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 ...

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.

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".

Transmembrane protein 135 (TMEM135) is a highly conserved 52 kDa protein with five predicted transmembrane domains that colocalizes with mitochondria and peroxisomes. Previous studies have shown that TMEM135 is involved in mitochondrial dynamics, thermogenesis, and lipid metabolism across multiple tissues and species; however, its role in the inner ear and auditory system remains unknown. We investigated the function of TMEM135 in hearing using wild-type (WT) and Tmem135 (FUN025) mutant mice on a CBA/CaJ background, a normal-hearing mouse strain. Although FUN025 mice displayed normal auditory brainstem response (ABR) thresholds at 1 month, we observed significantly elevated ABR thresholds at 8, 16, and 64 kHz by 3 months, which progressed to profound hearing loss by 12 months. Consistent with our auditory testing results, 13-month-old FUN025 mice exhibited a severe loss of outer hair cells and more modest changes in inner hair cell survival, spiral ganglion neuron density, and stria vascularis integrity in the cochlea. Our results using BaseScope RNA in situ hybridization indicate that TMEM135 is expressed in the inner hair cells, outer hair cells, supporting cells, and stria vascularis. Using Volocity software and Costes colocalization analysis, we found that TMEM135 closely colocalizes with mitochondria in hair cells. Together, these results demonstrate that the FUN025 mutation in Tmem135 causes progressive sensorineural hearing loss, and suggest that TMEM135 is crucial for maintaining key cochlear cell types and normal sensory function in the aging cochlea.

A mutation in Tmem135 causes progressive sensorineural hearing loss.

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.

Article	Year
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
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
A mutation in Tmem135 causes progressive sensorineural hearing loss. Hearing research\| PubMed ID: 39970612	2025
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

Wei-Wen Chen

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