Biodistribution of Nano-drug Carriers: Applications of SEM

1. Nanoparticle Injection and Organ Harvesting

1. Inject nanoparticles into an anesthetized mouse intravenously to allow passive targeting.
2. At the desired time points, i.e., 1, 4 and 8 weeks, post-injection, humanely euthanize the mice according to American Veterinary Medical Association (AVMA) Guidelines.
3. Open the body cavity and surgically remove the organs of interest. Place the organs in 10% phosphate buffered formalin in a polypropylene container until sample preparation.

2. Tissue Sample Preparation

1. Use forceps to transfer the mouse tissue from the fixative into phosphate buffered saline (PBS). Rock the sample for 30 min, replacing the PBS every 10 min.
2. Remove the tissue and dry with a kimwipe. Then place it in a plastic mold containing optimal cutting temperature compound (OCT). Store at -80 °C overnight.
3. The next day, transfer the sample to the cryostat and set the temperature to -23 °C.
4. Label slides with the organ type and nanoparticle size, and place them on a shelf in the cryostat.
5. Cover the cryostat chuck with OCT and place the sample on top. Lower the extractor plunger over the sample and allow it to equilibrate for 3-5 min.
6. Mount the chuck onto the specimen holder and orient it so that the blade can cut straight across the frozen sample. Bring the sample close to the blade for rough facing. Set the thickness to 30 μm and slice several sections until an evenly cut slice is produced.
7. Switch to fine facing by decreasing the section thickness to 7-8 μm. Collect a sliced section by pressing a labeled glass slide on the slice. Place two slides onto each slide, and store in a slide rack. Allow to dry at room temperature.
8. Once dry, dehydrate the samples by dipping the slide rack in 50% ethanol for 3 min to remove OCT. Then transfer the rack to 80% ethanol for 3 min before placing the rack in a 1:1 ratio of cold methanol to acetone for 10 min at -20 °C.
9. Remove the slide rack and drain the excess solvent on a paper towel. After 20–30 min, place the slides in a slide box and store in a freezer at -20 °C until imaging.

3. High Resolution Imaging using SEM and EDS

1. Prepare the sample as described in “SEM Imaging of Biological Samples.” Then load the sample into the SEM.
2. Turn the SEM on, and adjust the working distance to around 5 mm and the accelerating voltage and beam current to 25 keV, which would normally be too high for a biological sample. However, the sample is coated for conductivity and protection.
3. Start imaging and zoom to around 1,000-2,000X magnification to see the structures that would contain the nanoparticles. Note that without back-scatter detection (BSD) one cannot distinguish them below a certain depth.
4. Insert the BSD under the same parameters and move the stage in the z-direction to the same working distance as before.
5. Start imaging at around the same magnification and check that you are able to see high contrast in the presence of nanoparticles. Save the images.
6. Use different BSD configurations (where the charges on the detector align) to choose the one that shows the highest contrast for the nanoparticles.
7. Zoom-in on a high contrast area showing a nanoparticle or clump of nanoparticles.
8. Open the 2^nd camera of the chamber and watch as you insert the EDS into the system by pressing the down button on the SEM attachment. Once the EDS is close but not touching the BSD or the gun, release the button.
9. Open the Aztec program on the EDS computer (still at workstation) and acquire an image from the SEM. Use the “point and shoot” method to click on an area very dense in contrast and nanoparticles. 
10. The EDS will show the spectrum of characteristic X-rays from that point. Look for both barium and titanium peaks to be identified on the graph. This confirms that what you are looking at are indeed the nanoparticles and not any kind of contamination. 
11. Return to the sample and use the Atlas software to map the borders of the organ on the slide. Select the “Organ” protocol for creating a mosaic image of the area, and let it run (this can take a few hours at most). 
12. Once the composite image is created and stitched by the software, export it as a Tif file. 
13. Open the Tif file in ImageJ, an open source software, and adjust contrast threshold values to highlight the areas of very high contrast (i.e., the nanoparticles). Use built-in functions to quantify the volume of nanoparticles using the pixel size defined in the Organ protocol (should be around 100 nm). 
14. While this procedure refers only to the 1-week sample of the mouse lung, this procedure is repeated with the samples from other weeks and other organs to compile a graph showing distribution.
15. After calculating the bio-distribution for each organ for each week, biodistribution graphs will show the changes in biodistribution and concentration of nanoparticles over the course of the 8 weeks. These show the peak concentration and also provide information on how long it takes for the nanoparticles to clear from the organ.