John F. Lesoine,
OPT407: Practical Electron Microscopy Spring 2006 Final Project
Fig. 1. A colorized image showing white blood cells in yellow.
blood contains all of the different cell types that are found in the arteries
and veins. Red blood cells (RBCs) or
erythrocytes are the most abundant cell type in whole blood and the RBCs take up roughly 40-50% of the volume of the
blood. The exact percentage of the volume that RBCs
takes up is called hematocrit. One of the main
responsibilities of the RBC is to transport oxygen throughout the body and to
transport CO2 to the lungs in the form of bicarbonate HCO3.
RBCs are extremely elastic and if their cell wall is
distorted they will spring back to their native shape in a very short amount of
time. The RBC's cytoskeleton holds the cell
membrane in a specific shape which is that of a concave disk. This
increases the cells surface area to volume ratio and this allow the RBC to
squeeze through very small holes including capillaries and plaque
buildup. White blood cells (WBCs) come in
different types but their main function is as a defense mechanism. Platelests are yet another type of cell found in the blood
and they are generally disk shaped unless they become activated.
Platelets are responsible for the clotting process and this process can be seen
when a scab forms over an open wound.
Fig. 2. 3-D image of RBCs and a fibrin chord that is attaching two cells. The fibrin chord was created by a platelet and this chord is used to snare cells to aid in the clotting process.
Blood cells will be imaged with the light microscope, the SEM and the TEM. Image colorization will be applied to some images in order to increase the information content of those images. 3-D images will also be created that can be viewed with 3-D stereo glasses. The project will examine the cell's structures.
Fixing, drying and embedding, centrifuging, staining, microtoming, sputtering, light microscopy, secondary electron imaging, transmission electron microscopy, image colorization, creating 3-D images.
Whole blood was drawn from a donor and the blood was fixed in a 2% buffered gluteraldehyde solution. RBCs were seperated from WBCs using a centrifuge. The RBCS are denser so they were found at the bottom of the centrifuge tube while the WBCs were found floating on the top. The cells were then washed and chilled for the next step.
Biological samples present problems for electron microscopy because they are often nonconductors and they are hydrated. In order to make meaningful measurements these samples need to be dried and made conductive in some way that does not produce many artifacts. For the SEM work the samples were simply washed with ddH2O and then allowed to dry on disks of aluminum foil mounted onto SEM sample stubs. Because the fixing process made the cell walls so rigid more exotic drying techniques such as critical point drying or HMDS drying were not required. In the case of the TEM sample RBCs were spun into a pellet and transition fluids were used to move from the buffer solution and the RBCs were stained with osmium to facilitate imaging in the TEM.
The sample was then mixed with an epoxy and this epoxy was allowed to dry in an oven to become hard.
To obtain a cross section of the cells an ultra microtome was used to cut 100 nm sheets from the sample embedded in the epoxy. The thin sheets were then picked up using a copper TEM grid.
many biologicals are nonconductors so in order to
image them in conventional EM they need to be made conductive. Five-six
nm of gold was sputtered onto the cell samples in order to image them in the
Biological samples are diverse and different imaging techniques are complimentary to each other in that they can reveal information that could be hidden from other techniques.
microscope imaging is often a useful first step in microscopic studies simply
because it is the easiest and the cheapest imaging method that will be
described here. Often times researchers wish to
distinguish between different components in a cell. This can be
accomplished through the use of different stains that will appear different in
light microscopy or through the use of fluorescent probes that target specific
parts of the cell. It is also possible to image living and wet cells so
artifacts due to sample processing should be less of a concern for the light
microscope. Light microscopy does suffer from a short depth of field at
high resolution and this can be seen in the light microscope image of the red
Fig. 3. Light microscopy image of the SEM RBC sample that was sputtered with gold.
To image the blood cells in the SEM the chamber secondary electron detection was used. A low accelerating voltage of 3-5 kV was used because the samples were only coated with a 5-6 nm layer of gold and the electrons would not penetrate that far through the gold layer. If the electrons had the energy to penetrate through the gold layer then they would pass straight through the biological samples because the electron density in the biological samples dealt with here is much less than that of gold. Higher energy electrons in the case of these measurements would have produced images with lower resolution because the interaction volume with the sample would actually be larger.
Fig. 4. SEM image of a red blood cell and a white blood cell stacked on top of the red blood cell. It is possible that the RBC is being eaten by the growth that is wrapping onto its surface.
Fig. 5. The porous cell may be eating the cell that is standing up. Notice how the structure of the porous cell is similar to the structure which is wrapping around the RBC in Fig. 4.
Fig. 6. An SEM image of WBCs and RBCs. It may be that the larger structures are eating the smaller cells and removing them from circulation.
Fig. 7. An SEM image showing two different stages of a platelet becoming activated. The disk is shown morphing into an activated platelet and the spiky cell is even further along the pathway to becoming an activated platlet. To see a fully activated platelet please see Figs. 14 and 16.
Fig. 8. An SEM image of WBCs.
Fig. 9. An SEM image that shows an activated WBC as the wrinkly cell.
Fig. 10. An SEM image showing cells with some interesting structure.
Fig. 11. An SEM image possibly showing aggregated blood cells or cells which are being removed from circulation.
Fig. 12. A horn like structure is seen on this cell and this may be a microvillae.
Fig. 13. An image showing white blood cells.
Higher accelerarting voltages lead to higher spatial resolution at the focus of the electron beam. Because TEM samples are only ~100 nm thick the interaction volume is actually limited mainly by the accelerating voltage and the sample thickness. The samples did not need to be coated with gold but they did need to be stained in order to have areas with higher electron density for imaging. Osmium was used to stain the RBCs and this provides a uniform staining of the RBCs. There were also other cell types which were found while the TEM measurements were being performed.
Fig. 14. A TEM micrograph is shown of a slice through RBCs. The center RBC shows the characteristic concave disk shape.
Fig. 15. A white blood cell is shown in the TEM micrograph. The osmium did not stain this cell uniformly and this may be because the osmium did not penetrate certain intracellular organelles.
SEM images are grayscale so colorization can be used as an effective tool in adding more information to an image. The addition of the red color the the RBCs makes them easier to recognize because most people associate the color red to them.
Fig. 16. The clotting process is shown with RBCs red, the clot as yellow and the activated platelet, orange, is found near the bottom of the clot.
Fig. 17. A 3-D image of an activated platelet.
Fig. 18. A 3-D image of white blood cells possibly being eaten or being aggregated together.
Fig. 19. A 3-D image of an RBC, WBCs and an unknown larger object. It is possibly the larger object was a cell such as a macrophage which did not survive the drying process.
Fig. 20. A 3-D image of an unspecified object. The small circles on the sample holder and on the object are blood cells. It is possible that this larger structure plays a role in removing the smaller cells from circulation but this is only a guess.
Cells were successfully imaged using the different imaging techniques. The drying of the cells using air drying did not appear to produce noticeable artifacts for most cell types.
Thanks go to Brian McIntyre for his help and guidance and to Karen Bentley for providing guidance with the TEM sample preparation.