The present invention relates to a transmission electron microscope system for observing an obtained specimen under a microscope to determine whether it contains microorganisms and, if it does, to identify their types in order to identify the cause of a disease or carry out food sanitation supervision. The present invention also relates to an inspection method using this transmission electron microscope system.
When dealing with a disease or a food poisoning case caused by a virus or bacterium, the first thing to do is to identify the cause of the symptoms that the people or livestock are suffering. In such a case, a sample of fecal matter, etc. is gathered from a patient, etc., and this sample is checked to see if it contains any virus or bacterium. Since viruses and bacteria are very small, observing them usually requires a microscope which allows observation of minute samples at high resolution, such as a transmission electron microscope. Since transmission electron microscopes form an image using electrons transmitted through the sample, they are also suitable for observing substances having a minute three-dimensional structure, such as protein.
For example, the H-7600 transmission electron microscope catalog from Hitachi High-Technologies Corporation lists transmission electron microscopes for such a purpose. A transmission electron microscope is made up of an electron gun, an irradiation system electron lens, a sample holder stage, an imaging system lens, a camera, a vacuum pumping system, a control system, etc.
When viruses in a specimen are observed using such a microscope, fecal matter or living tissue, for example, is gathered from a test subject and pretreated so that it can be actually observed under a transmission electron microscope as a specimen.
Samples for observation are broadly classified into three types: (1) negatively stained samples, (2) stained sliced samples, and (3) frozen sliced samples.
(1) A negatively stained sample is prepared by purifying and concentrating living tissue, fecal matter, etc. by use of a reagent or a centrifugal separator and then mounting it on a mesh for electron microscopes. Representative examples of negatively stained samples are fine granular specimens of viruses, etc. Tungstic acid is usually used as the staining agent. When this type of sample contains viruses, a levee (or a low wall) is formed around each virus, providing contrast between each virus and its levee.
(2) A stained sliced sample is prepared by slicing living tissue of an animal or plant with a diamond cutter, etc. to produce a slice having a thickness of a few tens of nanometers and then mounting the slice on a mesh for electron microscopes. Before and when slicing the living tissue, it must be subjected to processes such as fixing, dehydrating, embedding, and cutting. Generally, the tissue must be stained so as to provide enough contrast to observe its structure under an electron microscope. Suitable staining agents include reagents containing a heavy metal, such as uranium acetate, lead citrate, lead hydroxide, and lead acetate. Usually, the tissue is double-stained with uranium and lead. Staining is required since a living body is primarily made up of light elements, such as hydrogen, oxygen, carbon, and nitrogen. That is, these elements only exhibit a small scattering power with respect to electron beams and, furthermore, they differ only a little in such power, resulting in very low image contrast. What is stained in the tissue is its protein; the higher the protein concentration, the more heavily the tissue is stained. As a result, the obtained electron microscope image has a contrast according to the protein concentration.
(3) A frozen sliced sample is prepared by bring living tissue into contact with a copper block, which has been cooled down by liquid helium or liquid nitrogen so as to freeze the tissue and then slicing the frozen tissue by use of a cooling stage and a microtome. This type of sample was devised to observe a tissue structure in an active state. Therefore, the tissue is neither fixed nor stained, and it is observed under a cryo-electron microscope equipped with a cooling sample stage. Since the sample tissue is not stained, the contrast of the obtained image is low.
A sample pretreated as described above for observation is mounted on the stage of an electron microscope and observed by magnifying its image a few tens of thousands to a few hundreds of thousands of times. When identifying microorganisms, such as viruses contained in the sample, a person observes the magnified image to determine whether it includes virus images based on the characteristic shape and internal structure of each virus, and, if the image contains any virus image, to identify the type of virus. Further, when analyzing the structure of a protein(s) in the sample, several magnified images are taken of the sample to be observed while the sample stage of the electron microscope is being tilted. The taken images are subjected to CT (computer tomography) processing to provide a fine three-dimensional structure having dimensions on the order of a few tens of nanometers.
Recently, a transmission electron microscope has been provided with (or to include) a control personal computer having autofocus and imaging (control) capabilities to increase the efficiency of the observation work. However, the user must still determine whether a sample contains microorganisms, such as viruses, and to identify their types based on captured images. Actually, the user compares each possible virus image against images of previously found viruses listed in books and other documents to identify each imaged microorganism (virus).
A problem with such a method is that it takes considerable time to identify each virus. This is because the captured image includes images of various things other than microorganisms to be identified, making it necessary to discriminate each target microorganism from them. Furthermore, manually searching a huge amount of past microorganism data requires substantial time and labor. Further, there is another problem in that virus identification may or may not be accurate depending on the skill of the observer or the degree of fatigue suffered by the observer.
Further, still another problem arises when it is necessary to handle a new virus, etc. That is, viruses and microbes grow and mutate into new forms, meaning that a new (undiscovered) virus may suddenly appear. As a result, the documents at hand might not list a virus currently under observation, which may make it necessary to consume a large amount of time to identify the virus.
A similar problem arises, for example, when it is necessary to determine whether a case of a disease found in Japan has been caused by a microorganism which usually exists locally in some other part of the world, such as Africa. When this occurs, a sample is usually sent to large research institutes in the world to identify the microorganism which has caused the case. This may lead to a delay in identifying and handling the microorganism.