Lub hom phiaj, tus cwj pwm, thiab kev siv lub fluorescence microscope
Lub hauv paus ntsiab lus thiab cov yam ntxwv ntawm lub fluorescence microscope: lub fluorescence microscope siv lub teeb pom kev zoo nrog cov luminous efficiency kom emit ib co wavelength ntawm lub teeb (xws li ultraviolet lub teeb 3650 nyob rau hauv los yog liab doog xiav lub teeb 4200 nyob rau hauv) los ntawm lub lim system raws li cov excitation. lub teeb los txhawb cov fluorescence hauv cov qauv. Tom qab cov khoom emits fluorescence ntawm ntau yam xim, nws yog pom los ntawm lub magnification ntawm lub hom phiaj lens thiab eyepiece. Nyob rau hauv txoj kev no, nyob rau hauv ib tug muaj zog sib piv keeb kwm yav dhau, txawm yog hais tias tus fluorescence yog heev qaug zog, nws yog ib qho yooj yim los txheeb xyuas thiab muaj siab rhiab heev. Nws yog tsuas yog siv rau kev tshawb fawb ntawm cov qauv ntawm tes thiab kev ua haujlwm thiab tshuaj lom neeg muaj pes tsawg leeg. Cov qauv yooj yim ntawm fluorescence microscope yog tsim los ntawm ib lub tshuab kho qhov muag zoo tib yam ntxiv rau qee yam khoom siv (xws li lub teeb fluorescent, lub tshuab lim dej, ob lub xim kab sib cais thiab cov lim lim, thiab lwm yam). Fluorescent teeb qhov chaw-feem ntau yog siv ultra-siab siab mercury teeb (50-200W), uas muaj peev xwm emit lub teeb ntawm ntau lub wavelengths, tab sis txhua yam fluorescent tshuaj muaj ib tug excitation wavelength uas ua rau cov fluorescence muaj zog tshaj, yog li ib tug excitation lim (Feem ntau, muaj ultraviolet, ntshav, xiav thiab ntsuab excitation lim), uas tsuas yog tso cai rau lub teeb excitation ntawm ib co wavelength los ntawm thiab irradiate tus qauv, thaum absorbing lwm lub teeb. Tom qab txhua yam khoom yog irradiated los ntawm excitation lub teeb, nws emits pom fluorescence nrog ib tug ntev wavelength tshaj lub irradiation wavelength nyob rau hauv ib tug luv luv lub sij hawm. Fluorescence yog qhov tshwj xeeb thiab feem ntau tsis muaj zog dua li lub teeb pom kev zoo. Txhawm rau saib xyuas cov fluorescence tshwj xeeb, yuav tsum muaj kev thaiv (lossis suppressing) lim tom qab lub hom phiaj lens.
Nws muaj ob txoj haujlwm: ib qho yog txhawm rau nqus thiab thaiv lub teeb pom kev zoo los ntawm kev nkag mus rau qhov muag, kom tsis txhob cuam tshuam cov fluorescence thiab ua rau lub qhov muag puas; Lwm qhov yog xaiv thiab cia qhov tshwj xeeb fluorescence hla dhau, uas qhia cov xim fluorescent tshwj xeeb. Ob lub lim yuav tsum tau siv ua ke.
Muaj ob hom fluorescence microscopes raws li lawv txoj kev kho qhov muag:
1. Transmission fluorescence microscope: Lub teeb pom kev zoo tau dhau los ntawm cov khoom siv los ntawm lub lens condenser kom ua rau muaj fluorescence. Ib qho chaw tsaus nti yog feem ntau siv, thiab ib qho khoom siv zoo tib yam kuj tuaj yeem siv los kho daim iav kom lub teeb pom kev zoo raug xa rov qab thiab hla mus rau cov qauv. Qhov no yog qhov qub fluorescent microscope. Qhov kom zoo dua yog tias cov fluorescence muaj zog ntawm qhov tsis tshua muaj qhov loj, tab sis qhov tsis zoo yog qhov fluorescence txo qis nrog qhov nce ntawm qhov loj. Yog li ntawd, nws yog qhov zoo dua los soj ntsuam cov ntaub ntawv loj dua.
2. Epi-fluorescence microscope yog ib hom tshiab ntawm fluorescence microscope tsim nyob rau niaj hnub no. Qhov txawv yog hais tias lub teeb excitation ntog los ntawm lub hom phiaj lens mus rau saum npoo ntawm cov qauv, uas yog, tib lub hom phiaj lens yog siv raws li lub illumination condenser thiab lub hom phiaj lens rau sau fluorescence. Lub dichroic beam splitter yuav tsum tau ntxiv rau hauv txoj kev teeb, uas yog 45 degree deb ntawm lub teeb uranium. Lub teeb excitation yog reflected rau hauv lub hom phiaj lens thiab sau rau ntawm tus qauv. Cov fluorescence tsim los ntawm cov qauv thiab lub teeb excitation tshwm sim los ntawm lub lens nto ntawm lub hom phiaj lens thiab lub npog iav nto nkag mus rau lub hom phiaj lens tib lub sij hawm, thiab rov qab mus rau ob-xim beam splitter kom lub teeb excitation sib cais los ntawm fluorescence. , residual excitation teeb yog absorbed los ntawm thaiv cov ntxaij lim dej. Xws li kev hloov mus rau kev sib xyaw ua ke ntawm kev sib txawv excitation lim / ob-xim beam splitter / thaiv lim, nws tuaj yeem ua tau raws li qhov xav tau ntawm cov khoom sib txawv fluorescent. Qhov kom zoo dua ntawm hom fluorescence microscope no yog qhov pom ntawm qhov pom kev zoo ib yam, cov duab pom tseeb, thiab qhov loj dua qhov loj dua, qhov muaj zog fluorescence.
(2) Yuav siv cov fluorescence microscope li cas.
1. Tig lub teeb ci, thiab lub teeb ci ultra-siab mercury yuav tsum sov li ob peb feeb kom ncav cuag qhov ci ntsa iab.
2. Cov kis tau tus mob fluorescence microscope yuav tsum tau nruab qhov yuav tsum tau lim excitation ntawm lub teeb qhov chaw thiab lub condenser, thiab nruab ib tug sib thooj lim lim qab lub hom phiaj lens. Epi-fluorescence microscopes yuav tsum tau ntxig rau qhov yuav tsum tau lim excitation / dual-xim beam splitter / thaiv lim ntxig rau hauv cov qhov nyob hauv txoj kev kho qhov muag.
3. Saib xyuas nrog lub lens qis, thiab kho qhov chaw ntawm lub teeb pom kev zoo kom nws nyob hauv nruab nrab ntawm tag nrho cov teeb pom kev zoo raws li kev hloov kho ntawm cov qauv sib txawv ntawm fluorescence microscopes.
4. Muab cov qauv coj mus kuaj thiab saib tom qab tsom. Kev saib xyuas yuav tsum tau them thaum siv: tsis txhob saib ncaj qha nrog qhov kawg lim, thiaj li tsis ua rau qhov muag puas; thaum soj ntsuam cov qauv nrog lub lens roj, koj yuav tsum siv lub lens roj tshwj xeeb uas tsis muaj fluorescence; Tom qab lub teeb hluav taws xob siab mercury raug kaw, nws tsis tuaj yeem tig rov qab tam sim ntawd, nws yuav tsum tau sim. Nws tuaj yeem rov pib dua tom qab 5 feeb, txwv tsis pub nws yuav tsis ruaj khov thiab cuam tshuam rau lub neej ntawm lub teeb mercury.
(3) Kev soj ntsuam Siv lub teeb ci xiav-violet nyob rau hauv lub fluorescent microscope ntawm lub platform qhia, nws tuaj yeem pom tias o. 01 feem pua ntawm cov hlwb stained nrog acridine txiv kab ntxwv fluorescent dye, lub nucleus thiab cytoplasm zoo siab los tsim ob xim sib txawv ntawm fluorescence (ntsuab ntsuab thiab txiv kab ntxwv liab).
