Ni H et al. (2021) Anal Chem "Multiwindow SRS Imaging Using a Rapid Widely Tunable Fiber Laser."

Ni H, Cheng JX, Zhang M, Zhu Y, Tan Y, Lin P, Wang Z, Zhan Y

[Anal Chem, 2021]

Spectroscopic stimulated Raman scattering (SRS) imaging has become a useful tool finding a broad range of applications. Yet, wider adoption is hindered by the bulky and environmentally sensitive solid-state optical parametric oscillator (OPO) in a current SRS microscope. Moreover, chemically informative multiwindow SRS imaging across C-H, C-D, and fingerprint Raman regions is challenging due to the slow wavelength tuning speed of the solid-state OPO. In this work, we present a multiwindow SRS imaging system based on a compact and robust fiber laser with rapid and wide tuning capability. To address the relative intensity noise intrinsic to a fiber laser, we implemented autobalanced detection, which enhances the signal-to-noise ratio of stimulated Raman loss imaging by 23 times. We demonstrate high-quality SRS metabolic imaging of fungi, cancer cells, and <i>Caenorhabditis elegans</i> across the C-H, C-D, and fingerprint Raman windows. Our results showcase the potential of the compact multiwindow SRS system for a broad range of applications.

Li M et al. (2013) Anal Chem "Stable isotope probing and Raman spectroscopy for monitoring carbon flow in a food chain and revealing metabolic pathway."

Huang WE, Jousset A, Li M, Gibson CM, Fowler PW

[Anal Chem, 2013]

Accurately measuring carbon flows is a challenge for understanding processes such as diverse intracellular metabolic pathways and predator-prey interactions. Combined with stable isotope probing (SIP), single-cell Raman spectroscopy was demonstrated for the first time to link the food chain from carbon substrate to bacterial prey up to predators at the single-cell level in a quantitative and nondestructive manner. Escherichia coli OP50 with different (13)C content, which were grown in a mixture of (12)C- and fully carbon-labeled (13)C-glucose (99%) as a sole carbon source, were fed to the nematode. The (13)C signal in Caenorhabditis elegans was proportional to the (13)C content in E. coli. Two Raman spectral biomarkers (Raman bands for phenylalanine at 1001 cm(-1) and thymine at 747 cm(-1) Raman bands), were used to quantify the (13)C content in E. coli and C. elegans over a range of 1.1-99%. The phenylalanine Raman band was a suitable biomarker for prokaryotic cells and thymine Raman band for eukaryotic cells. A biochemical mechanism accounting for the Raman red shifts of phenylalanine and thymine in response to (13)C-labeling is proposed in this study and is supported by quantum chemical calculation. This study offers new insights of carbon flow via the food chain and provides a research tool for microbial ecology and investigation of biochemical pathways.

Charan S et al. (2011) Chemistry "Development of lipid targeting Raman probes for in vivo imaging of Caenorhabditis elegans."

Kuo CW, Chien FC, Charan S, Singh N, Chen P

[Chemistry, 2011]

A simple, sensitive, and highly specific lipid targeting Raman probe (Nile red coated silver nanoparticles) has been developed to image living nematode Caenorhabditis elegans (C. elegans). Our idea of imaging lipids in C. elegans is to combine the specificity of the fluorescent dye, Nile red, and the highly enhanced Raman scattering on the silver nanoparticles. Our strategy involves the fabrication of a lipid targeting probe, which is incorporated into the intracellular intestinal granules of C. elegans by incubating these worms in the solution containing Raman probes, resulting in an uptake and subsequent incorporation of these Raman probes into the intestinal granule, thus allowing fast visualization of lipid droplets through a conventional confocal imaging technique.

Shi L et al. (2018) Nat Commun "Optical imaging of metabolic dynamics in animals."

Zhang L, Chen Z, Silveira ES, Targoff K, Shi L, Zheng C, Min W, de Sena-Tomas C, Shen Y, Wei M, Liu C

[Nat Commun, 2018]

Direct visualization of metabolic dynamics in living animals with high spatial and temporal resolution is essential to understanding many biological processes. Here we introduce a platform that combines deuterium oxide (D₂O) probing with stimulated Raman scattering (DO-SRS) microscopy to image in situ metabolic activities. Enzymatic incorporation of D₂O-derived deuterium into macromolecules generates carbon-deuterium (C-D) bonds, which track biosynthesis in tissues and can be imaged by SRS in situ. Within the broad vibrational spectra of C-D bonds, we discover lipid-, protein-, and DNA-specific Raman shifts and develop spectral unmixing methods to obtain C-D signals with macromolecular selectivity. DO-SRS microscopy enables us to probe de novo lipogenesis in animals, image protein biosynthesis without tissue bias, and simultaneously visualize lipid and protein metabolism and reveal their different dynamics. DO-SRS microscopy, being noninvasive, universally applicable, and cost-effective, can be adapted to a broad range of biological systems to study development, tissue homeostasis, aging, and tumor heterogeneity.

Wang MC et al. (2011) Nat Methods "RNAi screening for fat regulatory genes with SRS microscopy."

Wang MC, Min W, Freudiger CW, Xie XS, Ruvkun G

[Nat Methods, 2011]

Identification of genes regulating fat accumulation is important for basic and medical research; genetic screening for those genes in Caenorhabditis elegans, a widely used model organism, requires in vivo quantification of lipids. We demonstrated RNA interference screening based on quantitative imaging of lipids with label-free stimulated Raman scattering (SRS) microscopy, which overcomes major limitations of coherent anti-Stokes Raman scattering microscopy. Our screening yielded eight new genetic regulators of fat storage.

Weeks T et al. (2010) J Biophotonics "Signal generation and Raman-resonant imaging by non-degenerate four-wave mixing under tight focusing conditions."

Weeks T, Wachsmann-Hogiu S, Huser T, Schie IW

[J Biophotonics, 2010]

The authors demonstrate Raman-resonant imaging based on the simultaneous generation of several nonlinear frequency mixing processes resulting from a 3-color coherent anti-Stokes Raman scattering (CARS) experiment. The interaction of three coincident short-pulsed laser beams simultaneously generates both 2-color (degenerate) CARS and 3-color (non-degenerate) CARS signals, which are collected and characterized spectroscopically - allowing for resonant, doubly-resonant, and non-resonant contrast mechanisms. Images obtained from both 2-color and 3-color CARS signals are compared and found to provide complementary information. The 3-color CARS microscopy scheme provides a versatile multiplexed modality for biological imaging, which may extend the capabilities of label-free non-linear microscopy, e.g. by probing multiple Raman resonances.

Rocha-Mendoza I et al. (2015) Biomed Opt Express "Rapid spontaneous Raman light sheet microscopy using cw-lasers and tunable filters."

Marro M, Licea-Rodriguez J, Olarte OE, Plata-Sanchez M, Rocha-Mendoza I, Loza-Alvarez P

[Biomed Opt Express, 2015]

We perform rapid spontaneous Raman 2D imaging in light-sheet microscopy using continuous wave lasers and interferometric tunable filters. By angularly tuning the filter, the cut-on/off edge transitions are scanned along the excited Stokes wavelengths. This allows obtaining cumulative intensity profiles of the scanned vibrational bands, which are recorded on image stacks; resembling a spectral version of the knife-edge technique to measure intensity profiles. A further differentiation of the stack retrieves the Raman spectra at each pixel of the image which inherits the 3D resolution of the host light sheet system. We demonstrate this technique using solvent solutions and composites of polystyrene beads and lipid droplets immersed in agar and by imaging the C-H (2800-3100cm(-1)) region in a C. elegans worm. The image acquisition time results in 4 orders of magnitude faster than confocal point scanning Raman systems, allowing the possibility of performing fast spontaneous Raman-3D-imaging on biological samples.

Zhang D et al. (2011) J Phys Chem Lett "Highly Sensitive Vibrational Imaging by Femtosecond Pulse Stimulated Raman Loss."

Slipchenko MN, Cheng JX, Zhang D

[J Phys Chem Lett, 2011]

Nonlinear vibrational imaging of live cells and organisms is demonstrated by detecting femtosecond pulse stimulated Raman loss. Femtosecond pulse excitation produced a 12 times larger stimulated Raman loss signal than picosecond pulse excitation. The large signal allowed real-time imaging of the conversion of deuterated palmitic acid into lipid droplets inside live cells, and three-dimensional sectioning of fat storage in live C. elegans. With the majority of the excitation power contributed by the Stokes beam in the 1.0 to 1.2 m wavelength range, photodamage of biological samples was not observed.

Mugova F et al. (2017) Environ Toxicol Chem "PHENOTYPIC RESPONSES IN CAENORHABDITIS ELEGANS FOLLOWING CHRONIC LOW-LEVEL EXPOSURES TO INORGANIC AND ORGANIC COMPOUNDS."

Riding MJ, Spurgeon D, Mugova F, Read DS, Tyne W, Martin FL, Svendsen C

[Environ Toxicol Chem, 2017]

Responses of organisms to sub-lethal exposure of environmental stressors can be difficult to detect. We investigated phenotypic changes in the tissue of Caenorhabditis elegans via Raman spectroscopy, as well as survival and reproductive output when exposed to chronic low dose of exposure metals (copper, zinc or silver), an herbicide (diuron) and a pesticide (imidacloprid). Raman spectroscopy measures changes in phenotype by providing information about the molecular composition and relative abundance of biomolecules. Multivariate analysis was used to evaluate the significance of treatment phenotype segregation plots compared to controls. Dose-dependent responses were observed for copper, zinc, silver and diuron while imidacloprid exposure resulted in a small response over the tested concentrations. Concentration-dependent shifts in nematode biomolecular phenotype were observed for copper. Despite having a dose-dependent reproductive response, silver, diuron and imidaclorprid had inconsistent biological phenotype patterns. In contrast, there was a clear stepwise change between low concentrations (0.00625 - 0.5mg/L) and higher concentration (1-2mg/L) of ionic zinc. The findings demonstrate that measuring phenotypic responses via Raman spectroscopy can provide an insight into the biomolecular mechanisms of toxicity. Despite the lack of consistency between survival and Raman-measured phenotypic changes, the results supports the effectiveness of Raman spectroscopy and multivariate analysis to detect sub-lethal responses of chemicals on whole organisms and to identify toxic effect thresholds. This article is protected by copyright. All rights reserved.

Freudiger CW et al. (2011) Nat Photonics "Highly specific label-free molecular imaging with spectrally tailored excitation stimulated Raman scattering (STE-SRS) microscopy."

Freudiger CW, Xie XS, Dantus M, Xu B, Min W, Holtom GR

[Nat Photonics, 2011]

Label-free microscopy with chemical contrast and high acquisition speed up to video-rate has recently been made possible by stimulated Raman scattering (SRS) microscopy. While SRS imaging offers superb sensitivity, the spectral specificity of the original narrowband implementation is limited, making distinguishing chemical species with overlapping Raman bands difficult. Here we present a highly specific imaging method that allows mapping of a particular chemical species in the presence of interfering species based on tailored multiplex excitation of its vibrational spectrum. This is done by spectral modulation of a broadband pump beam at a high-frequency (>1MHz), allowing detection of the stimulated Raman gain signal of the narrowband Stokes beam with high sensitivity. Using the scheme, we demonstrate quantification of cholesterol in the presence of lipids, and real-time three-dimensional spectral imaging of protein, stearic acid and oleic acid in live C. elegans.