The protocol presents how the combination of liquid cell TEM and light illumination can be used to observe the structural changes in the bacteria encapsulated with photosensitizer upon light illumination. The main advantage of this technique is its simpleness. The protocol does not require difficult sample preparation and provides sufficient encapsulation of bacteria with photosensitizer solution.
To begin, modify the transmission electron microscope by connecting the optical fiber to the microscope column at the top of the objective lens. Allow a few centimeters of space between the objective lens and the condenser lens, plus a free slot for accessories. Place two untreated TEM grids covered with amorphous carbon between two crossed tweezers, facing the carbon coated side to the top.
Hold the grids as close to the edge as possible. Put four microliters of bacteria solution on one of the grids. The optical density of the sample must be in the range of five to 10.
Wait for 60 seconds, and carefully blot the liquid using filter paper. Try to spread the liquid throughout the entire grid surface during the process. Put four microliters of photosensitizer on the same grid.
After five seconds, blot out the liquid. Leave a very thin layer of liquid on the substrate. Quickly place the dry grid on top of the one covered with liquid.
Gently move the upper grid to the edge of the second one, and squeeze the substrates at the edges using tweezers. Carefully repeat the squeeze. Leave the liquid cell between the tweezers for one minute.
Insert the sample into the TEM holder right after finishing the preparation. After inserting the sample into the microscope, start the observations in low magnification mode to find a suitable observation area. During the observations keep the electron dose as low as possible.
Expand the exposure time. Find the cells surrounded by liquid at the appropriate magnification with extended exposure time, and capture the first image immediately. Keep the electron dose constant, and collect images every 30 seconds to observe the changes.
Carefully examine the images and calculate the total electron dose that corresponds to the first visible changes in the cells. Before starting the experiment, set the laser light intensity by adjusting the current value. Insert the sample into the microscope and find the observation area.
Capture the first image of the cell before starting illumination of the sample, and use the beam blanker to turn off the electron beam to avoid the effects of electron irradiation. Turn on the laser. Start with a short illumination time, as the laser light has relatively high intensity.
After that time, turn off the light source. Turn off the beam blanker, and capture the image immediately. If possible, use an automatic algorithm to expose the sample only for the time needed for taking the image.
Carefully measure the electron irradiation time to calculate the electron dose used for imaging. A notable degradation of the outer layer of the cell caused by the reactive oxygen species generated by lighter radiation of the photosensitizer after one minute is observed. Further light illumination made the changes more visible.
Proper observation spots with enough liquid can be easily found at low magnification, as these areas appear darker and blurred. The liquid covered bacteria are less visible than the dry ones, but can still be distinguished. It's useful to look at the liquid boundary during the imaging, and its movement can be easily detected, which implies that the chosen area might be unsuitable and unstable.
Another issue during observations is the evaporation of the water and precipitation of the solution which can occur in random areas of the sample, including the areas surrounding the bacteria. Encapsulation of bacteria can be also performed using graphene and silicon nitrate membranes. The sample preparation is more difficult, but the stability of the liquid cell will be better.
The presented method can be used for observations of light-induced reactions on liquid. For example, the influence of light on metal materials, light-induced catalysts and photochemical reactions.
