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

Place a microscope glass slide on a flat horizontal surface, and arrange two glass slides topped with two layers of adhesive tape around it. Pipette a 70-microliter droplet of 1% melted agarose onto the glass slide, and place a fourth slide on top to flatten the agarose droplet. Press down gently for about a minute.

Take off the upper slide and drop about three microliters of bacteria in three to four spots at different locations on the agarose pad. Cover the agarose pad with a microscopy glass coverslip, and fix it with melted paraffin to seal it onto the glass slide, starting with the four corners.

Place the microscopy slide on the stage, with the coverslip facing the objective. Turn the filter cube turret to select the eGFP cube, and open the fluorescence lamp shutter. Then, send the fluorescence light towards the eyepiece of the microscope. Focus the objective on the bacteria using the microscope knob.

Select a region of interest in the sample using the joystick that controls the motorized stage. Send the fluorescence emission path back towards the detector, then, turn back the filter cube turret to select the dichroic cube for the 930-nanometer laser, and set the laser power to 20 milliwatts. Set the size of the region of interest to 30 micrometers, which adjusts the voltage operating the galvo-mirrors and defines the range of their movements.

Turn on the detector, and start scanning the sample. Choose the field of view for imaging, by finally moving the stage from the computer interface. This can be done on the setup by moving the cross on the image in the microscope control software which will define the new center of the image, and pressing "Move stage."

A good field of view for acquisition should contain 10 to 30 immobile bacteria, all in focus. If interested in extracting single cells' FLIM-FRET data, ensure that the bacteria are well individualized.