The overall goal of this procedure is to image biological assemblies and liquid in order to observe their dynamic behaviors at nanometer resolution. This is accomplished by first preparing the silicon nitride microchips with an affinity capture surface to enable macromolecules to be tethered to the microchip surface, preventing diffusion in liquid. The second step is to load biological samples onto the microchip in order to recruit rotavirus particles.
Next, the microchip containing the virus particles is washed with a contrast agent and then loaded onto the microfluidic specimen holder. The final step is to load the top chip and face plate to form a closed vessel encasing the liquid specimen. Ultimately, images are recorded in the TEM to reveal features of the virus sample within the liquid micro chamber.
The main advantage of this technique over other existing methods to examine entities in liquid is that our specimen do not rapidly diffuse over great distances while imaging due, do the affinity capture coating applied to the microchips? The implications of this technique extend towards real-time assessments of nanoparticle based drug delivery systems or vaccine development against viral pathogens because we can see for the first time how dynamic nano machines change in solution. Though this method can provide insight into visualizing biological macromolecules, it can also be applied to other systems such as electrochemistry applications.
A visual demonstration of this method is critical. Since preparing affinity capture devices and loading the microfluid specimen holder may present a problem to the novice user. To begin clean the silicon nitride e chips by incubating them in 15 milliliters of acetone for two minutes, followed by 15 milliliters of methanol for two minutes.
To remove residual photoresist used in the manufacturing process, allow the chips to quickly dry under laminar airflow. Once dry, incubate the chips on a plate heated to 150 degrees Celsius for 1.5 hours. Then allow them to cool to room temperature.
Next, use Hamilton syringes to make up a lipid mixture in a small glass tube. By first adding 25%chloroform, then add 55%DLPC in chloroform at one milligram per milliliter. And finally, using a separate syringe, add 20%nickel NTA lipid in chloroform at one milligram per milliliter for a total volume of 40 microliters.
Then cut a piece of param to fit inside a 100 by 15 millimeter glass Petri dish, and add nine 15 microliter drops of Milli Q water on the surface of the param. Apply a one microliter aliquot of the lipid mixture over each 15 microliter drop of Milli Q water and place the plate on ice in a humid environment for at least 60 minutes while a lipid monolayer forms on the surface of the drops. Place an EIP with a 150 nanometer integrated spacer on top of the monolayer droplet and incubate them together for one minute.
Next, gently lift the chip off of the droplet and add three microliters of hist tagged protein A at 0.01 milligrams per milliliter directly to the surface of the chip. Incubate the chip and protein A for one minute at room temperature. Then blot away the excess liquid using watman number one filter paper and immediately add three microliters of IgG antibody solution at 0.01 milligrams per milliliter.
Incubate the sample for one minute at room temperature. Next, remove the excess solution using a Hamilton syringe and immediately add a one microliter aliquot of rotavirus, double layered particles at 0.1 milligrams per milliliter in a heaps buffer solution, and incubate the chip for at least two minutes at room temperature. A second flat IP is prepared by glow discharging for one minute, assemble the microfluidic chamber using a ST.Stereoscope by first loading the tip of the specimen chamber with O-rings.
Then load the wet E IP containing the viral sample into the tip of the microfluidic specimen holder. Next, add the glow discharge flat EIP over the top of the specimen sandwich the entire assembly together to form a sealed enclosure held in place mechanically within the holder by three brass screws. Following assembly, pump the tip of the holder down to 10 to the minus six tor.
Using a turbo pump dry pumping station before placing the holder inside the TEM, load the I two specimen holder into a transmission electron microscope equipped with a lab six filament operating at 120 kilovolts. Next, turn on the TEM filament and adjust the centric height of the microscope stage with respect to the specimen by using the wobbler function to tilt the sample for minus 15 degrees to plus 15 degrees back and forth. In the column record 6, 000 to 30, 000 times magnification images along the edges and in the corner regions of the microfluidic chamber.
First image using a CCD camera at low dose conditions of one to three electrons per angstrom squared. Determine the proper defocus value by focusing at the edge of the fluidic chamber. In general, use a defocus value of minus 1.5 micrometers to record images at 30, 000 times magnification to ensure the solution is contained in the microfluidic chamber.
Throughout the experiments, focus the electron beam until the bubbles are formed in the liquid within the device. The image shown here was taken after two minutes at five electrons per angstrom squared. After five minutes, even more bubbles formed glow discharge, microchips bind.
Very few double layered particles in solution presumably due to diffusion. In comparison, the double layered particles are enriched on affinity decorated microchips shown here are representative images and a 3D reconstruction of double layered particles in liquid containing a contrast reagent. The diameter of the reconstruction is 80 nanometers.
Class averages of the double layered particles and liquid revealed nicely defined features along their outer surface. Individual panels are 110 nanometers. When performing this procedure, it's important to remember to center the E IPS in the tip of the specimen holder and PrepU the holder prior to inserting it into the electron microscope.
Following this procedure, other methods like assessing the transcriptional activity can be performed in order to answer additional questions like, to what extent did the electron beam affect the functional capacity of the viral complexes? After watching this video, you should have a good understanding I how to produce affinity capture devices, decorate them with molecular adapters, load virus specimens into the microfluidic TEM holder and image the specimens using cche microscopy procedures. Don't forget that working with viral pathogens can be extremely hazardous, and precautions such as wearing gloves and protective eyewear should always be taken when performing procedures with BSL two pathogens.
