need for fluorescent markers. Our findings demonstrate that DqOBM can effectively characterize microbial activity and provide insights into plant-microbe interactions , offering a valuable tool for advancing our understanding of sustainable agriculture.

Label-Free Functional Analysis of Root-Associated Microbes with Dynamic Quantitative Oblique Back-illumination Microscopy.

The increasing global demand for food, coupled with concerns about the environmental impact of synthetic fertilizers, underscores the urgency of developing sustainable agricultural practices. Nitrogen-fixing bacteria, known as diazotrophs, offer a potential solution by converting atmospheric nitrogen into bioavailable forms, reducing the reliance on synthetic fertilizers. However, a deeper understanding of their interactions with plants and other microbes is needed. In this study, we introduce a recently developed label-free 3D quantitative phase imaging technology called dynamic quantitative oblique back-illumination microscopy (DqOBM) to assess the functional dynamic activity of diazotrophs in vitro and in situ. Our experiments involved three different diazotrophs (Sinorhizobium meliloti, Azotobacter vinelandii, and Rahnella aquatilis) cultured on media with amendments of carbon and nitrogen sources. Over 5 days, we observed increased dynamics in nutrient-amended media. These results suggest that the observed bacterial dynamics correlate with their metabolic activity. Furthermore, we applied qOBM to visualize microbial dynamics within the root cap and elongation zone of Arabidopsis thaliana primary roots. This allowed us to identify distinct areas of microbial infiltration in plant roots without the need for fluorescent markers. Our findings demonstrate that DqOBM can effectively characterize microbial dynamics and provide insights into plant-microbe interactions in situ, offering a valuable tool for advancing our understanding of sustainable agriculture.

Label-free functional analysis of root-associated microbes with dynamic quantitative oblique back-illumination microscopy.

The success of the mRNA vaccine against COVID-19 has garnered significant interest in the development of mRNA therapeutics against other diseases, but there remains a strong need for a stable and versatile delivery platform for these therapeutics. In this study, we report on a family of robust hybrid lipid nanocapsules (hLNCs) for the delivery of mRNA. The hLNCs are composed of kolliphore HS15, labrafac lipophile WL1349, 1,2-dioleoyl--glycero-3-phosphoethanolamine (DOPE), and a conjugate of oleic acid (OA) and polyethylenimines of varying size (PEI─0.8, 1.8, and 25 kDa). They are prepared by a solvent-free, temperature-phase inversion method, yielding an average size of ∼40 nm and a particle distribution index (PDI) < 0.2. We demonstrate that the PDI remains <0.2 over a wide pH range and in a wide range of medium. We further show that the PDI and the functionality of mRNA condensed on the particles are robust to drying in a sugar glass and subsequent rehydration. Finally, we demonstrate that mRNA-loaded hLNCs yield reasonable transfection in vitro and in vivo settings.

Hybrid Lipid Nanocapsules: A Robust Platform for mRNA Delivery.

Broadband coherent anti-Stokes Raman scattering (BCARS) microscopy is a label-free imaging approach that provides detailed chemical information at high spatial resolution in a sample through nonlinear, coherent excitation of molecular vibrations and detection of Raman spectra. While its utility for biological imaging has been demonstrated, many aspects of this technique must mature before it can be widely adopted. One of the areas of required improvement is imaging speed-most BCARS implementations involve sample rastering, which limits imaging speed. Beam scanning can provide faster BCARS imaging but presents some unique challenges. Here, we describe a beam-scanning BCARS microscopy system that improves spatial resolution twofold and imaging speed by fivefold over a previous beam-scanning implementation. These enhancements were enabled by an improvement in supercontinuum power and the use of a sCMOS camera for its high data transfer rate and low read noise. Implementation of the sCMOS camera required correction for the significant pixel-to-pixel background and photon response nonuniformity. We report on the method that we implemented for calibrating and correcting the pixel-to-pixel differences in sCMOS camera noise.

Broadband coherent anti-Stokes Raman scattering (BCARS) microscopy for rapid, label-free biological imaging.

Information about the environment can, in some cases, be transmitted to an organism's offspring by epigenetic inheritance. Here, we describe a novel form of epigenetics in  where information is transmitted between generations not by alterations in DNA, chromatin, or RNA, but by changes in the composition of lipids. Specifically, we delineate an environment-to-neuron-to-intestine-to-oocyte signalling axis that alters progeny thermotolerance by remodelling lipid provisioning to oocytes when animals detect social pheromones. Intergenerational information transmission via 'lipid memories' may represent an underappreciated form of epigenetics.

An intergenerational lipid memory of the social environment in .

One of the outstanding questions regarding liquid dynamics is the cause of the apparent mechanistic changes in relaxation at the characteristic temperatures  and , where, as the temperature is lowered, α relaxation times become super-Arrhenius, and the β relaxation bifurcates from α, respectively. Based on system-averaged picosecond-time scale dynamic signatures in five molecular liquids and a Kob-Andersen (KA) model system, we propose that these mechanistic changes arise from the percolation of distinct dynamic environments in the liquid, where the dynamic environment of a particle is defined by the number of structural excitations in its first solvation shell. Analysis of the KA system trajectories supports this idea and suggests that the most prominent effects can be understood in terms of environments that are mobile or immobile on a picosecond time scale. Further, the existence and percolation of these dynamic environments can account for many of the characteristic dynamic signatures of glass-forming liquids.

Article	Year
The polarizability response of a glass-forming liquid reveals intrabasin motion and interbasin transitions on a potential energy landscape. Soft matter\| PubMed ID: 32057068	2020
Spectroscopic coherent Raman imaging of Caenorhabditis elegans reveals lipid particle diversity. Nature chemical biology\| PubMed ID: 32572275	2020
Low-aberration high-speed-compatible optical delay line. Optics letters\| PubMed ID: 32630963	2020
Witnessing the survival of time-energy entanglement through biological tissue and scattering media. Biomedical optics express\| PubMed ID: 34221686	2021
Discriminating cell line specific features of antibiotic-resistant strains of Escherichia coli from Raman spectra via machine learning analysis. Journal of biophotonics\| PubMed ID: 35238159	2022
Excitations follow (or lead?) density scaling in propylene carbonate. The Journal of chemical physics\| PubMed ID: 36456241	2022
Identifying lipid particle sub-types in live with two-photon fluorescence lifetime imaging. Frontiers in chemistry\| PubMed ID: 37123874	2023
Toward Gene-Correlated Spatially Resolved Metabolomics with Fingerprint Coherent Raman Imaging. The journal of physical chemistry. B\| PubMed ID: 37311254	2023
Correction to "Toward Gene-Correlated Spatially Resolved Metabolomics with Fingerprint Coherent Raman Imaging". The journal of physical chemistry. B\| PubMed ID: 37555814	2023
Label-Free Functional Analysis of Root-Associated Microbes with Dynamic Quantitative Oblique Back-illumination Microscopy. Research square\| PubMed ID: 37961396	2023
Label-free functional analysis of root-associated microbes with dynamic quantitative oblique back-illumination microscopy. Scientific reports\| PubMed ID: 38461279	2024
Hybrid Lipid Nanocapsules: A Robust Platform for mRNA Delivery. ACS applied materials & interfaces\| PubMed ID: 38507686	2024
Broadband coherent anti-Stokes Raman scattering (BCARS) microscopy for rapid, label-free biological imaging. The Review of scientific instruments\| PubMed ID: 40257323	2025
An intergenerational lipid memory of the social environment in . bioRxiv : the preprint server for biology\| PubMed ID: 40501915	2025
A Case for Dynamic Percolation Underlying Mechanistic Crossovers in the Relaxation of Liquids. The journal of physical chemistry. B\| PubMed ID: 40527856	2025

Marcus T. Cicerone

Biography

JoVE Journal Publications

Other Publications