Ib qho kev sib piv ntawm txawv Super-Resolution Microscopy Techniques
Rau cov pa microscopy, diffraction ntawm lub teeb txwv kev daws teeb meem rau kwv yees li 250 nm. Niaj hnub no, cov txheej txheem daws teeb meem super tuaj yeem txhim kho qhov no los ntawm ntau tshaj li ntawm 10. Cov txheej txheem no tsuas yog ua tiav los ntawm peb txoj hauv kev: ib leeg-molecule localization microscopy, suav nrog photosensitive localization microscopy (PALM) thiab stochastic optical reconstruction microscopy (STORM); Structured illumination microscopy (SIM); thiab stimulated emission depletion microscopy (STED). Yuav ua li cas xaiv lub tshuab daws teeb meem super yog qhov uas txhua tus neeg mob siab txog. "Hmoov tsis zoo, tsis muaj cov hauv paus ntsiab lus yooj yim rau kev txiav txim siab siv txoj kev twg," hais Mathew Stracy, tus kws tshawb fawb tom qab kawm tiav ntawm University of Oxford, UK. "Txhua tus muaj nws qhov zoo thiab qhov tsis zoo." Cov kws tshawb fawb tseem tab tom nrhiav seb yuav xaiv txoj hauv kev zoo li cas rau ib qhov project tshwj xeeb. "Nyob rau hauv cov ntsiab lus ntawm bioimaging, cov ntsiab lus tseem ceeb uas yuav tsum tau txiav txim siab suav nrog: kev daws teeb meem ntawm lub cev thiab lub cev, kev nkag siab zoo rau photodamage, lub peev xwm sau ntawv, cov qauv tuab, thiab keeb kwm yav dhau los fluorescence lossis cell autologous Fluorescence." Nws ua haujlwm li cas Qhov ntau yam kev daws teeb meem microscopes ua haujlwm sib txawv. Nyob rau hauv cov ntaub ntawv ntawm PALM thiab STORM, tsuas yog ib feem me me ntawm cov cim fluorescent zoo siab lossis photoactivated ntawm ib lub sijhawm, ua rau lawv muaj kev ywj pheej hauv zos nrog qhov siab. Los ntawm cov txheej txheem no nrog tag nrho cov ntawv fluorescent ua rau ua tiav cov duab daws teeb meem. Stefan Hell, yog ib tus yeej ntawm 2014 Nobel nqi zog hauv Chemistry thiab tus thawj coj ntawm Max Planck lub koom haum ntawm Biophysical Chemistry, tau hais tias: "PALM / STORM system yog qhov yooj yim rau kev teeb tsa, tab sis nws nyuaj rau kev thov, vim tias cov fluorescent. Cov pab pawg yuav tsum muaj peev xwm photoactivation. Cov kev txwv qhov tsis zoo yog tias lawv yuav tsum tau kuaj pom ib qho fluorescent molecule nyob rau hauv cov ntsiab lus ntawm ib lub xovtooj, thiab tsis tshua muaj kev ntseeg siab tshaj STED. " STED siv lub laser mem tes los ua kom cov fluorophore thiab lub nplhaib zoo li lub laser kom tua cov fluorophore, tawm hauv qhov nruab nrab nanometer-loj Fluorescence rau kev daws teeb meem loj. Kev txheeb xyuas tag nrho cov qauv tsim cov duab. "Qhov zoo ntawm STED yog tias nws yog lub tshuab thawb-khawm," Hell piav qhia. "Nws ua haujlwm zoo li tus qauv confocal fluorescence microscope." Nws tuaj yeem ua cov duab nyob hauv hlwb siv fluorophores xws li ntsuab lossis daj fluorescent proteins thiab rhodamine-derived dyes. Parametric kev sib piv Txawm hais tias tag nrho cov txheej txheem daws teeb meem super tshaj qhov pom kev pom lub teeb pom kev zoo ntawm kev daws teeb meem, lawv txawv ntawm ib leeg. SIM kwv yees ob npaug ntawm qhov kev daws teeb meem mus txog 100 nm. PALM thiab STORM tuaj yeem daws 15 nm lub hom phiaj. Raws li ntuj raug txim, STED muab qhov kev daws teeb meem ntawm 30 nm hauv cov hlwb nyob thiab 15 nm hauv cov hlwb. Thaum nws los txog rau cov ntawv thov tshwj xeeb, peb kuj yuav tsum xav txog cov teeb liab-rau-nruab nrab piv. Qee zaum, kev daws teeb meem qis dua tab sis siab dua SNR tuaj yeem ua rau cov duab zoo dua li qhov sib txawv (kev daws teeb meem siab dua tab sis qis dua SNR). Kev nrawm ntawm cov duab tau los kuj tseem ceeb heev, tshwj xeeb tshaj yog rau cov hlwb nyob. Stracy tau hais tias "Txhua txoj kev daws teeb meem super yog qeeb dua li cov txheej txheem fluorescence imaging ib txwm muaj," Stracy tau hais. "PALM / STORM yog qhov qeeb tshaj plaws, nws xav tau kaum tawm txhiab tus thav ntawv kom tau txais ib daim duab, SIM xav tau kaum ob lub thav ntawv, thiab STED yog lub tshuab luam ntawv, yog li qhov kev tau txais ceev yog nyob ntawm qhov loj ntawm qhov kev pom." Ntxiv nrog rau cov cell nyob los yog cov imaging cell ruaj khov, qee cov kws tshawb fawb kuj xav nkag siab tias cov khoom txav mus li cas. Stracy txaus siab rau kev nkag siab txog kev hloov pauv ntawm cov kab mob lom neeg hauv cov hlwb, tsis yog cov duab zoo li qub. Nws ua ke PALM nrog ib qho particle taug qab los txheeb xyuas qhov muaj zog hauv cov hlwb nyob. Nyob rau hauv txoj kev no, nws tuaj yeem taug qab cov cim molecules ncaj qha thaum lawv ua lawv txoj haujlwm. Txawm li cas los xij, nws ntseeg tias SIM tsis tsim nyog rau kev kawm cov txheej txheem dynamic ntawm qib molecular, tab sis vim nws qhov nrawm nrawm, nws yog qhov tshwj xeeb tshaj yog tsim nyog rau kev soj ntsuam cov qauv ntawm cov qauv loj, xws li tag nrho cov chromosomes. Qhov kawg tshwm sim Hauv xyoo 2017, Hell's team tau tshaj tawm MINFLUX super-resolution microscope hauv Science. Raws li ntuj raug txim, txoj kev daws teeb meem super no ua tiav qhov kev daws teeb meem ntawm 1 nm thawj zaug. Tsis tas li ntawd, nws tuaj yeem taug qab ib tus neeg molecules hauv cov cell nyob yam tsawg kawg 100 npaug sai dua li lwm txoj hauv kev. Lwm cov kws tshawb fawb kuj tau hais lus zoo heev ntawm MINFLUX microscope. Shechtman tau hais tias "Cov ntawv thov tshiab thiab cov kev coj ua tau tsim kho tas li, tab sis ob qho kev nce qib sawv rau kuv," Shechtman tau hais. Ib qho yog MINFLUX. "Nws siv txoj kev txawj ntse kom tau txais qhov tseeb ntawm qhov chaw molecular." Hais txog kev loj hlob thib ob, Shechtman tau hais txog WE Moerner thiab nws cov npoj yaig ntawm Stanford University. Moerner kuj yog tus tau txais 2014 Nobel nqi zog hauv Chemistry. Ib tug ntawm cov yeej. Txhawm rau daws qhov kev txwv ntawm kev daws teeb meem tshwm sim los ntawm anisotropic tawg ntawm fluorescent ib qho molecules, cov kws tshawb fawb siv cov excitation polarizations sib txawv los txiav txim qhov kev taw qhia thiab txoj hauj lwm ntawm cov molecules. Tsis tas li ntawd, lawv tau tsim cov menyuam kawm ntawv qhov chaw. Cov txheej txheem no txhim kho lub peev xwm los tsim cov qauv hauv zos. Txog cov ntawv fluorescent Hauv ntau daim ntawv thov daws teeb meem, cov ntawv tseem ceeb heev. Kuj tseem muaj qee lub tuam txhab uas muab cov khoom muaj feem xyuam. Piv txwv li, lub teb chaws Yelemees Miltenyi tau koom tes nrog Abberior, ib lub tuam txhab tsim los ntawm Stefan Hell, los muab kev cai tshuaj tiv thaiv kab mob sib txuas rau kev daws teeb meem microscopy dyes. Ib tug xov tooj ntawm lwm lub tuam txhab kuj muab cov cim cim. Christoph Eckert, tus kws tshaj lij kev lag luam ntawm ChromoTek hais tias "Peb cov Nano-Boosters tsawg heev, tsuas yog 1.5 kDa, thiab tshwj xeeb heev," Cov proteins no khi ntsuab thiab liab fluorescent proteins (GFP thiab RFP). Lawv tau muab los ntawm alpaca antibody fragments, hu ua VHH los yog nanobodies, nrog zoo heev khi zog thiab ruaj khov zoo yam tsis muaj batch-rau-batch variation. Cov cim no tsim nyog rau ntau yam kev daws teeb meem loj xws li SIM, PALM, STORM thiab STED. Ai-Hui Tang, tus pab xibfwb ntawm University of Maryland School of Medicine, thiab cov npoj yaig tau siv ChromoTek's GFP-Booster thiab STORM los tshawb nrhiav cov ntaub ntawv nthuav tawm hauv lub paj hlwb. Lawv pom molecular nanoclusters, hu ua nanocolumns, hauv presynaptic thiab postsynaptic neurons. Cov kws tshawb fawb ntseeg tias cov qauv no qhia tau hais tias lub paj hlwb hauv nruab nrab siv cov hauv paus ntsiab lus yooj yim los tswj thiab tswj cov synaptic efficiency. Ntau cov qauv ntawm kev daws teeb meem zoo tshaj plaws thiab ntau txoj hauv kev tau coj cov kws tshawb fawb txawm tob rau hauv kev paub tsis meej txog kev lom neeg. Los ntawm kev rhuav tshem qhov sib txawv ntawm qhov pom ntawm lub teeb pom kev, cov kws kho tsiaj muaj peev xwm txawm tias "zoo saib xyuas" cov haujlwm ntawm cov hlwb.
