For those dental students reading along, in class yesterday, Dr. Bick discussed transmission electron microscopy (TEM). Cell structures can be imaged using this technique to obtain magnifications of up to the maximum of 25,000,000X in some microscopes. Of course with this kind of power, these magnifications would be like looking at an elephant with a light microscope and that wouldn't make very much sense. Typically magnifications in the range of 2000x to 40,000x are useful for examining things like cells (eukaryotic and prokaryotic), viruses and protein complexes.
For tissue structure, specimens are stained with heavy metals (osmium tetroxide for lipids, Uranyl Acetate for proteins and lipids) and embedded in a resin. The resin is then sectioned thinly and mounted on a metal grid which is then stained again with Uranyl acetate and lead citrate. The grid is placed in the microscope where electrons are passed through the sample where the heavy metal stains interact with the beam giving the image its characteristic contrast.
The way this happens is that electrons hit a structure and change course (because of the heavy metal), some pass through with minimal interference (no heavy metal or just a little). A charged coupled device (CCD or digital camera) camera records the image by measuring the electron density at each pixel, thus creating a black and white EM image like we see in class. Electrons do not have color so images are black and white.
Since samples need to be sectioned for this technique, this doesn't really tell us much about the 3D structure of cells. For that analysis, we need to use a scanning electron microscope. Cells are coated with a very gold palladium in a sputter coater, and the electron beam is bounced off the surface. The resulting image shows only the cell surface.
Recently, however, advances in computing have allowed scientists to further develop electron cryomicroscopy, or cryoEM. This is a technique that is used to image very small biological specimens (< 1 micron) frozen and perfectly preserved in amorphous ice. NOT resin! Why is that important? The specimen that is imaged is perfectly preserved and, in the case of virus, the virus can later be removed from the microscope, thawed from the ice, and used to infect more cells - illustrating that the images recorded were indeed of infectious virus.
The process begins by applying 3-4 microliters of specimen (let's just say we're looking at a virus in this example) in buffer onto a 3 mm copper EM grid coated with a net-like layer of carbon holes. The virus is blotted with filter paper creating a thin layer of buffer in which the specimen is briefly suspended. The EM grid is then quickly plunged into a liquid ethane cup at liquid nitrogen temperature where the virus is flash frozen so quickly ice crystals cannot form and the specimen becomes embedded in a layer of glass-like ice. The virus is then placed into an electron microscope specifically designed to image these frozen specimens. Remember, this is TEM, so electrons are passing through the sample. We get a black and white picture that looks something like this:
These are alphaherpesvirus particles!
For the next step, it is important to understand that for homogenous particles such as viruses, all the particles embedded in the ice are in all possible orientations and can be considered different views of the same object. Using a computer, the digitized images from the microscope and image analysis software, the orientation of each particle is determined relative to the electron beam. Once all the orientations are determined, the computer merges all the different views into one 3D model which then represents the specimen. Because the specimen is reproduced in 3 dimensions, it can be dissected and studied by imaging software. Behold the modeled structure of Sindbis virus (an alphaherpesvirus):
A few words about resolution, from the expert:
"Ok. You would have to ask about resolution. Resolution in the world we live in means essentially the minimum distance that two small elements in an image (dots) are distinguishable as two independent elements or dots.
(Also remember from Dr. Bick's lecture that the distance (d) between points that can be resolved increases as the wavelength of light increases, i.e. the resolving power goes down.)
In cryoEM you relate everything to your pixel size and image size. The pixel is a measurement of a grey value of the specimen you scanned. The pixel size is dependent on the mag. The higher the mag, the smaller the pixel size when you image it with the digital camera on the microscope.
Ok, now you have pixel size and image size. Say you have a pixel size that is 2 angstroms/pixel. This means that the absolute best resolution you can get is 4 angstroms resolution because you cannot achieve a resolution that is equal to your scanning step size or pixel. The best you can do is twice the pixel size.
So, what does resolution mean? In cryoEM resolution is quantifiable. You have an image. The image has noise and you refine the data to achieve the best resolution and that is when the noise and signal equal each other and the ration is 1. You cannot get better than that. With the image and pixel size you know exactly at what resolution this occurs. For my newest virus image, I get to 13 ansgtroms resolution. That does not mean that I can see two dots no closer than 13 angstroms. In fact I can see detail that is smaller than 13 angstroms. The resolution in this case is a quantifiable measure of how far out I can signal from the data in terms of resolution with the maximum being 2 times the pixel size.
I know it's complicated but YOU ASKED."
sindbis is an alpahvirus not an alphaherpes virus. they are very different.
ReplyDeleteThanks for pointing that out. For the record, alphaviruses are single-stranded RNA viruses, while alphaherpesviruses (such as HSV-1) are DNA viruses.
ReplyDelete