title: Specific sorting of single bacterial cells with microfabricated fluorescence-activated cell sorting and tyramide signal amplification fluorescence in situ hybridization.
abstract: When attempting to probe the genetic makeup of diverse bacterial communities that elude cell culturing, researchers face two primary challenges: isolation of rare bacteria from microbial samples and removal of contaminating cell-free DNA. We report a compact, low-cost, and high-performance microfabricated fluorescence-activated cell sorting (μFACS) technology in combination with a tyramide signal amplification fluorescence in situ hybridization (TSA-FISH) to address these two challenges. The TSA-FISH protocol that was adapted for flow cytometry yields a 10-30-fold enhancement in fluorescence intensity over standard FISH methods. The μFACS technology, capable of enhancing its sensitivity by ~18 dB through signal processing, was able to enrich TSA-FISH-labeled E. coli cells by 223-fold. The μFACS technology was also used to remove contaminating cell-free DNA. After two rounds of sorting on E. coli mixed with λ-phage DNA (10 ng/μL), we demonstrated over 100,000-fold reduction in λ-DNA concentration. The integrated μFACS and TSA-FISH technologies provide a highly effective and low-cost solution for research on the genomic complexity of bacteria as well as single-cell genomic analysis of other sample types.
authors.affiliation: Department of Bioengineering, University of California, San Diego, La Jolla, California 92093, USA. chc008@gmail.com
authors.forename: Chun H
authors.initials: CH
authors.lastname: Chen
authors.forename: Sung H
authors.initials: SH
authors.lastname: Cho
authors.forename: Hsin-I
authors.initials: HI
authors.lastname: Chiang
authors.forename: Frank
authors.initials: F
authors.lastname: Tsai
authors.forename: Kun
authors.initials: K
authors.lastname: Zhang
authors.forename: Yu-Hwa
authors.initials: YH
authors.lastname: Lo
mesh_terms: D004926:Escherichia coli / Q000166:cytology*; D005434:Flow Cytometry; D017404:In Situ Hybridization, Fluorescence; D014439:Tyramine / Q000032:analysis* / Q000737:chemistry
chemical_list: D014439:Tyramine