Cov Ntsiab Cai ntawm Fluorescence Microscopy
Fluorescence microscopes txawv ntawm cov khoom siv kho qhov muag zoo tib yam. Hloov chaw ntawm kev soj ntsuam cov qauv hauv qab lub teeb pom kev zoo tib yam, lawv siv lub teeb ntawm qee qhov wavelength (feem ntau yog lub teeb ultraviolet, lub teeb xiav-violet) los ua kom cov khoom fluorescent nyob rau hauv cov qauv hauv qab lub tshuab ntsuas kom lawv tawm fluorescence. Yog li ntawd, lub teeb qhov chaw ntawm lub fluorescence microscope tsis ua hauj lwm raws li illumination ncaj qha, tab sis raws li ib tug lub zog qhov chaw uas excites cov fluorescent tshuaj nyob rau hauv cov qauv. Qhov laj thawj yog vim li cas peb tuaj yeem soj ntsuam cov qauv yog vim lub teeb pom kev ntawm lub teeb, tab sis qhov tshwm sim fluorescence tshwm sim tom qab cov tshuaj fluorescent nyob rau hauv cov qauv absorbs lub teeb ci zog.
Nws tuaj yeem pom tias tus yam ntxwv ntawm lub tshuab hluav taws xob fluorescence yog tias nws lub teeb ci tuaj yeem muab ntau lub teeb pom kev zoo nyob rau hauv ib qho kev sib txawv wavelength, kom cov khoom siv fluorescent hauv cov qauv kuaj tuaj yeem tau txais lub teeb pom kev zoo ntawm qhov tsim nyog siv. Nyob rau tib lub sijhawm, fluorescence microscope yuav tsum muaj cov lim dej sib thooj.
Fluorescence microscopy yog ib qho cuab yeej tseem ceeb hauv immunofluorescence histochemistry. Nws yog tsim los ntawm ultra-high-voltage light source, lim system (xws li excitation thiab suppression lim phaj), optical system thiab photography system thiab lwm yam tseem ceeb Cheebtsam. Nws siv lub teeb ntawm ib lub wavelength los ua kom cov qauv kom tawm fluorescence.
Txoj kev ua kom muaj zog fluorescence: Raws li lub wavelength ntau ntawm lub teeb, nws tuaj yeem muab faib ua ob hom: UV excitation method (siv txoj kev teeb pom kev zoo ultraviolet) thiab BV excitation method (siv xiav-violet lub teeb). Txoj kev UV excitation siv ze-ultraviolet lub teeb luv dua 400nm rau excitation. Tsis muaj lub teeb pom kev zoo nyob rau hauv txoj kev no, yog li qhov pom fluorescence nthuav tawm cov fluorescence ntawm cov dye, thiab nws yog ib qho yooj yim kom paub qhov txawv cov fluorescence ntawm cov qauv ntawm autofluorescence ntawm cov ntaub so ntswg tom qab.
BV excitation method yog raws li 404nm thiab 434nm rau excitation los ntawm ultraviolet mus rau xiav lub teeb. Txoj kev no siv lub teeb xiav rau irradiate cov qauv, yog li cov lim txiav tawm ntawm lub fluorescence soj ntsuam system yuav tsum siv lub lim uas tuaj yeem thaiv lub teeb xiav tag nrho thiab dhau qhov xav tau ntsuab thiab daj fluorescence. Fluorescent dyes rau fluorescent tshuaj tiv thaiv kab mob. Qhov siab tshaj plaws nqus wavelength ntawm lub teeb excitation yog ze rau qhov siab tshaj plaws emission wavelength ntawm fluorescence, yog li cov lim siv hauv BV excitation txoj kev yuav tsum tau siv cov lim txiav tawm ntse. Txoj kev no tuaj yeem siv lub teeb xiav raws li lub teeb pom kev zoo, yog li kev nqus ntawm cov xim fluorescent siab dua, thiab cov duab ci tuaj yeem tau txais. Nws qhov tsis zoo yog qhov fluorescence hauv qab 500nm tsis tuaj yeem pom, thiab cov fluorescence saum 500nm ua rau tag nrho cov duab tshwm daj. Hauv txoj kev fluorescent antibody, qhov tshwj xeeb ntawm fluorochrome feem ntau txiav txim los ntawm cov xim tshwj xeeb rau fluorochrome. Yog li ntawd, thaum tham txog kev hloov maj mam, qhov tsis txaus ntawm cov lus hais saum toj no ntawm BV excitation txoj kev feem ntau muaj kev cuam tshuam zoo.
Txhawm rau ua tiav, lub teeb pom kev ntawm fluorescence microscope tuaj yeem txiav txim siab raws li peb lub ntsiab lus hauv qab no raws li cov qauv ntawm lub condenser thiab lub wavelength ntawm lub teeb excitation.
① Los ntawm qhov kev xav ntawm qhov sib txawv ntawm cov duab fluorescent, UV excitation dark field concentrator yog siv rau illumination.
② Xav txog qhov ci ntawm daim duab, BV excitation lim muaj qhov ua tau zoo tshaj plaws ntawm qhov tsaus ntuj pom.
③Cov yam ntxwv ntawm UV excitation lim tsaus qhov kev soj ntsuam thiab BV excitation tsaus nti concentrator illumination tuaj yeem suav hais tias yog ntawm ob txoj kev teeb pom kev zoo no, tab sis yav dhau los muaj zog dua qhov tsaus ntuj thiab pom cov duab ci dua. Qhov sib txawv me me; qhov tom kawg khaws cov khoom ntawm qhov tsaus ntuj lub teeb txoj hauv kev, yog li cov duab pom tau tsaus dua, thiab qhov sib txawv zoo dua. Thaum siv lub tshuab hluav taws xob fluorescence tiag tiag, txoj kev teeb pom kev zoo tshaj plaws yuav tsum tau siv rau kev soj ntsuam.
Nws yuav tsum tau taw qhia tias txawm tias UV-excited dark field condenser nrog qhov zoo sib xws yog illuminated, ib feem ntawm ultraviolet excitation lub teeb refracted los yog tawg los ntawm cov qauv yuav nkag mus rau lub hom phiaj lens. Autofluorescence tuaj yeem ua rau cov duab teb. Yog li ntawd, lub lim dej UV-absorbing yuav tsum tau siv nyob rau hauv pem hauv ntej ntawm eyepiece raws li ib tug txiav-tawm lim.
