Fluorescence microscopy: hauv paus ntsiab lus ntawm txoj kev

Haum thiab re-emission ntawm lub teeb ntxiv inorganic thiab organic kua yog lub txiaj ntsim ntawm phosphorescence los yog fluorescence. Qhov txawv ntawm qhov tshwm sim yog lub duration ntawm lub caij nyoog nruab nrab haum thiab emission ntawm lub teeb flux. Thaum lub fluorescence ntawm cov dab tshwm sim yuav luag ib txhij, thaum phosphorescence - nrog ib co kev ncua.

keeb kwm ntaub ntawv

Nyob rau hauv 1852, British paub txog Stokes, thawj piav fluorescence. Nws qhia ib tug tshiab lub sij hawm raws li ib tug tshwm sim ntawm lub thwmsim nrog fluorspar, uas emit teeb liab nyob rau hauv ultraviolet lub teeb. Stokes sau tseg ib qho kev nthuav tshwm sim. Nws yog pom tias lub wavelength ntawm lub fluorescent tawg yog ib txwm ntau dua qhov ndlwg ntawm lub excitation teeb.

Yuav kom paub meej tias cov kev xav nyob rau hauv lub xyoo pua puv 19 muaj ntau thwmsim. Lawv pom tias ib tug ntau yam kev kuaj fluoresce nyob rau hauv tus ntawm ultraviolet lub teeb. Ntawm cov ntaub ntawv, cov lwm yam, tau muaju, resins, minerals, chlorophyll, crude tshuaj, inorganic tebchaw, vitamins, roj. Qhov ncaj kev siv cov dyes rau lom ntsuam xyuas pib xwb nyob rau hauv xyoo 1930

Fluorescence microscopy: piav qhia

Ib txhia ntawm cov ntaub ntawv siv nyob rau hauv thawj ib nrab ntawm lub xyoo pua 20th kev tshawb fawb tshaaj siab things. стал важнейшим инструментом и в биомедицинских, и в биологических исследованиях. Tsaug rau qhov kev kawm, uas yuav tsis ua tau tiav los ntawm contrasting txoj kev, cov qauv ntawm cov fluorescence microscopy tau ua ib qho tseem ceeb lub cuab tam nyob rau hauv biomedical thiab lom tshawb fawb. Attendance tseem ceeb tau raug tau, thiab rau cov ntaub ntawv.

? Yuav ua li cas zoo li cas cov qauv ntawm cov fluorescence microscopy? Siv cov ntaub ntawv tshiab tau ua tau thiab cov kev xaiv ntawm mas tej cell submicroscopic Cheebtsam. Fluorescence microscopy yuav ntes ib molecules. Ib tug ntau yam ntawm dyes cia cov cim ntawm ntau yam khoom ntawm ib zaug. Dua li ntawm qhov tsawg spatial kev daws teeb meem ntawm lub diffraction txwv ntawm cov khoom, uas, nyob rau hauv lem, yog nyob ntawm cov kev thaj chaw ntawm tus qauv, cov cim ntawm molecules hauv qab no rau theem no yog tseem heev sai tau. Ntau kuaj tom qab irradiation sau autofluorescence. Qhov no tshwm sim yog dav siv nyob rau hauv petrology, botany, lub semiconductor kev lag luam.

nta

Kawm tsiaj ntaub so ntswg los yog pathogens feem ntau nyuab heev los yog tsis muaj zog los yog zog heev unspecific autofluorescence. Txawm li cas los, tus nqi nyob rau hauv cov kev tshawb fawb acquires kev taw qhia mus rau hauv cov ntaub ntawv uas lub Cheebtsam zoo siab heev hauv ib lub wavelength thiab emitting qhov tsim nyog teeb txaus siv. Fluorochromes ua raws li dyes muaj peev xwm ntawm nws tus kheej uas txuas mus rau lug (pom los yog tsis pom). Yog li lawv muaj lub siab selectivity rau lub hom phiaj, thiab lub quantum tawm los.

стала широко применяться с появлением естественных и синтетических красителей. Fluorescence microscopy tau lug siv txij thaum lub advent ntawm yam thiab hluavtaws dyes. Lawv muaj tej yam kev siv profiles ntawm lub emission thiab excitation thiab tsom kom meej lom lub hom phaj.

Qhia txog ib tug neeg molecules

Feem ntau, nyob rau hauv zoo tagnrho tej yam kev mob, koj muaj peev xwm sau npe ib tug nyias muaj nyias ib lub caij ci ntsa iab. Rau qhov no, cov lwm yam, nws yog tsim nyog los muab ib tug txaus tsis muaj suab nrov ntawm lub ntes thiab tus kho qhov muag tom qab. Fluorescein molecule rau tsis ua hauj lwm vim photobleaching yuav emit mus txog rau 300 txhiab. Photons. Nyob rau hauv 20% sau efficiency ntawm tus txheej txheem thiab yuav sau npe rau lawv nyob rau hauv ib tug npaum li ntawm txog 60 txhiab.

, основанная на лавинных фотодиодах или электронном умножении, позволяла исследователям наблюдать поведение отдельных молекул на протяжении секунд, а в ряде случаев и минут. Fluorescence microscopy raws li avalanche photodiodes los yog hluav taws xob multiplication, tso cai rau kev soj ntsuam mus soj ntsuam cov cwj pwm ntawm ib tug neeg molecules los ntawm lub thib ob, thiab nyob rau hauv tej rooj plaub txawm feeb.

complexity

Ib tug tseem ceeb qhov teeb meem nyob rau hauv dej siab ntawm tus tsuj ntawm optical suab nrov tom qab. Vim lub fact tias muaj ntau yam ntawm cov ntaub ntawv siv nyob rau hauv lub tsim ntawm cov ntxaij lim dej thiab lo ntsiab muag muaj ib co autofluorescence, lub zog ntawm zaum nyob rau hauv thaum ntxov ua sawv tau taw qhia kom paub mus rau lub zus tau tej cov Cheebtsam muaj ib tug uas tsis muaj fluorescence. Txawm li cas los, tom ntej thwmsim tau coj mus rau tshiab cov lus xaus. , основанная на полном внутреннем отражении, позволяет достичь низкого фона и высокоинтенсивного возбуждающего светового потока. Nyob rau hauv kev, nws pom hais tias fluorescence microscopy, raws li nyob rau hauv tag nrho cov sab hauv rog xav txog, nws tso cai rau mus cuag ib tug uas tsis muaj keeb kwm yav dhau thiab siab siv excitation teeb.

mechanism

, основанной на полном внутреннем отражении, заключаются в использовании быстрозатухающей или нераспространяющейся волны. Lub hauv paus ntsiab lus ntawm fluorescence microscopy, raws li nyob rau hauv tag nrho cov sab hauv thiaj yog siv cov evanescent yoj los yog evanescent. Nws tshwm sim nyob rau ciam ntawm xov xwm uas sib txawv refractive indices. Nyob rau hauv cov ntaub ntawv no, lub teeb beam kis tau los ntawm cov plaub. Nws muaj ib tug high refractive index parameter.

Cov plaub uas nyob ib sab mus rau lub aqueous tov los yog iav nrog tsawg parameter. Yog hais tias ib tug nqaj ntawm lub teeb qhia rau nws nyob rau ntawm ib lub uas muaj ntau ib qho tseem ceeb, lub beam yog lig reflected los ntawm cov interface. Qhov no tshwm sim, nyob rau hauv lem, ua nonpropagating tsis. Nyob rau hauv lwm yam lus, cov generated electromagnetic teb uas penetrates mus rau hauv ib tug nruab nrab nrog ib tug tsawg dua refractive index parameter rau ib tug deb tsawg tshaj 200 nanometers.

Lub evanescent yoj kev siv ntawm lub teeb yuav txaus ua kom muaj siab fluorophores. Txawm li cas los, vim hais tias ntawm nws tsis tshua me me tob, nws volume yuav heev me me. Cov no ib tug tsis tshua muaj qib tom qab.

kev hloov kho

Fluorescence microscopy yog raws li nyob rau hauv tag nrho cov sab hauv rog xav txog, yuav muab los siv nrog epi-illumination. Qhov no yuav tsum tau lo ntsiab muag nrog siab txog zauv aperture (yam tsawg kawg 1.4, tab sis nws yog ntshaw tias nws mus txog 1.45-1.6), thiab cov illuminated teb apparatus. Cov yav tas yog tiav nrog ib tug me me qhov chaw loj. Rau ntau dua uniformity siv ib tug nyias nplhaib, uas yog sim los ntawm ib feem ntawm tus kwj. Rau ib qho tseem ceeb lub, tom qab uas muaj ib tug tag nrho cov rog xav txog, peb yuav tsum tau ib theem siab ntawm refraction ntawm lub immersion nruab nrab nyob rau hauv lub lens thiab lub hau khob ntawm lub tshuab kuaj kab mob.