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Optical Photothermal Infrared - Fluorescence In Situ Hybridization (OPTIR-FISH).
- Source :
-
Journal of visualized experiments : JoVE [J Vis Exp] 2024 Feb 23 (204). Date of Electronic Publication: 2024 Feb 23. - Publication Year :
- 2024
-
Abstract
- Understanding the metabolic activities of individual cells within complex communities is critical for unraveling their role in human disease. Here, we present a comprehensive protocol for simultaneous cell identification and metabolic analysis with the OPTIR-FISH platform by combining rRNA-tagged FISH probes and isotope-labeled substrates. Fluorescence imaging provides cell identification by the specific binding of rRNA-tagged FISH probes, while OPTIR imaging provides metabolic activities within single cells by isotope-induced red shift on OPTIR spectra. Using bacteria cultured with <superscript>13</superscript> C-glucose as a test bed, the protocol outlines microbial culture with isotopic labeling, fluorescence in situ hybridization (FISH), sample preparation, optimization of the OPTIR-FISH imaging setup, and data acquisition. We also demonstrate how to perform image analysis and interpret spectral data at the single-cell level with high throughput. This protocol's standardized and detailed nature will greatly facilitate its adoption by researchers from diverse backgrounds and disciplines within the broad single-cell metabolism research community.
Details
- Language :
- English
- ISSN :
- 1940-087X
- Issue :
- 204
- Database :
- MEDLINE
- Journal :
- Journal of visualized experiments : JoVE
- Publication Type :
- Academic Journal
- Accession number :
- 38465924
- Full Text :
- https://doi.org/10.3791/66562