An imaging platform for investigating microbiomes in medical and environmental samples can perform high-throughput metabolism and identity analyses with single-cell resolution. Called SRS-FISH, or stimulated Raman scattering two-photon fluorescence in situ hybridization, the technique is the result of a collaborative effort among researchers at Boston University, the University of Vienna, and Aalborg University. The imaging speed of SRS-FISH is 10 to 100 ms per cell. Using SRS-FISH, the researchers detected metabolic responses of more than 30,000 individual cells to various mucosal sugars in the human gut microbiome. To detect low concentrations of metabolites inside cells with diameters around 1 μm, the researchers developed a protocol that maximizes the isotope label content in cells and exploits the intense stimulated Raman scattering (SRS) signal from the Raman band used for isotope detection. They created a system that uses a single laser source to implement SRS metabolic imaging with two-photon FISH. These improvements collectively led to SRS-FISH, an integrative, high-throughput platform that combines the advantages of SRS for single-cell stable isotope probing with two-photon FISH for identifying cells quickly and with a high level of sensitivity. An international team of researchers has introduced an integrative, high-throughput platform that combines the advantages of Raman scattering microscopy for single-cell stable isotope probing with two-photon fluorescence in situ hybridization for identifying cells quickly and with a high level of sensitivity. Courtesy of Xiaowei Ge, Fatima et al., PNAS 2022,119,e2203519119. Due to its exceptionally high imaging speed, SRS-FISH could fill a gap among the tools available for linking metabolism and identity in complex microbial communities. “SRS-FISH enables correlative imaging of cell identity and metabolism at a speed of 10 to 100 ms per cell. In comparison, it takes about 20 s to record a Raman spectrum from a single cell in a conventional spontaneous Raman FISH experiment,” professor Ji-Xin Cheng said. “Until now, microbiologists have only been able to study the function of the most abundant microbes at the single-cell level,” professor Michael Wagner, who led the research on the Austrian side, said. “This enormous increase in speed means that less abundant representatives of microbiomes are now also accessible for single-cell isotope measurements.” To demonstrate the capability of SRS-FISH to link phylogenetic identity (genotype) with metabolic activity (phenotype), the researchers incubated samples in heavy water (i.e., deuterium oxide), enabling the deuterium to be incorporated into metabolically active cells. Deuterium incorporation from heavy water can be combined with Raman-based approaches like SRS-FISH to track metabolic activity at the single-cell level in response to a variety of compounds. SRS-FISH enabled fast, sensitive determination of the deuterium content of individual cells, while simultaneously revealing their phylogenetic identity. The researchers applied this approach to complex microbial communities and locally tracked the metabolic responses of two phylogenetic groups of microbes in the human gut, Bacteroidales and Clostridia. Metabolism and identity analyses of multiple samples revealed that Clostridia may contribute more to mucosal sugar degradation than previously thought. Diagram of the SRS and FISH combination. The high-speed, high-solution chemical mapping delivered by SRS provides information on metabolism, and the method provides quantitative analysis and fluorescence technique compatibility. FISH delivers in situ capabilities and is a nondestructive method. Courtesy of Xiaowei Ge, Fatima et al., PNAS 2022,119,e2203519119. The results demonstrate the potential of SRS-FISH to identify the metabolism of specific microbes in microbiomes. “The application of SRS-FISH to the gut microbiome demonstrates the suitability of our approach to link identity to metabolism within complex microbial communities. At the same time, it revealed interesting findings related to mucosal sugar foraging in the human gut,” Cheng said. Wagner said that insights into the function of the human microbiome are essential to better understand its role in human health and develop more targeted probiotics. The researchers believe that the SRS-selective scanning of FISH-targeted cells could further improve the throughput of SRS-FISH, especially when the number of taxa of interest is extremely low. Laser equipment with upgraded wavelength tuning could also potentially increase throughput. The sensitivity and resolution of SRS-FISH could be improved by implementing visible SRS. SRS-FISH can be applied to a broad range of environmental samples, from marine sediments to soil. It can be used with samples where some autofluorescence background is present, because SRS is resilient to sample autofluorescence. Additionally, the technique is applicable to eukaryotes in addition to microbiome samples. “Conventional approaches to imaging individual microbes in their environment, such as single-cell isotope probing, allow only a few cells or samples to be analyzed at one time,” Cheng said. “SRS-FISH allows multiple samples to be scanned with high speed and sensitivity, revealing details that existing low-throughput methods might miss and providing a more complete understanding of the function of microbes in their natural environment.” The research was published in PNAS (www.doi.org/10.1073/pnas.2203519119).