title: Using total internal reflection fluorescence microscopy to visualize rhodopsin-containing cells.
abstract: Sunlight is captured and converted to chemical energy in illuminated environments. Although (bacterio)chlorophyll-based photosystems have been characterized in detail, retinal-based photosystems, rhodopsins, have only recently been identified as important mediators of light energy capture and conversion. Recent estimates suggest that up to 70% of cells in some environments harbor rhodopsins. However, because rhodopsin autofluorescence is low-comparable to that of carotenoids and significantly less than that of (bacterio)chlorophylls-these estimates are based on metagenomic sequence data, not direct observation. We report here the use of ultrasensitive total internal reflection fluorescence (TIRF) microscopy to distinguish between unpigmented, carotenoid-producing, and rhodopsin-expressing bacteria. Escherichia coli cells were engineered to produce lycopene, β-carotene, or retinal. A gene encoding an uncharacterized rhodopsin, actinorhodopsin, was cloned into retinal-producing E. coli. The production of correctly folded and membrane-incorporated actinorhodopsin was confirmed via development of pink color in E. coli and SDS-PAGE. Cells expressing carotenoids or actinorhodopsin were imaged by TIRF microscopy. The 561-nm excitation laser specifically illuminated rhodopsin-containing cells, allowing them to be differentiated from unpigmented and carotenoid-containing cells. Furthermore, water samples collected from the Delaware River were shown by PCR to have rhodopsin-containing organisms and were examined by TIRF microscopy. Individual microorganisms that fluoresced under illumination from the 561-nm laser were identified. These results verify the sensitivity of the TIRF microscopy method for visualizing and distinguishing between different molecules with low autofluorescence, making it useful for analyzing natural samples.
authors.affiliation: Department of Civil and Environmental Engineering, University of Delaware, Newark, Delaware, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Keffer
authors.affiliation: Delaware Biotechnology Institute, Newark, Delaware, USA.
authors.forename: C R
authors.initials: CR
authors.lastname: Sabanayagam
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: M E
authors.initials: ME
authors.lastname: Lee
authors.affiliation: Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: E F
authors.initials: EF
authors.lastname: DeLong
authors.affiliation: Research Institute for Limnology, University of Innsbruck, Mondsee, Austria.
authors.forename: M W
authors.initials: MW
authors.lastname: Hahn
authors.affiliation: Department of Civil and Environmental Engineering, University of Delaware, Newark, Delaware, USA jmaresca@udel.edu.
authors.forename: J A
authors.initials: JA
authors.lastname: Maresca
mesh_terms: D003116:Color; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D012243:Rhodopsin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D012243:Rhodopsin