Microscopic Imaging Classroom yog daim ntawv thov ntawm Fluorescence Microscopy
Tag nrho cov kev xav sab hauv fluorescence microscopy (TIRFM) yog ib qho txheej txheem uas siv cov evanescent nthwv dej uas tsim los ntawm lub teeb beam propagating ntawm ob qhov sib txawv refractive index media los soj ntsuam qhov saum npoo ntawm fluorescently sau cov hlwb nyob. Nyob rau hauv kev xyaum, thaum ib tug teeb meem laser beam ntsib lub interface ntawm lub coverlip thiab lub aqueous nruab nrab uas muaj cov hlwb, nws yog reflected ntawm lub kaum sab xis tseem ceeb (tag nrho cov sab hauv xav). Vim hais tias lub zog ntawm evanescent nthwv dej txo qis exponentially nrog qhov kev ncua deb ntawm cov ntaub npog, tsuas yog fluorophores nyob rau hauv 10 nanometers ntawm qhov chaw (nruab nrab ntawm 10 thiab 200 nanometers) zoo siab los ntawm evanescent yoj, thaum fluorophores nyob deb heev yog unexcited. Muaj kev cuam tshuam. Yog li, TIRFM ua rau muaj cov teeb liab siab los ntawm fluorophores nyob ze ntawm daim npog npog, muab tso rau ntawm qhov tsaus ntuj heev, muab cov teeb liab-rau-suab nrov piv. Qhov kev txwv tsis pub tshaj plaws ntawm excitation qhov tob yog qhov zoo tagnrho rau kev kawm ib qho molecules los yog daim nyias nyias thiab cov organelle muaj pes tsawg leeg nyob rau hauv cov hlwb uas nyob ze ntawm daim npog npog (saib daim duab 8(e)). Txij li thaum excitation raug txwv rau cov cheeb tsam nyias nyob ze ntawm daim npog npog, photobleaching thiab phototoxicity kuj raug txwv rau cov cheeb tsam no, ua TIRFM yog ib txoj hauv kev zoo tshaj plaws rau kev soj ntsuam mus sij hawm ntev. Cov txheej txheem tau dhau los ua cov cuab yeej tseem ceeb rau kev kawm ntau yam tshwm sim hauv cell thiab molecular biology.
Deconvolution yog ib qho algorithm siv rau ib txheej ntawm tag nrho cov duab tsom xam tau raws qhov kho qhov muag (Z) axis los txhim kho cov teeb liab photon hauv ib pawg ntawm cov duab rau ib lub dav hlau duab los yog ntau lub dav hlau focal. Microscopes yuav tsum tau nruab nrog high-precision motorized focus drives los lav cov duab tau los ntawm lub sij hawm uas tau hais meej meej ntawm cov dav hlau focal ntawm cov qauv. Hauv daim ntawv thov raug (saib daim duab 8(f)), cov txheej txheem deconvolution yog siv los deblur thiab tshem tawm lub teeb pom kev ntawm lub dav hlau focal uas siv dav fluorescence excitation thiab emission. Daim ntawv thov nyuaj tshaj plaws yog siv cov txheej txheem deconvolution rau tag nrho cov duab pawg los tsim cov kev xav pom lossis 3D qauv. Ib txheej ntawm cov duab widefield siv rau deconvolution captures theoretical siab tshaj tus naj npawb ntawm photons emitted los ntawm cov qauv. Cov txheej txheem deconvolution redistributes qhov "blurred" intensities ntawm photons emissions saum toj no thiab hauv qab lub focal dav hlau rov qab mus rau lub qub dav hlau. Yog li, deconvolution siv ze li ntawm tag nrho cov muaj emission intensities thiab muab ib qho kev pom lub teeb pob nyiaj siv, ua cov txheej txheem no txoj kev xaiv rau heev light-sensitive qauv.
Ib qho kev hloov pauv ntawm lub zog resonance hloov pauv tshwm sim ntawm fluorescence microscopy, fluorescence lossis Förster resonance zog hloov (FRET) yog siv kom tau txais cov ntaub ntawv ntau ntawm lub cev thiab qhov chaw ntawm kev sib txuas thiab kev sib cuam tshuam ntawm cov proteins, lipids, enzymes thiab nucleic acids hauv cov hlwb nyob. FRET tuaj yeem ua haujlwm tsis tu ncua hauv xeev lossis lub sijhawm daws teeb meem, tab sis lub sijhawm daws teeb meem FRET imaging muaj qhov zoo dua ntawm qhov tseeb dua daim duab qhia tus neeg txais nyiaj nyob deb. Tus qauv widefield fluorescence microscope nruab nrog tsim nyog excitation thiab emission lim thiab rhiab lub koob yees duab yuav siv tau rau FRET imaging. Biosensors uas qhaub cij ib puag ncig rhiab protein lossis peptide ntawm ob FRET-tswj cov fluorescent proteins tam sim no tau siv dav hauv cell biology. Cov kev sojntsuam no tau yooj yim pom nyob rau hauv qhov dav-teeb fluorescence microscopy siv sensitized emission FRET cov txheej txheem ua ke nrog kev txheeb xyuas qhov sib piv. Tsis tas li ntawd, spectral imaging thiab linear unmixing siv laser scanning confocal microscopy tuaj yeem pab saib xyuas FRET phenomena hauv biosensors thiab lwm yam kev siv fluorescent protein.
Fluorescence lifetime imaging microscopy (FLIM) yog cov txheej txheem sophisticated muaj peev xwm ntawm ib txhij sau cov fluorescence lub neej thiab qhov chaw nyob ntawm fluorophore ntawm txhua qhov chaw hauv daim duab. Txoj kev no muab cov txheej txheem los kawm txog ib puag ncig tsis zoo xws li pH, ionic concentration, hnyav polarity, noncovalent kev sib cuam tshuam, viscosity, thiab oxygen nro nyob rau hauv tib lub hlwb thiab nthuav tawm cov ntaub ntawv nyob rau hauv spatial thiab temporal arrays. FLIM kev ntsuas ntawm lub xeev zoo siab lub neej ntawm nanosecond scale yog ywj siab ntawm lub zos fluorophore concentrations, photobleaching teebmeem, thiab txoj kev ntev (piv txwv li thickness), tab sis yog rhiab heev rau lub xeev kev xav xws li resonance zog hloov. Qhov tseeb, kev sib koom ua ke FLIM nrog FRET los ntawm kev saib xyuas lub neej hloov pauv ntawm cov neeg pub fluorescent ua ntej thiab tom qab lub zog hloov pauv tau suav tias yog ib txoj hauv kev zoo tshaj plaws los kawm txog qhov tshwm sim no.
Kev txav mus los (transverse diffusion coefficient) ntawm fluorescently sau macromolecules thiab cov fluorophores me tuaj yeem txiav txim siab los ntawm fluorescence rov qab tom qab photobleaching (FRAP) cov txheej txheem. Nyob rau hauv FRAP, ib cheeb tsam me me heev (ob peb lub micrometers hauv txoj kab uas hla) yog illuminated, feem ntau nrog lub laser, los ua tiav photobleaching ntawm fluorophore hauv cheeb tsam ntawd. Qhov tshwm sim yog qhov txo qis lossis kev puas tsuaj ntawm fluorescence. Tom qab lub photobleaching pulse, tus nqi thiab qhov ntev ntawm cov fluorescence siv rov qab nyob rau hauv cov cheeb tsam bleached tau saib xyuas raws li lub sijhawm ua haujlwm ntawm qhov qis excitation intensities los tsim cov ntaub ntawv ntawm kinetics ntawm fluorophore repopulation thiab rov qab (Daim duab 9). FRAP feem ntau yog siv EGFP lossis lwm yam fluorescent proteins. Cov txheej txheem photoactivation cuam tshuam yog ua raws li cov khoom siv tshwj xeeb uas muaj fluorophores lossis cov khoom siv fluorescent zoo sib xws uas tuaj yeem qhib tau los ntawm luv luv pulses ntawm UV lossis violet. Photoactivation thiab FRAP tuaj yeem siv los ua cov txheej txheem ntxiv los txiav txim siab txog kev txav mus los.
Hauv cov txheej txheem cuam tshuam nrog FRAP, hu ua fluorescence poob tom qab photobleaching (FLIP), ib cheeb tsam fluorescent uas tau hais tseg hauv ib lub xov tooj ntawm tes tau rov ua dua photobleaching los ntawm khaus irradiation. Yog hais tias tag nrho cov fluorophores muaj peev xwm diffuse mus rau hauv cheeb tsam uas tau photobleached thaum lub sij hawm lub sij hawm ntsuas, qhov no yuav ua rau tag nrho cov teeb liab fluorescent nyob rau hauv lub cell. Los ntawm kev xam tus nqi ntawm cov fluorescence ploj ntawm tag nrho cov xovtooj ntawm tes, lub diffusional txav ntawm lub hom phiaj fluorophore tuaj yeem txiav txim siab. Tsis tas li ntawd, FLIP tuaj yeem txheeb xyuas qhov chaw thiab qhov xwm txheej ntawm ib qho kev cuam tshuam ntawm cov kab mob ntawm cov hlwb, xws li cov teeb meem ntawm soma thiab axon ntawm neuron.
Fluorescence correlation spectroscopy (FCS), feem ntau yog siv rau hauv laser scanning confocal microscopy lossis multiphoton microscopy, yog ib txoj hauv kev tsim los txiav txim siab cov ntaub ntawv kinetic xws li tshuaj tiv thaiv tus nqi, diffusion coefficients, Cov txheej txheem rau qhov hnyav molecular, txaus tus nqi thiab sib sau ua ke. Hauv FCS, lub ntim me me (kwv yees li ib lub femtometer; qhov cuam tshuam-txhim kho ntawm lub laser) yog illuminated nrog lub teeb pom kev laser los sau cov autofluorescence siv zog hloov pauv vim yog kev ua haujlwm ntawm cov fluorescent molecules raws li kev ua haujlwm ntawm lub sijhawm hauv ntim ntim los ntawm fluorescent. molecules (Daim duab 10). Cov me me fluorophores diffuse sai heev nyob rau hauv lub illuminated ntim, ua luv luv tawg ntawm random siv. Hloov pauv, cov complexes loj dua (fluorophores khi rau macromolecules) txav qeeb dua, ua kom ntev dua, siv sijhawm ntev dua cov qauv siv fluorescence.
Thaum fluorescented cov qauv qauv yog densely tau ntim thiab sib tshooj hauv cov cheeb tsam uas muaj sia nyob, lawv cov kev faib khoom tsis txaus ntseeg yog qhov nyuaj los soj ntsuam. Fluorescence spot microscopy (FSM) yog cov txheej txheem sib xws nrog yuav luag txhua qhov kev ntsuas kev ntsuas uas siv qhov zoo ntawm qhov tsis tshua muaj siab ntawm cov fluorescently sau npe subunits, txo cov fluorescence tawm ntawm lub ntsej muag, thiab txhim kho kev pom ntawm cov ntawv sau thiab lawv cov dynamics hauv cov cheeb tsam tuab. FSMs yog siv los ntawm kev sau npe tsuas yog ib feem ntawm tag nrho cov qauv ntawm kev txaus siab. Nyob rau hauv qhov kev nkag siab no, FSM zoo ib yam li kev ua FCS nyob rau hauv tag nrho cov kev pom, txawm hais tias nws muab ntau qhov tseem ceeb ntawm cov qauv spatial es tsis yog qhov kev ntsuam xyuas ntawm lub cev. Fluorescent spot microscopy yog tshwj xeeb tshaj yog pab tau nyob rau hauv kev txiav txim ntawm kev mus los thiab sib sau ua ke ntawm cytoskeletal ntsiab xws li actin thiab microtubules nyob rau hauv lub hlwb hyperactive.
Stimulated emission depletion microscopy (STED) yog ib qho kev daws teeb meem zoo tshaj plaws nrog kev daws teeb meem spatial deb tshaj qhov kev txwv tsis pub dhau, siv lub nplhaib-zoo li lub teeb depleted los ncig lub teeb me me ntawm lub teeb ci kom tau txais sub-50 nm ntawm-axis rau kev daws teeb meem. Cov txheej txheem tso siab rau kev zoo siab ntawm fluorophores nrog synchronized laser pulses thiab spatially sib koom tes ncig STED pulses uas deplete lub teeb emitted, suppressing fluorescence ntawm zoo siab molecules nyob ib ncig ntawm lub laser scanning tsom xam. Fluorescence generated nyob rau hauv periphery ntawm qhov chaw yog suppressed, tab sis tsis nyob rau hauv nruab nrab ntawm qhov chaw, yog li no ho txo qhov luaj li cas ntawm qhov chaw fluorescent thiab correspondingly nce qhov kev daws teeb meem. STED tau ua pov thawj tias yog ib qho cuab yeej muaj txiaj ntsig zoo rau kev kuaj pom cov hlwb nyob. Lwm cov kev daws teeb meem zoo tshaj plaws, xws li photoactivated localization microscopy (PALM) thiab teeb pom kev teeb pom kev zoo microscopy (SIM), kuj tseem yuav dhau los ua cov cuab yeej tseem ceeb rau kev yees duab ntawm tes nyob rau yav tom ntej.
Kev siv cov noob caj noob ces encoded fluorescent proteins thiab cov hluavtaws fluorophores siab heev rau cov duab nyob hauv cell qhib lub qhov rooj rau cov kev kho qhov muag tshiab rau kev saib xyuas lub cev lub cev thiab kev sib raug zoo. Microscopists tam sim no muaj cov cuab yeej ua tiav los soj ntsuam thiab sau cov duab cov ntaub ntawv ntawm cov txheej txheem cellular tshwm sim nyob rau ntau lub sijhawm thiab ntawm ntau qhov kev daws teeb meem. Cov xwm txheej qeeb tuaj yeem pom tau yooj yim thiab kaw siv laser scanning confocal microscopy, thaum cov xwm txheej ceev nrawm tuaj yeem tau txais los ntawm kev siv tshuab spinning disk. Tsis tas li ntawd, multiphoton microscopy ua rau pom kev sib sib zog nqus hauv cov ntaub so ntswg tuab, thiab tag nrho cov txheej txheem kev xav sab hauv tuaj yeem ua rau kev sojntsuam ntawm daim nyias nyias nrog qhov tseeb. Advanced fluorescence txoj kev, xws li FRET, FLIM, FRAP, FCS, FSM, SIM, PALM, thiab STED, tuaj yeem siv los saib xyuas cov protein-protein kev sib cuam tshuam ntawm kev daws teeb meem feem ntau zoo dua li cov uas tau tso cai los ntawm qhov txwv tsis pub muaj. Nrog rau kev nce qib hauv fluorophore, lub tshuab tsom iav thiab cov cuab yeej kuaj xyuas, lub ntiaj teb dav dua yuav raug coj mus rau "nyob rau hauv lub tshuab microscope".
