Mid-Infrared Photothermal–Fluorescence In Situ Hybridization for Functional Analysis and Genetic Identification of Single Cells

Abstract

Simultaneous identification and metabolic analysis of microbes with single-cell resolution and high throughput are necessary to answer the question of "who eats what, when, and where" in complex microbial communities. Here, we present a mid-infrared photothermal−fluorescence in situ hybridization (MIP−FISH) platform that enables direct bridging of genotype and phenotype. Through multiple improvements of MIP imaging, the sensitive detection of isotopically labeled compounds incorporated into proteins of individual bacterial cells became possible, while simultaneous detection of FISH labeling with rRNA-targeted probes enabled the identification of the analyzed cells. In proof-of-concept experiments, we showed that the clear spectral red shift in the protein amide I region due to incorporation of 13 C atoms originating from 13 C-labeled glucose can be exploited by MIP−FISH to discriminate and identify 13 C-labeled bacterial cells within a complex human gut microbiome sample. The presented methods open new opportunities for single-cell structure− function analyses for microbiology.

Stable Isotope Probing-nanoFTIR for Quantitation of Cellular Metabolism and Observation of Growth-dependent Spectral Features

“…In a similar fashion, other isotope-labelling based analytical techniques have also provided insights into the ecological function of the individual cells that comprise complex microbial communities. [1,35,54] Additionally, quantification of cellular heterogeneity using SIP-nanoFTIR has applications in biopharmaceutical and medicinal fields, complementing studies such as the characterization of high-value eukaryotic proteins, [55] the determination of patient-specific drug effectivity, [56] in-situ detection of antibiotic-resistant bacteria or biofilms, [57,58] and the metabolic tracing of cancer cells [59,60] or the human microbiota. [61,62] Isotopically induced changes in the amide I peak shape arose both in response to environmental factors and were dependent on the cellular growth phase of harvested cultures.…”

“…[32,33] Metabolic profiling of microorganisms has been achieved at the single-cell level using both SIP-Raman spectroscopy [34][35][36] and mid-IR photothermal-SIPfluorescence in situ hybridization. [1] To further expand the range of single-cell spectroscopic techniques, here we introduce SIP-nanoFTIR as a novel technique that combines the super-resolution and molecular specificity of nanoFTIR with the integration of detectable stable isotopes into metabolically active cells. High spectroscopic contrast between sample and substrate allows for resolution at the single-cell level.…”

“…Single-cell analyses have made tremendous progress in recent years, providing new insights into fundamental aspects of cell behavior such as differential growth, responses to external stimuli, cell sensing, intercellular communication and interactions. Intercellular heterogeneity (both phenotypic and metabolic) is a consistent feature of cellular populations, ranging from different individuals within clonal populations, [1] multi-species ensembles, [2, 3] and of course multicellular organisms. Even when genetically identical cells are cultured under the same conditions, they may display different phenotypes.…”

Rapid Detection of Broad-Spectrum Pathogenic Bacteria Based on Highly Sensitive Proton Response of the Nucleic Acid Amplification SPQC Platform

“…6,7 Nucleic acid-based molecular biology methods such as gene sequencing demand bulky instruments and trained operators, restricting their application to centralized laboratories. 8,9 Despite the ongoing evolution of molecular hybridization, 10,11 DNA chips, 12,13 and nucleic acid biosensors, 14,15 technologies are advancing toward expedited response, reduced costs, and simplified miniaturization, the absence of a universal target compels patients with bacterial infections to undergo a complex pathogen identification procedure, thus delaying treatment.…”

“…The culture method continues to be the gold standard for the clinical detection of bacteria in hospitals. However, this method has significant limitations including a slow process time, low detection rates, which are a result of generational time delay, and the inability to cultivate certain bacteria. , Time-of-flight mass spectrometry provides faster bacterial identification but necessitates pure cultured bacteria and expensive equipment, hindering point-of-care testing (POCT). , Furthermore, immunological methods pose challenges due to low detection sensitivity and difficulties in procuring monoclonal antibodies. , Nucleic acid-based molecular biology methods such as gene sequencing demand bulky instruments and trained operators, restricting their application to centralized laboratories. , Despite the ongoing evolution of molecular hybridization, , DNA chips, , and nucleic acid biosensors, , technologies are advancing toward expedited response, reduced costs, and simplified miniaturization, the absence of a universal target compels patients with bacterial infections to undergo a complex pathogen identification procedure, thus delaying treatment.…”