FLIM-FRET Imaging for Characterization of Protein-Protein Interactions in Live Bacteria

Abstract

Source: Manko, H. et al., FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria. J. Vis. Exp. (2020)

The video describes the FLIM-FRET imaging technique to determine the protein-protein interaction in live bacteria expressing cytoplasmic proteins labeled with fluorescent proteins, a donor eGFP, and acceptor mCherry. The combined technique also allows the quantification of the interacting proteins.

Protocol

1. Preparation of agarose pad (Figure 1) Place a microscope glass slide on a flat horizontal surface. Arrange two glass slides topped with two layers of adhesive tape on each side of the initial slide. NOTE: Keep a 1-2 mm space between the three aligned slides to prevent the melted agarose from eventually spreading on the slides with adhesive tape. Pipette and pour a droplet of 70 µL of 1% melted agarose on the glass slide. Add a fourth slide on the top to flatten the ag…

Representative Results

Figure 1.  Agarose pad preparation. Figure 2. Schematic representation of the interface of microscope control software

Materials

525/50 nm band-pass filter	F37-516, AHF, Germany
680 nm short pass filter	F37-516, AHF, Germany
Agarose	Sigma-Aldrich	A9539
Fiber-coupled avalanche photo-diode	SPCM-AQR-14- FC, Perkin Elmer
Glass coverslips (Thickness No. 1.5, 20×20mm	Knitel glass	MS0011
Microscope slides (25×75mm)	Knitel glass	MS0057
TCSPC module	SPC830, Becker & Hickl, Germany
Ti:Sapphire laser	Insight DeepSee, Spectra Physics