Txoj kev soj ntsuam tseem ceeb ntawm optical microscope yog kev soj ntsuam fluorescence
Fluorescence yog hais txog cov txheej txheem uas cov tshuaj fluorescent emits lub teeb nrog lub wavelength ntev yuav luag ib txhij thaum nws yog irradiated nrog lub teeb ntawm ib lub wavelength (Daim duab 1). Thaum lub teeb ntawm ib lub wavelength tshwj xeeb (excitation wavelength) ntaus lub molecule, xws li cov nyob rau hauv ib tug fluorophore, lub zog photon yog absorbed los ntawm lub molecule lub electrons. Tom ntej no, cov hluav taws xob hloov pauv ntawm lub xeev hauv av (S0) mus rau qib siab zog, lub xeev zoo siab (S1'). Cov txheej txheem no hu ua excitation①. Cov hluav taws xob nyob twj ywm hauv lub xeev zoo siab rau 10-9–10-8 vib nas this, thaum lub electron poob qee lub zog②. Thaum lub sij hawm tus txheej txheem ntawm electrons tawm hauv lub xeev excitation (S1) thiab rov qab mus rau hauv av xeev ③, lub zog seem absorbed thaum lub sij hawm excitation txheej txheem yog tso tawm.
Lub sijhawm nyob ntawm cov fluorescent molecule hauv lub xeev zoo siab yog lub neej fluorescence, uas feem ntau yog nyob rau theem nanosecond, thiab yog tus yam ntxwv ntawm cov fluorescent molecule nws tus kheej. Fluorescence Lifetime Imaging (FLIM), uas siv fluorescence lifetime imaging technology, hu ua fluorescence lifetime imaging (FLIM). Ntxiv nrog rau kev siv tshuaj fluorescence, kev ntsuas qhov tob thiab qhov tseeb tuaj yeem tau txais kom tau txais kev sib haum xeeb ntawm cov molecular, kev sib cuam tshuam ntawm cov molecular, thiab microenvironment ntawm molecules. Cov ntaub ntawv uas nyuaj kom tau txais nrog cov qauv kho qhov muag pom.
Lwm cov cuab yeej tseem ceeb ntawm fluorescence yog Stokes hloov, qhov sib txawv ntawm lub wavelength ntawm excitation thiab emission peaks (Daim duab 2). Feem ntau cov emission wavelength ntev dua li qhov excitation wavelength. Qhov no yog vim hais tias electrons yuav poob ib feem ntawm lawv lub zog los ntawm cov txheej txheem so tom qab cov tshuaj fluorescent zoo siab thiab ua ntej tso photons. Cov tshuaj fluorescent nrog qhov loj dua Stokes hloov tau yooj yim dua los soj ntsuam nyob rau hauv lub fluorescence microscope.
Fluorescence Microscopy thiab Fluorescence Filter Cubes
Fluorescence microscope yog ib qho khoom siv kho qhov muag uas siv cov khoom fluorescence rau kev soj ntsuam thiab kev ntsuam xyuas, thiab tau siv dav hauv ntau yam xws li cell biology, neurobiology, botany, microbiology, pathology, thiab noob caj noob ces. Fluorescence imaging muaj qhov zoo ntawm siab rhiab heev thiab siab tshwj xeeb, thiab yog heev haum rau kev soj ntsuam ntawm kev faib cov proteins thiab organelles tshwj xeeb hauv cov ntaub so ntswg thiab hlwb, kev kawm ntawm colocalization thiab kev sib cuam tshuam, kev taug qab ntawm lub neej dynamic txheej txheem xws li ion concentration hloov. , lwm.
Feem ntau cov molecules hauv hlwb tsis fluoresce, thiab kom pom lawv lawv yuav tsum tau fluorescently sau npe. Muaj ntau txoj hauv kev ntawm fluorescent labeling, xws li daim ntawv lo ncaj qha (xws li siv DAPI los sau npe DNA), lossis kev tiv thaiv kab mob siv cov tshuaj tiv thaiv kab mob antigen, lossis siv cov fluorescent proteins (xws li GFP, ntsuab fluorescent protein) los sau cov hom phiaj proteins. , thiab reversible binding. Synthetic dyes (xws li Fura-2) thiab lwm yam.
Tam sim no, lub fluorescence microscope tau dhau los ua cov cuab yeej ntsuas tus qauv ntawm ntau lub chaw soj nstuam thiab cov duab thaij duab, thiab yog tus pab zoo rau peb cov kev sim niaj hnub. Fluorescence microscopes feem ntau muab faib ua peb pawg: upright fluorescence microscopes (tsim rau slicing), inverted fluorescence microscopes (tsim rau cov hlwb nyob, noj rau hauv tus account slicing), fluorescent stereoscopes (tsim rau cov qauv loj, xws li cov nroj tsuag, zebrayofish (tus neeg laus) ), medaka, nas/rat organs, etc.).
Fluorescence filter block yog lub hauv paus tseem ceeb ntawm kev siv tshuaj tua kab mob fluorescence. Nws muaj peb ntu: lim excitation, emission lim thiab dichroic beam splitter. Nws yog ntsia rau hauv lub log lim. Piv txwv li, Leica DMi8 yog nruab nrog 6- txoj haujlwm lim log (Fig. 3 ). Tus naj npawb ntawm txoj haujlwm ntawm lub tshuab tsom iav sib txawv yuav txawv, thiab qee lub tshuab tsom iav siv lim thaiv cov swb.
Lub lim thaiv ua lub luag haujlwm tseem ceeb hauv kev fluorescence imaging: lub lim excitation xaiv lub teeb excitation kom excite tus qauv, thiab thaiv lub teeb ntawm lwm cov wavelengths; Lub teeb dhau los ntawm kev lim dej excitation dhau los ntawm daim iav dichroic (nws txoj haujlwm yog los cuam tshuam lub teeb excitation thiab xa cov fluorescence), Tom qab kev xav, nws yog tsom los ntawm lub hom phiaj lens, irradiates tus qauv, thiab excites cov fluorescence sib xws, uas yog , tawm lub teeb. Lub teeb emitted yog sau los ntawm lub hom phiaj lens, dhau los ntawm dichroic beam splitter, thiab ncav cuag lub emission lim. Raws li pom nyob rau hauv daim duab 4: lub excitation wavelength yog 450-490nm, daim iav dichroic qhia lub teeb luv dua 510nm, xa lub teeb ntev dua 510nm, thiab qhov tau txais ntau yam ntawm lub teeb emitted yog 520-560nm.
