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    US_2005_0051725_A1

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    US_2005_0051725_A1

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    US_2005_0051725_A1

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    Rusifar R

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    IM US 200505172541 c») United States 2 Patent Application Publication 0) Pub. No.: US 2005/0051725 A1 Nakagaki et al, 3) Pub, Date: Mar. 10, 2005 6» Publication Classeation 6) Ine Gown 23/00 (2) US.cL 250311 (76) tavestors: Ryo Nakagakt, Kawasaki OP), Yu Takagh, Kamakura (P); Hiroto 6 ABSTRACT Okada, Yokohama (IP); Hiroshi een ee) Lis possible to reliably and efficiently determine whether a specimen contains viruses, bacteria, etc. and, if it does, iden thee types, regardless ofthe observer. Furthermore, DATABASE! ss Aas RECRERTEAMET sroor a xnaus, _ Sersumitlseedtncernm nw eed {300 NORTH SEVENTEENTH STREET . -- — — Ba en) and an imaging condition used when the microscopic thing Ce api Nos a0oi8ado ‘Sopra me dt se Ay ie te oF ——Lr— {nt cated ne compas hinges sed J 18 101 107 M16 M16 ‘17 2 OVERALL STORAGE ConTROL| |S™ONA IMAGING ott CONTROL, UNIT 110 PROCESSING | | INPUT/OUTPUT 4 UNIT. UNIT CAMERA 112 113 Patent Application Publication Mar. 10,2005 Sheet 1of7 US 2005/0051725 Al FIG.1 18 107 -—_— Ms 16 a7 OVERALL STORAGE | comet] | unit | |paTaBase IMAGING — Si CONTROL] UNIT [ 110 112 CAMERA PROCESSING UNIT INPUT/OUTPUT, UNIT 113 Patent Application Publication Mar. 10,2005 Sheet 2 of 7 US 2005/0051725 AL FIG.2 TET OPERATOR ENTER KEYWORD |—~~201 INTO INPUT UNIT SELECT ONE OR MORE 202 REFERENCE MICROBES BASED [~~~ ON KEYWORD RETRIEVE PRETREATMENT 203 METHOD, IMAGING CONDITIONS, }——~ AND CAPTURED IMAGE OF EACH ‘SELECTED MICROBE DISPLAY RETRIEVED 204 INFORMATION ON OUTPUT UNIT ¥ LET OPERATOR SELECT ONE OR 205 MORE OF SELECTED REFERENCE ~~ MICROBES FOR COMPARISON HAVE ALL REFERENCE MICROBES SELECTED ‘AT STEP 205 BEEN COMPARED. WITH SAMPLE? PRETREAT SAMPLE OBSERVE AND CAPTURE IMAGE OF SAMPLE UNDER RETRIEVED IMAGING CONDITIONS COMPARE CAPTURED IMAGE a WITH THAT RETRIEVED FROM | —_-209 DATABASE AND DISPLAY COMPARISON RESULT ee) Patent Application Publication Mar. 10,2005 Sheet 3 of 7 US 2005/0051725 AL FIG.3 IMAGE] IMAGING |PRETREAT- NAME] 'Sata | CONDITIONS | MENT |SYMPTOMS| FEATURES. ACCELERATION | STEP ‘| | VOLTAGE: 50kV- MIUS||G) | MAGNIFICATION a FEVER | SPHERICAL: ‘ABOUT 20 NM IN DIAMETER VOMITING | ELLIPSOIDAL [=] [ACCELERATION |STEP1 DIARRHEA | SPECKLED VIRUS| VOLTAGE:40kV_ @ MAGNIFICATION: a Ge 404 401 PRETREATMENT METHOD: IMAGE CONDITIONS: .. Patent Application Publication Mar. 10,2005 Sheet 4 of 7 US 2005/0051725 AL FIG.5A 128 PIXELS mS 505 : 508 128 PIXELS a }r28 PIXELS CALCULATE FEATURE VALUE VECTOR Mt \ \ 503 502 — FIG.5B CALCULATE CALCULATE FEATURE FEATURE VALUE VECTOR | VALUE VECTOR Ma 2 Patent Application Publication Mar. 10,2005 Sheet 5 of 7 US 2005/0051725 AL FIG.6 START 601 RECEIVE CAPTURED IMAGE [~~~ SE See RECEIVE IMAGE OF REFERENCE 602 VIRUS (OR MICROBE) RETRIEVED FROM DATABASE Beams see tees tee tee seems tes cee ees ieee FIND EACH VIRUS IMAGE WITHIN | —_-603 CAPTURED IMAGE J CALCULATE FEATURE VALUE 604 VECTOR OF epee VIRUS |} ed CALCULATE FEATURE VALUE 605 VECTOR OF EACH VIRUS IMAGE [~~ FOUND AT STEP 603 a CALCULATE DEGREE OF SIMILARITY BETWEEN FEATURE | __606 VALUE VECTOR OF EACH FOUND VIRUS IMAGE AND THAT OF REFERENCE VIRUS IMAGE OUTPUT CALCULATED DEGREE 607 OF SIMILARITY a Patent Application Publication Mar. 10,2005 Sheet 6 of 7 US 2005/0051725 AL FIG.7 706 REFERENCE VIRUS. TO LS COMPARED: VIRUS Z PRETREATMENT METHOD: IMAGING. CONDITIONS: DEGREE OF SIMILARITY: 703 [NUMBER DEGREE OF] 708 PRETREATMENT SIMILARITY, METHOD: 502 S| XxX IMAGING 2 yy CONDITIONS: 705 704 Patent Application Publication Mar. 10,2005 Sheet 7 of 7 US 2005/0051725 AL FIG.8 801 B01, . C4 ~ E_| -800 _ 117 118 FIG.9 901(A) 901(8) , 901(C) 902(A) 903(C) I \ \_ | 903(A) '902(B) *903(B) ‘902(C) US 2005/0051725 AL "TRANSMISSION ELECTRON MICROSCOPE, SYSTEM AND METHOD OF INSPECTING A. ‘SPECIMEN USING THE SAME, BACKGROUND OF THE INVENTION The present invention relates to 4 transmission croscope system for observing an obtained speci= ten under 1 microscope to determine whether it contains microorganisms and, iF it does, 10 identify theit types in ‘order to identify the cause of a disease or cary out food jon supervision. The present invention also relates to an inypection method using this transmission electton miero- scope system, {0002} When dealing with a disease or a food poisoning ‘case caused by a virus or bacterium, the frst thing to do is, to identify the ease of the symptoms that the people oF livestock are suffering, In such a ease, a Sample of fecal matter, ele i gathered from a patient, et. and this sample is checked tose if t contains aay vins or bacterium. Since viruses and bacteria are very small, observing them usvally requires a microscope which allows observation of minute ‘samples at high resalution, such asa transmission electron microscope, Since transmission electron microscopes form fan image using electrons transmitted through the sample, they’ ate also suitable for observing substances having & minute thee-dimensional stucture, sich a8 prot [0003] For example, the H-7600 transmission electron microscope catalog from Hitachi High-Techoologies Cote poration lists traasmission eleciron microscopes for such & Porpose. A transmission electon microscope i made up of anelectron gun, an radiation system elect lens, a sample holder stage, an imaging system lens, a camera, a vacuum pumping system, 4 contol system, ete [0004] When viruses in a specimen are observed using. ‘such microscope fecal matter or living tise, forexample, 's gathered ffom a test subject and pretreated 80 that it can he actually observed under transmission electron micro- scope a6 4 specimen. [0005] Samples for observation are broadly classified into three types: (1) negatively stained samples, 2) stained sliced ‘samples, and (8) frozen sliced samples. [0006] (1) A acgatively stained sample is prepared by purifying and concentrating living tissue, fecal mater, ete hy use of a reagent or 4 centrifugal separator and then ‘mounting it on a mesh for eletron micrascopes. Represen- tative examples of negatively stained samples ae fine pranv lar specimens of viruses, et. Tungstic acid is usually used as the stining agent. When this type of sample contains Viruses, a levee (or a low wall) formed around each virus, providing contrast between each virus and its levee. [0007] (2) A stained sliced sample is prepared by slicing living tssie of an animal o plat with a diamond cate, te to produce 1 slice having 1 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, ad cutting. Generally, the tissue ris he sttined so as to provide enough contrast Io observe its survcture under an electroa microscope, Suitable staining agents include reagents containing a heavy metal, sich a6 uranium acetate, lead citrate, lead hydroxide, and lead Mar. 10, 2005 scctate. Usually, the tissue is double-stained with uranium Sod lead. Staining is required since a living body is primarily made up of light elements, such as hydrogen, oxygen, tathon, and nitrogen, That is these elements only exhibit a small scattering power with fespect to electron beams and, forthermore, they differ oaly a lille 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 resull, the obtained electron microscope image has a conteast according t0 the protein concentration, [0008] (3) A frozen sliced sample is prepared by bring living tissue into contact with a copper block, which has. been eooled down by liquid helium or liquid nitrogen so a8 to feze the tissue and then slicing the frozen iss by use ff oooling stage and a microtome. This ype of sample was Sevised to observe a tissue structure in an active state Therefor, the tissue is neither fixed nor sained, aad itis ‘observed under #eryo-elecron microscope equipped with cooling sample stage. Since the sample tissue i not stained, the contrast of the obiained image is low. [0009] A sample pretreated as described above for obser= vation is mounted onthe stage of anclectron microscope and observed by magnilyng its image a few tens of thousands to ' fow hundreds of thousinds of times. When identifying ‘microorganisms, suchas viruses contained in the simple, 4 person observes the magnified image to determine whether it includes virus images based on the characteristic shape snd internal structure of each vis, and, if the image contains any virus image, to identify the type of virus Further, when analyzing the structure of 2 protei(s) in the sample, several magnified images are taken ofthe sample to bre observed while the sample stage ofthe electron micro- ‘cope is being tilted. The taken images are subjected to CT (computer tomogeaphy) processing to provide a fine three dlimensionalstractare having dimensions on the order of & few tens of nanometers [0010] Recently, a cansmission electron microscope bas been provided with (orto include) a contol personal com puter having autofocus and imaging (contol) capabilities to increase tbe eficency of the observation work. However, the user must sill determine whether a sample contains riiccoorganisms, sich a vires, snd to identify thei Iypes bused on captured images. Acta, the user compares cach possible virus image agains! images of previously found Viruses listed in books and other documents o identify each Jmaged microorganism (wins). [0011] A problem with such » method is that it takes ‘onserable time to identify each virus, This is because the Caphired image includes images of various things other than microorganisms to be identified, making it necessary to iseriminate each target microorganism from them. Further ‘more, manually searching a huge amount of past microoe anism dlta requires substantial time and labor. Further, there is another problem in that virus idenification may or ‘may not he accurate depending on the skill of the observer or the degree of fatigue suffered by the observer [0012] Furter, stil another problem arises when itis necessary to handle a new virus, ete. 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 lista virus curently under US 2005/0051725 AL ‘observation, which may make it necessary to consume a Targe amount of time t identity the views. [0013] A similar problem arses, for example, when it is recessary to determine whether a ease ofa 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 seat large research institutes in the world to identify the microorganism which has caused the ease. This may lead fo delay in identifying tnd handling the microorganism, ‘SUMMARY OF THE INVENTION [0014] The present invention provides. transmission elec tson microscope systom that is capable of easily determining ‘whether a specimen contains mieroorganisms sad, if it does, ‘enifying their types, thereby solving the above-described an problems. The present invention alsa proves an inspee- tion method using sucha transmission electron microscope system, [0015] Further, the present invention also provides 3 net- work system capable of quickly determining whether a virus in specimen is identical to a vies nowly discovered at 3 remote location by gathering information on the new virus through a wide area network, [0016] _A transmission electron microscope systom of the present invention comprises: means for accessing a database Which stores transmitted electron images of difieent types ‘of microorganisms and specimen pretreatment methods and aging conditions for capturing the transmitted electron images; an input unit for inputting « keyword for selecting a microscopic thing whose presence ar absence ina specie men isto be determined; means for receiving a transmited ‘leetron image of the mietoscopie thing and specimen prireatment method and an imaging condition selected based om the input keyword; an imaging unit for capturing. an image of the specimen under the received imaging ‘condition after the specimen is pretreated by’ the received pretreatment method; a comparison tit for comparing the image captured by the imaging unit with the image received from the database; and an output unit for outputting @ ‘comparison result reacived from the comparison uni [0017] Further, the present invention also provides 4 net- work system in whieh the above-mentioned. transmission ‘electron microscope system and one or more database ‘reation sites ate comnected fo 4 wide area network. This system is characterized in that the transmission electron microscope system accesses a database created from all or ‘some of the original databases stored in the above-men- tioned database creation sites [0018] The transmission electron microscope system of the present invention makes it easy to determine whether 8 specimen contains microorganisms and, iit does, to identify their types. Furthermore, iis possible to quickly determine whether a virus in a specimen is identical to a vis newly

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