The overall goal of liquid phase scanning transmission electron microscopy is to observe structures and phenomena of nanomaterials in biological samples fully embedded in a liquid layer of up to several micrometers in thickness. This method can help answer key questions in the Materials Science field such as the behavior of nanomaterials in liquid and the study of biological samples in the natural liquid environment. The main advantage of this technique is that it provides nanoscale morphological information about specimens in liquid.

Generally, individuals new to this method will struggle because the loading of the specimen holder and image acquisition are not as simple as first done with electron microscopy. To begin the procedure, in a clean laminar flow hood, clean a light microscopy glass slide with a fiber-free tissue and pure ethanol. Place the slide on a cleanroom tissue in a Petri dish with a lid.

To manipulate the SiN microchips, using carbon-coated tweezers, grab the microchip firm but carefully on the long sides, keeping the SiN membrane facing upwards at all times. Using this technique, place five microchips without a spacer and five microchips with a 200-nanometer spacer on the glass slide. Close the Petri dish and bring the microchips to a fume hood.

Place the microchips in a beaker of HPLC-grade acetone with the membrane side up. Gently swirl the beaker for two minutes to remove the protective coating, being careful not to flip over the microchips. Then, quickly transfer the microchips to a beaker of pure ethanol.

Cover the beaker with aluminum foil. Gently swirl the beaker for two minutes to finish removing the coating and bring it in a closed Petri dish to the laminar flow hood. Place the microchips on a fresh cleanroom tissue, being careful not to flip over the microchips as they are released from the tweezers.

Allow the microchips to dry for a few minutes. And then place the microchips on the glass slide in the Petri dish. Close the Petri dish and bring the microchips to a plasma cleaner.

Place the glass slide and microchips in the plasma cleaner and run a five-minute cleaning program to remove hydrocarbons from the SiN membrane. Using a light microscope, inspect the microchips for ruptured membranes or dirt particles. Discard damaged or dirty microchips.

In the laminar flow hood, immobilize the microchips in a clean transport box with a sticky inner surface. Apply a one microliter droplet of a three molar citrate stabilized aqueous gold nanoparticle solution to the SiN membrane of each microchip without a spacer, and allow the solution to dry. Then, apply one microliter of deionized water to the membrane to wash away salt and surfactants.

After 30 seconds, use filter paper to carefully dab away the water and allow the microchips to dry. Place the tip of the liquid flow TEM holder under a binocular light microscope. Remove the titanium lid from the holder tip and place it on a sheet of aluminum foil.

Set up a microfluidic syringe pump with a one milliliter glass syringe containing 0.5 milliliters of HPLC-grade water. Connect the syringe to the flow system and start the pump. As the water is flushed through the system, check the lines for leaks or flow constrictions.

When pumping is complete, remove the rinse from the liquid cell compartment and dry the holder tip with filter paper. Inspect the holder tip with the light microscope. Dry the tip with cleanroom tissue and remove dust or fibers with clean PTFE-coated tweezers.

Inspect the holder tip lid, the O-ring, and the screws, and remove dust or fibers with PTFE-coated tweezers. Place the O-ring in the holder groups. Place the first screw and turn it a few times so that it just stays.

With clean curved tweezers, place the sample microchip in the pocket of the holder tip with the SiN membrane facing upwards. Use the binocular light microscope to check that the microchip is seated correctly. Place a 0.3 microliter drop of pure filtered water onto the sample microchip.

Holding the microchip in place with tweezers. Then, pick up a spacer microchip with curved tweezers held upside down. Carefully rotate the tweezers so the microchip membrane faces down.

Place the spacer microchip on the sample microchip. Place light reflecting material under the holder tip and check the microchip alignment under the binocular light microscope. Use tweezers to carefully adjust the microchips if the SiN windows are out of alignment.

Then, pick up the specimen chamber lid with tweezers. Turn the lid upside down, and without touching the microchips, rest the rear side of the lid on the holder tip. Place the remaining screw using tweezers and tighten both screws in an iterative way.

Tighten carefully until they meet resistance. Windows can break easily if the tightening is too strong. Start a four-microliter per minute liquid flow through the system and check for leaks in the holder tip.

Then, bring the holder to a vacuum pump station and perform a leak check. Ensure that the pressure reaches at least 10 to the negative fifth mbar within five minutes. Place the holder in its enclosure.

And bring the holder to the electron microscope. Set up the microscope in STEM mode. Measure the current density of the electron beam with a thin carbon film coated with gold nanoparticles as a reference sample without water.

Start the flow of pure water at a rate of no greater than two microliters per minute. Insert the liquid flow TEM holder into the vacuum load lock and begin evacuation. Ensure that the pressure decreases normally.

And then insert the TEM holder fully into the microscope. Once the pressure is sufficiently low, open the beam valve and insert the ADF detector. Set the microscope to continuous acquisition mode and translate the sample stage in the X and Y directions to find the SiN window.

Adjust the contrast and brightness so the edges of the window are clearly visible. Translate the stage in the X and Y directions so a corner of the window is at the center of the field of view. Then reset the objective lens.

Adjust the vertical position of the sample stage to coarse focus the corner. Tilt the stage by five degrees back and forth to check that the sample is at the eucentric height. Center the window corner in the field of view and then store the stage position in the software.

Translate the stage in the X and Y directions until the gold nanoparticles are visible. And then focus the objective lens. Note the current density and calculate the liquid cell thickness.

Translate the stage in the X and Y directions to locate an area with at least 20 gold nanoparticles. Set the parameters and acquire an image. Gold nanoparticles we're immobilized on a silicon nitride membrane and imaged with liquid phase STEM.

In pure water, the gold nanoparticles maintained their shape throughout imaging. Radiolysis products from the water could oxidize individual gold atoms which could eventually alter the shape of the nanoparticles. In another experiment, chloride ions we're introduced into the liquid phase.

The gold nanoparticles slowly dissolved through the experiment as the oxidized gold atoms formed soluble tetrachloroaureat. To investigate the movements of gold nanoparticles in water, in the subsequent experiment, the nanoparticles were not fully immobilized on the sample membrane. The gold nanoparticles agglomerated and upon reaching a critical cluster size, moved out of the field of view.

Once mastered, this technique can be done in two hours if it is performed properly. Several weeks of training will be required. While attempting this procedure, it's important to remember to work calmly and check vacuum tightness and liquid thickness.

After its development, this technique paved the way for researchers in Material Science, Chemistry, and Biology to explore nanoparticle growth and movement in liquid, the structure of nanoscale materials in liquid environments, and the functionality of proteins in mammalian cells. After watching this video, you should have a good understanding of how to conduct scanning transmission electron microscopy of gold nanoparticles embedded in a water layer including correct loading of the specimen holder and adjustment of the microscope. Don't forget that working with liquid in electron microscope can cause damage if the specimen holder is not correctly loaded.

So it is important to check for vacuum leakage prior to loading.