PMID- 38465924
OWN - NLM
STAT- MEDLINE
DCOM- 20240312
LR  - 20240312
IS  - 1940-087X (Electronic)
IS  - 1940-087X (Linking)
IP  - 204
DP  - 2024 Feb 23
TI  - Optical Photothermal Infrared - Fluorescence In Situ Hybridization (OPTIR-FISH).
LID - 10.3791/66562 [doi]
AB  - 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 (13)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.
FAU - Guo, Zhongyue
AU  - Guo Z
AD  - Department of Biomedical Engineering, Boston University.
FAU - Bai, Yeran
AU  - Bai Y
AD  - Neuroscience Research Institute, Department of Molecular, Cellular, and 
      Developmental Biology, University of California Santa Barbara; Photothermal 
      Spectroscopy Corp.; yrbai@ucsb.edu.
FAU - Pereira, Fatima C
AU  - Pereira FC
AD  - School of Biological Sciences, University of Southampton.
FAU - Cheng, Ji-Xin
AU  - Cheng JX
AD  - Department of Biomedical Engineering, Boston University; Department of Electrical 
      & Computer Engineering, Photonics Center, Boston University; jxcheng@bu.edu.
LA  - eng
PT  - Journal Article
PT  - Video-Audio Media
DEP - 20240223
PL  - United States
TA  - J Vis Exp
JT  - Journal of visualized experiments : JoVE
JID - 101313252
RN  - 0 (RNA, Ribosomal)
RN  - 0 (Oligonucleotide Probes)
RN  - 0 (Isotopes)
SB  - IM
MH  - Humans
MH  - In Situ Hybridization, Fluorescence/methods
MH  - *RNA, Ribosomal
MH  - *Bacteria/genetics
MH  - Oligonucleotide Probes
MH  - Isotopes
EDAT- 2024/03/11 12:42
MHDA- 2024/03/12 06:42
CRDT- 2024/03/11 09:03
PHST- 2024/03/12 06:42 [medline]
PHST- 2024/03/11 12:42 [pubmed]
PHST- 2024/03/11 09:03 [entrez]
AID - 10.3791/66562 [doi]
PST - epublish
SO  - J Vis Exp. 2024 Feb 23;(204). doi: 10.3791/66562.