Using total internal reflection fluorescence microscopy to visualize rhodopsin-containing cells.

Area of Science:

• Microbiology
• Biophysics
• Microscopy

Background:

• Retinal-based photosystems (rhodopsins) are increasingly recognized for light energy capture, potentially present in up to 70% of cells in some environments.
• Previous estimates relied on metagenomic data due to rhodopsins' low autofluorescence, hindering direct observation and differentiation from other molecules like carotenoids.

Purpose of the Study:

• To develop and validate a sensitive method for directly visualizing and distinguishing rhodopsin-expressing microorganisms.
• To assess the utility of this method in analyzing natural environmental samples.

Main Methods:

• Engineered Escherichia coli to express carotenoids or the uncharacterized actinorhodopsin.
• Utilized ultrasensitive total internal reflection fluorescence (TIRF) microscopy with a 561-nm excitation laser.
• Applied PCR and TIRF microscopy to analyze water samples from the Delaware River.

Main Results:

• TIRF microscopy successfully differentiated between unpigmented, carotenoid-producing, and rhodopsin-expressing E. coli cells.
• The 561-nm laser specifically illuminated rhodopsin-containing cells, confirming actinorhodopsin production and correct membrane incorporation.
• Rhodopsin-containing microorganisms were identified in Delaware River water samples using TIRF microscopy.

Conclusions:

• Ultrasensitive TIRF microscopy is a sensitive and effective method for visualizing and distinguishing molecules with low autofluorescence, such as rhodopsins.
• This technique enables direct observation of rhodopsin-containing microorganisms in environmental samples, overcoming limitations of previous estimation methods.