In Situ Characterization of Boehmite Particles in Water Using Liquid SEM

The overall goal of this in situ liquid scanning electron microscopy imaging procedure is to provide technical knowhow for efficient imaging and analysis of particles in liquids in conventional high-vacuum SEM using a vacuum-compatible microfluidic module. This method can help answer key questions in the microscopy and microanalysis field such as how to characterize evolving particle size and morphology responding to the environmental changes. The main advantage of this technique is that we can directly visualize polydispersed particles in liquid without drying or freezing the sample in high-vacuum mode and also without resorting to environmental SEM.

The implication of this technique extend toward the efficient analysis or the various liquid material and the biological samples using SEM because many of them only function or exist in liquid. Visual demonstration of this method is critical as simple mounting and in situ imaging steps are difficult to learn because they are different from regular solid samples. Begin this procedure with carbon coating of the system for analysis at the liquid vacuum interface, or SALVI device, as described in the tech's protocol.

Mounting the microfluidic device on the SEM stage is quite critical for the success of in situ liquid SEM analysis. Make sure the device is properly grounded and is secured to minimize the charging effect before the experiment. To mount the SALVI device onto the SEM stage, first open the SEM specimen chamber door carefully once venting has completed.

Select the SEM specimen holder. Fix the holder onto the center of the stage using the appropriate bolt and hex wrench. Place two strips of double sided carbon tape on the specimen holder.

Then stick the SALVI device onto the carbon tape placed on the holder with the silicon nitride membrane side facing up. Securely immobilize the SALVI onto the specimen holder using an additional two strips of single sided copper tape to bind the SALVI PDMS block to the SEM specimen holder. In addition use the copper tapes to connect the silicon nitride frame and the metal specimen holder.

Make sure that the tape does not completely cover the silicon nitride membrane. To pump down the specimen chamber, close the specimen chamber door, select the high vacuum mode on the SEM software GUI under the beam control page, click the pump button on the beam control page to start vacuuming. Apply pressure by hand to the chamber door until the desired vacuum is established.

Next make apertures in the silicon nitride membrane using focused ion beam, or FIB, as described in the tech's protocol. Following the procedure, turn off both the electron beam and ion beam to vent the chamber by clicking beam on when the corresponding beam imaging area is activated. Click vent on the beam control page to vent the specimen chamber.

Carefully open the SEM chamber door after it is fully vented leaving the SALVI device as it is on the stage. Draw one milliliter of deionized water into a sterile syringe and connect the syringe with the inlet of the microfluidic device using a polytetrafluoroethylene tubing adapter fitting. Slowly inject the liquid for three to five minutes.

Repeat this process three times using one milliliter of 10 micrograms per milliliter aluminum oxyhydroxide to ensure the concentration of the sample is not diluted by the preloaded deionized water. After the injection, remove the syringe. Connect the inlet and outlet of SALVI using polyether ether ketone union.

Dry any liquid outside the SALVI with lab wipes. If there are any bubbles within the tubing or microchannel, redo the sample injection until no bubbles are seen within the tubing. To take images using the ETD and secondary electron mode, first close the specimen chamber door.

After establishing the desired vacuum on the chamber door as before, activate the electron beam imaging area by clicking the pause icon on the toolbar. Turn on the electron beam by clicking the beam on button on the beam control page. Select the ETD and the secondary electron mode for imaging from the detector's drop-down menu.

In selecting the optimal conditions it is really important to be able to attain high-quality images. In this work, we will show you a set of conditions suitable for a secondary electron imaging of the light elements like aluminum silicon. When analyzing other elements like iron and nickel, operation conditions should be modified to optimize the secondary electron imaging.

Set the accelerating voltage to eight kiloelectron-volts and the beam current to approximately 0.47 nanoamps from the corresponding list boxes displayed on the GUI toolbar at the electron beam imaging area. Set the working distance as seven millimeters by typing seven into the text box of the coordinate Z on the navigation page when the actual distance is selected. Magnify the feature to 1, 000 X and twist the contrast, brightness, coarse and fine knobs on the manual user interface to optimize the image of the particle feature.

Center the first hole in the live image of the electron beam imaging area by twisting the X and Y shift knobs on the manual user interface board, enlarge the images with particles to magnification 200, 000 X by twisting the magnification knob on the manual user interface board. Select the screen resolution to 1, 024 by 884 from the list box on the tool bar. Then set the scan rate as 30 microseconds from the list box on the tool bar.

Press the F4 key to take the snapshot of the current image shown in the electron beam imaging area. Press the control and S keys to save the image as a TIF file to the desired location with the defined file name including an incremental number. Zoom out by twisting the magnification knob to locate the next adjacent hole.

Repeat these operations to image the aluminum oxyhydroxide particles in the rest of the holes. To conduct elemental analysis using energy-dispersive x-ray spectroscopy, or EDX, insert the energy-dispersive spectroscopy detectors into the chamber. Select the ETD on the microscope control monitor.

Then select the secondary electron mode for viewing the sample on the electron beam imaging area and set the parameters as before. Enlarge the aluminum oxyhydroxide particles in each hole with a magnification of 200, 000 X by twisting the magnification knob on the manual user interface board. Next open the associated EDAC software.

Click start recording new spectra in the user interface to collect the EDX spectrum. Select peak ID to choose the probable elements of the spectrum. Then type in the observed elements into the element field.

Click add to apply the element to the spectrum. Click on file and then click save as. Save the spectral data in CSV format using the desired file name for plotting using a graphing software.

After finishing the imaging and spectrum recording for each of the holes, turn off the electron beam by clicking the beam on button on the beam control page when the electron beam imaging area is on. Vent the SEM chamber by clicking vent on the same page. Carefully take the sample off the stage by removing all the tapes after the chamber door is open.

Repeat the procedure to conduct the control experiments using deionized water and an empty microchannel. Finally plot the EDX spectrum as described in the tech's protocol. Using the SALVI microfluidic interface, the electron beam can directly bombard the small, micrometer sized aperture with liquid underneath.

Boehmite particles in deionized water were compared with the deionized water control and empty channel control in their secondary electron images. This provides direct evidence of particle secondary electron imaging of Boehmite particles in the liquid. Visualization of the particle in deionized water within the one micrometer hole demonstrates the feasibility to observe particles in liquid in situ.

In the Boehmite sample, a strong aluminum peak is observed. In contrast, only a strong oxygen peak is observed in the deionized sample indicating the observation of water. In the empty channel, residual of the gallium-focused ion beam is seen.

The oxygen peak is quite small because there is not any water inside. Once mastered this technique can be done in a couple of hours if it is performed properly. Generally individuals new to this method will struggle because it is difficult to directly visualize liquids in high-vacuum SEM.

After watching this video, you should have a good understanding of how to characterize particles in liquid using in situ liquid SEM with SALVI. While attempting this procedure it is really important to remember that the microreactor should be free of leaks before mounting to the SEM stage. Following this procedure, other methods like TOF-SIMS or NMR can be performed in order to answer additional questions like molecular composition and fluidic field mapping.

After its development, this technique paved the way for researchers in the field of material sciences, chemical engineering and nuclear engineering to explore particle size and morphological changes in a caustic liquid environment. Don't forget that when working with surface analysis using SEM, gloves and goggles should be worn when performing this procedure.