Qhov yuav tsum tau ua rau kev npaj cov qauv niaj hnub rau kev siv microscopy
Confocal microscope yog ib lub sijhawm ua cov khoom siv high-tech tshiab tsim nyob rau xyoo 1980s, thiab nws yog ib qho ntawm cov cuab yeej tshuaj ntsuam xyuas tseem ceeb hauv molecular biology thiab kev tshawb fawb txog lub neej niaj hnub no. Confocal microscopy yog ua raws li fluorescence microscopy imaging nrog lub laser scanning ntaus ntawv ntxiv, siv lub computer rau cov duab ua kom tau fluorescent dluab ntawm lub microstructure nyob rau hauv lub hlwb los yog cov ntaub so ntswg, thiab soj ntsuam cov kev hloov nyob rau hauv lub morphology ntawm ntau lub cev lub cev teeb liab tshuab nyob rau hauv subcellular theem. Ua ib qho cuab yeej tshawb fawb muaj zog hauv kev ua haujlwm ntawm morphology, molecular cell biology, neuroscience, pharmacology, noob caj noob ces thiab lwm yam kev tshawb fawb.
Kev npaj ua piv txwv rau confocal microscopy yog ib kauj ruam tseem ceeb ua ntej kev kuaj pom. Cov qauv yuav tsum tau sau nrog fluorescent probes (ib zaug, ob npaug, triple).
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Cov qauv yuav raug kho los yog cov ntaub so ntswg nyob, los yog cov kab lis kev cai nyob ruaj khov. Cells yuav tsum tau coj los ntawm Confocal tshwj xeeb me me kab lis kev cai tais diav los yog cov ntawv npog. Tshem tawm cov hlwb, co los yog tso cov hlais, thiab npog nrog cov ntawv npog npog zaj duab xis. Zda thickness ntawm cov qauv yog hais txog 1 ~ 2 hli, lub thickness ntawm lub npog iav siv yuav tsum tsawg tshaj li 0.17 hli, thiab lub thickness ntawm lub iav swb yuav tsum nyob nruab nrab ntawm 0.8 ~ 1.2 mm, thiab saum npoo yog du, lub thickness yog sib xws, thiab tsis muaj teeb meem cuam tshuam fluorescence. Mounting nruab nrab feem ntau yog siv rau cov qauv ruaj khov, thiab glycerol npaj hauv PBS nrog pH ntawm 8. 5-9 feem ntau yog siv rau mounting.
Cov txheej txheem kev npaj ua ntu zus:
1. Xaiv cov dyes
Txij li thaum lub confocal microscope siv ib lub wavelength laser raws li lub teeb qhov chaw, kev xaiv ntawm fluorescent dyes yuav tsum tau raws li nyob rau hauv lub wavelength ntawm lub laser nruab nrog lub confocal microscope. Yog hais tias muaj ntau yam fluorescent dyes nyob rau hauv tib tus qauv, lawv emission wavelength kuj yuav tsum tau txiav txim siab ntau npaum li cas. Tsis txhob sib tshooj kom tsis txhob muaj teeb meem hla xim.
2. Kev xaiv tus qauv nqa khoom
Feem ntau, lub hom phiaj siab lub hom phiaj ntawm lub tshuab tsom iav yog roj lo ntsiab muag, nws cov lej aperture yog me me, thiab qhov kev ncua deb ntawm lub lens thiab cov qauv yuav tsum tsis pub ntau tshaj 0.17mm. Slides thiab coverlip yog zoo. Yog hais tias nws yog tshem tawm cov hlwb los yog tshem tawm cov khoom, ib lub tais petri rau lub confocal microscope yuav siv tau los nqa cov qauv rau kev soj ntsuam.
3. Xaiv qhov nruab nrab mounting
Yog tias tus qauv tsuas yog yuav tsum tau soj ntsuam ib zaug thiab cov fluorescence tsis yooj yim quenched, koj tuaj yeem xaiv ib qho kev sib xyaw ntawm glycerol sib tov los kaw cov swb. Yog tias tus qauv yuav tsum tau muab tso rau ib lub sijhawm thiab siv ntau zaus, yuav tsum xaiv qhov nruab nrab los tiv thaiv fluorescence quenching mounting los txo qhov poob ntawm cov teeb liab fluorescent.
