Imaging Lub raum: Los ntawm Lub Teeb rau Super-resolution Microscopy
Mar 17, 2022
Hu rau: ali.ma@wecistanche.com
Maria Lucia Angelotti1,2, Giulia Antonelli1,2 Carolina Conte1,2thiab Paola Romagnani1,2
TSAB NTAWV
Qhov tseem ceeb achievements nyob rau hauvraumLub physiological thiab pathophysiological mechanisms tuaj yeem ua rau muaj kev vam meej hauv kev siv tshuab microscopy. Muaj ntau yam peb paub txog lub architecture ntawm lubraumyog ua raws li cov lus piav qhia tseem ceeb ntawm cov kws kho mob lub cev siv lub teeb microscopy thiab tom qab ntawd los ntawm kev tsom xam ultrastructural muab los ntawm electron microscopy. Ob txoj kev no tau siv rau thawj qhov kev faib tawm ntawm cov kab mob raum thiab rau lawv cov kev hloov kho tas li. Tsis ntev los no, cov txheej txheem ntawm cov duab tshiab tau ntxiv cov kev tshuaj ntsuam ntxiv ntawm lub sijhawm thiab qhov chaw. Confocal microscopy tau tso cai rau peb kom pom cov duab kho qhov muag raws li z-axis thiab muaj cov software tsom xam tshwj xeeb tau muab peb-seem rendering ntawm cov ntaub so ntswg tuab. Multiphoton microscopy tso cai rau peb tshawb xyuas ib txhijraummuaj nuj nqi thiab qauv nyob rau hauv real-time. Fluorescence-lifetime imaging microscopy tso cai rau peb kawm txog kev faib tawm ntawm cov metabolites. Super-resolution microscopy nce rhiab heev thiab kev daws teeb meem mus txog qib nano-scale. Nrog cryo-electron microscopy, cov kws tshawb fawb tuaj yeem pom cov neeg biomolecules ntawm qib atomic ncaj qha rau hauv cov ntaub so ntswg thiab nkag siab txog lawv cov kev cuam tshuam ntawm cov qib subcellular. Thaum kawg, matrix-pab laser desorption / ionization imaging mass spectrometry tso cai rau kev ntsuas ntawm pua pua ntawm cov molecules sib txawv tib lub sijhawm ntawm cov ntaub so ntswg ntawm qhov kev daws teeb meem siab. Qhov kev tshuaj xyuas no muab cov ntsiab lus ntawm muajraumcov tswv yim kev yees duab, nrog rau kev tsom mus rau qhov muaj feem cuam tshuam ntawm kev txhim kho thev naus laus zis tsis ntev los no.
Ntsiab lus:immunohistochemistry,raumbiopsy, podocytes,nyob zecev, qia hlwb
Cistanche herba rau mob raum, nyem qhov no kom tau txais cov qauv
Taw qhia
Covlub cevfascinating structural complexity yuav tsum tau ua kom tiav nws ntau yam physiological functions. Thaum pib, kawm covraumtau txwv rau kev soj ntsuam macroscopic thiab lawv txoj kev koom tes rau kev kuaj mob ntawm cov kab mob hauv lub raum yog qhov txwv tsis pub muaj. Clinical manifestations tau piav qhia tab sis lub hauv paus pathogenetic mechanisms tsis paub. Cov kev ua tiav hauv kev nkag siab txog lub raum physiology thiab pathophysiology tuaj yeem ua rau muaj txiaj ntsig zoo rau kev kawm txuas ntxiv hauv microscopy teb. Kev sim ntawmraumdaim duab pib ntev dhau los [1]. Hauv xyoo 1666, tus kws kho mob anatomist Malpighi tau tshuaj xyuasraumcov ntaub so ntswg nrog lub teeb pom kev zoo uas muaj lub zog kwv yees li 25-30 thiab tau piav qhia thawj zaug lub glomerulus li 'ib caj pas uas tso zis tawm ntawm cov ntshav'[2]. Qhov kev pab cuam no raug tsis quav ntsej txog thaum xyoo 1842 thaum cov qauv nephron raug tshem tawm nrog 10 npaug ntau dua lub teeb pom kev zoo los ntawm Sir William Bowman. Bowman nrhiav pom lub periglomerular capsule (tom qab lub npe hu ua Bowman capsule) raws li anatomically txuas rau thawj feem ntawm lub tubule [3]. Xyoo 1862, Jacob Henle tau piav qhia txog lub voj-zoo li tubule ntu uas coj nws lub npe, txuas lub cortical tubule thiab lub raum papilla.
Ua tsaug rau cov kev taw qhia ntawm lub teeb microscopy, ib tug histomorphological faib ntawmraumkab mobua tau [1]. Lub caij nyoog los soj ntsuam qhov tshwm sim hauv cov kab mob raum tau tso cai rau ib tus neeg nkag siab tias cov kev hloov pauv pathological sib txawv ntawm cov neeg mob uas muaj qhov tshwm sim zoo sib xws. Txawm li cas los xij, thawj cov kev tshawb fawb txog kev hloov pauv pathological hauv lub raum cov ntaub so ntswg tau ua nyob rau hauv kev kuaj mob ntawm lub cev, ua rau feem ntau ntawm kev piav qhia ntawm cov kab mob ntev. Qhov tsis muaj ntaub ntawv hais txog kev hloov pauv ntawm lub raum pathologies thiab cov teeb meem ntawm autolytic phenomena, cuam tshuam nrog kev siv cov ntaub so ntswg tom qab, txwv txoj kev paub ntawm tib neeg cov kab mob. Nyob rau hauv 1951, qhov kev taw qhia ntawmraumbiopsy tso cai saib ncaj qha thiab kawm tsis tau tsuas yog mob ntev xwb tab sis kuj ntawm cov kab mob raum mob. Meanwhile, microscopy maj mam dhau los ua sophisticated nrog nce rhiab heev thiab kev daws teeb meem mus txog qib nanoscale. Hauv qhov kev tshuaj xyuas no, peb yuav muab cov ntsiab lus ntawm cov kev tshawb pom hauv lub raum uas ua tau los ntawm kev nce qib hauv kev siv tshuab microscopy, nrog rau kev tsom mus rau qhov muaj peev xwm hloov mus los ntawm cov kev tsim kho tshiab tshaj plaws.
VISUALIZING IMMUNOPATHOLOGY OF THE KIDNEY
Immunolabelling thiab fluorescence microscopy
Qhov ua tau kom tau txais cov neeg sawv cev ntawm cov ntaub so ntswg nyob tso cai siv cov txheej txheem tawm tshiab uas yuav tsum tau khaws cov ntaub so ntswg ntau tshaj plaws, xws li cov txheej txheem immunohistochemical (fluorescent ua ntej, tom qab ntawd enzymatic). Qhov tseeb, xyoo 1950, Coons tau tsim cov txheej txheem ntawm kev sau cov tshuaj tiv thaiv nrog fluorescein isocyanate, uas yog, immunofluorescence, tsis muaj kev puas tsuaj rau lub peev xwm los cuam tshuam nrog lawv cov ntaub so ntswg antigens. Txij thaum ntawd los, immunofluorescence tau muab tso rau hauv cov txheej txheem kuaj mob rau kev ntsuam xyuas ntawm cov khoom khov rau lub raum biopsy. Thawj thawj zaug, cov tswv yim no tso cai rau kev soj ntsuam ntawm intrarenal immunoglobulins (Igs) thiab ntxiv cov nyiaj tso rau hauv cov neeg mob uas cuam tshuam los ntawm glomerulonephritis, ua rau muaj kev ntxub ntxaug ntawm cov kab mob sib txawv hauv qab qee yam ntawm cov ntaub so ntswg tsis tshwj xeeb, piv txwv li, hauv crescentic glomerulonephritis [4]. Tsis tas li ntawd, qhov muaj peev xwm los soj ntsuam tus yam ntxwv staining qauv thiab kev sib txuas ntawm fluorescein rau cov tshuaj tiv thaiv uas tsis yog Immunoglobulin G (IgG) ua rau kev lees paub ntawm Immunoglobulin A (IgA) deposits hauv IgA nephropathy, nrog rau ntxiv cov deposits hauv post-infectious glomerulo. thiab membranoproliferative glomerulonephritis [4].
Confocal microscopy
Fluorescence microscopy underwent kev txhim kho kev txhim kho nrog kev qhia txog confocal microscopy thaum xyoo 1980s. Cov ntsiab lus tseem ceeb hauv confocal microscopy yog taw tes teeb pom kev zoo tsom thiab luam theej tawm ntawm cov qauv thiab qhov sib txawv ntawm confocal aperture (pinhole) nyob rau hauv pem hauv ntej ntawm lub ntes uas tso cai rau kev sau ntawm lub teeb los ntawm tsuas yog ib feem nyias nyob ib ncig ntawm lub dav hlau focal (hu ua ntu optical). Confocal microscopy tau qhib thawj zaug hauv vivo kev tshawb fawb ntawm lub raum hauv kev sim ua qauv thiab kev soj ntsuam ntawm kev nce thiab thauj cov fluorescent molecules los ntawm tubules tsis zoo.lub raumtom qab micropuncture. Qhov no tso cai rau kev tshawb fawb ntawm tubular muaj nuj nqi hauv physiological thiab pathological mob [5].
The possibility to sequentially image optical sections along the z-axis and the availability of specific analysis software permitted a three-dimensional (3D) rendering of thicker tissue specimens, showing the frequent inconsistency and misrepresentations provided by 2D analyses [6, 7] (Figure 1A and a'). In addition, the introduction of genetically encoded fluorescent proteins in transgenic mice permitted the labeling of specific cells and tracing them directly within tissues without immunostaining (lineage tracing strategy) [6, 7, 8]. However, confocal microscopy still did not resolve details >200 nm, vim qhov kev txwv tsis pub dhau ntawm lub teeb pom kev, photobleaching, thiab phototoxicity.
Nkag siab txog GLOMERULAR FILTRATION BARRIER: ELECTRON MICROSCOPY
Thawj qhov kev sim kom kov yeej qhov kev daws teeb meem ntawm lub teeb thiab fluorescence microscopes yog xyoo 1931 thaum Ernst Ruska thiab Max Knoll tau tsim lub tshuab hluav taws xob hluav taws xob (EM), uas siv lub teeb hluav taws xob nrawm, es tsis txhob siv lub teeb ci, ua qhov zoo siab, thiab muaj lub zog daws teeb meem ntau dua li lub teeb microscopes (txog 0.2 nm). Rau qhov kev tsim kho tshiab no, xyoo 1986 Ernst Ruska tau txais txiaj ntsig Nobel nqi zog hauv Physics. Ua tsaug rau qhov kev daws teeb meem ntau ntxiv, nws tau dhau los ua pov thawj tias glomerulus tsis yog ib qho kev lim dej nkaus xwb, tab sis lub cev tsis tshua muaj zog uas muaj peb txheej: endothelial hlwb, glomerular hauv qab daus daim nyias nyias (GBM), thiab podocytes [9]. Kev xa tawm EM (TEM) tso cai rau kev pom ntawm glomerular hlwb los ntawm sab hauv [10] thiab qhia tias endothelial hlwb muaj nyias fenestrated cell lub cev, whereas podocytes muaj lub cell loj, thiab txuas ntxiv thawj thiab theem nrab 'ko taw' txheej txheem anchored rau GBM thiab interconnected los ntawm linear junctions (qhov me me diaphragm) (Daim duab 1B). Tsis tas li ntawd, kev tshuaj ntsuam EM (SEM), kev ntes cov hluav taws xob rov qab, tso cai rau kev pom cov hlwb glomerular los ntawm lawv cov txheej txheem sab nrauv [10] thiab qhia tias cov txheej txheem ko taw ntawm cov neeg nyob sib ze podocytes interdigitated thiab npog GBM (Daim duab 1C). EM tseem yog ib qho cuab yeej muaj zog tsis yog rau kev nkag siab ntawm ib txwm glomerular ultrastructure tab sis kuj rau kev nkag siab.raumphysiology thiab pathophysiology [11]. Ntau cov kev tshawb fawb tau ua los ntawm tib neeg cov qauv los yog hauv cov qauv kev sim tau qhia tias cov txheej txheem sib txawv ntawm cov kab mob cuam tshuam nrog cov txheej txheem tshwj xeeb ntawm kev hloov pauv ntawm cov txheej txheem ultrastructural, uas txhais tau hais tias muaj kev hloov kho ntxiv ntawm kev hloov pauv ntawm kev faib tawm.raumkab mob. Kev muaj peev xwm ua kom pom qhov nyuaj ntxiv ntawm lub raum ultrastructure pom zoo qhov tshwj xeeb ntawm 'nephro'pathologists.
Cov txheej txheem SEM tsis ntev los no, raws li lub ntsuas ntsuas siab, tso cai rau kev tshawb fawb ntawm qhov tob tshaj plaws ntawm lub qhov diaphragm thiab qhia tias nws yog tus cwj pwm los ntawm qhov pores nrog ntau qhov sib txawv [12]. Hauv cov nas, qhov pib ntawm cov proteinuria yog txuam nrog cov pores loj, nce qhov muaj peev xwm ntawm cov kev hloov pauv ntawm cov kab diaphragm tuaj yeem ua rau muaj kev hloov pauv hauv glomerular permselectivity [12]. Raws li kev txhim kho ntxiv, thaiv-ntsej muag SEM tso cai dhau los ntawm 2D mus rau 3D pom ntawm glomerular filtration barrier nrog kev daws teeb meem txaus los ua raws li nanostructure ntawm cov txheej txheem ntawm tes nyias nyias hauv cov nas noj qab haus huv, cov nas muaj kab mob, thiab thaum lub sijhawmraumkev loj hlob [13, 14].
Thaum kawg, kev sib txuas ntawm EM mus rau X-ray microanalysis system (X-ray microscopy) tso cai rau kev tsom xam ntawm cov hlwb lossis cov hlwb ntawm qib ultrastructural. X-ray microscopy siv lub scanning TEM nrog lub nqaim electron beam kom excite tus qauv. Qhov cuam tshuam ntawm cov hluav taws xob hluav taws xob siab los ntawm cov nqaj nrog cov atoms hauv cov qauv tsim ua lub zog hauv daim duab X-ray photons. Qhov X-ray teeb liab, sau nrog lub zog-dispersive semiconductor detector, yog siv los txheeb xyuas cov ntsiab lus tam sim no hauv cov qauv, nrog kev daws teeb meem ntawm, qhov zoo tshaj plaws, 30 nm. Hauvraumteb, X-ray microanalysis tau ua tiav los ntawm Beck thiab Thurau los kawm txog kev thauj mus los ntawm transepithelial tubule ion [15] thiab intracellular elemental concentration hauv tubular cells [16].
VISUALIZING RENAL PHYSIOLOGY Nrog MULTIPHOTON MICROSCOPY
Multiphoton microscopy (MPM) muab kev nce qib ntxiv hauvraumkev tshawb fawb, uas yog, muaj peev xwm los tshawb xyuas lub raum ua haujlwm thiab cov qauv kev sib raug zoo hauv lub sijhawm [17]. Sib txawv ntawm cov qauv siv lub teeb pom kev zoo, ob-photon excitation nyob ntawm qhov nqus ib txhij ntawm ob lub photons nrog ob npaug ntawm qhov excitation wavelength uas tsim nyog rau excite lub fluorophore hauv classic ib-photon excitation (Daim duab 1D). Kev siv cov khoom lag luam muaj pulsed infrared, qis zog excitation lasers tau nce cov ntaub so ntswg tob thiab txo qis phototoxicity (Daim duab 1D). Qhov no tau ua rau cov kws tshawb fawb pom pom cov txheej txheem ntawm tes ntawm tes ncaj qha hauv cov tsiaj muaj sia, ib qho tshwj xeeb tsis tau ua tiav nrog lwm cov txheej txheem. MPM tso cai rau kom muaj nuj nqis ntawm lub raum kev ua haujlwm thiab kev hloov pauv pathological xws li glomerular permeability, vascular ntshav txaus, ib leeg-nephron glomerular filtration rate, thiab tubular flow [17] (Daim duab 1E thiab F). Tsis tas li ntawd, MPM tso cai rau txoj kev tshawb fawb ntawm cov txheej txheem intracellular hauv kev teb rau kev raug mob, suav nrog kev tuag ntawm tes thiab tso tawm, leukocyte dov, thiab kev nrhiav neeg ua haujlwm thiab cuam tshuam ntawm GBM [17]. Cov kev txhim kho ntxiv tau tso cai rau kev kuaj mob ntev ntev rau ntau hnub rau lub lis piam [18] uas, ua ke nrog cov nas transgenic qhia cov fluorescent proteins, tau siv los taug qab txoj hmoo ntawm tib lub hlwb ncaj qha hauv vivo hauv lub raum noj qab haus huv thiab raug mob [19] thiab nkag siab zoo dua cov txheej txheem ntawm lub raum cell turnover thiab regeneration.
Daim ntawv lo-dawb duab cov tswv yim kuj ua tau nrog MPM, siv lub ntuj autofluorescence ntawm nicotinamide adenine dinucleotide hydrate (NADH) los kawm txog redox lub xeev ntawm tubular hlwb tom qab raug mob [20]. Tsis tas li ntawd, thib ob-harmonic tiam los ntawm fibrillar collagen tau siv los ua kom muaj nuj nqis collagen deposition, ib qho cim ntawm lub raum fibrosis [21]. Qhov thib ob-harmonic tiam yog cov txheej txheem uas tsis yog-linear optical nyob rau hauv uas ob excitation photons nrog tib lub wavelength cuam tshuam nrog non-centrosymmetric qauv nyob rau hauv cov ntaub so ntswg lom, xws li collagen los yog microtubules, thiab tsim ib tug tshiab photon nrog ib nrab ntawm lub wavelength.
Ib qho kev hloov tshiab tsis ntev los no ntawm intravital microscopy yog qhov kev taw qhia ntawm ib qho miniaturized multiphoton endoscope tau siv los ua cov duab raum hauv cov nas uas muaj tshuaj loog [22]. Qhov minimally invasive MPM tuaj yeem ua txoj hauv kev rau lub sijhawm tiag tiag hauv vivo kuaj mob, tsis muaj cov ntaub so ntswg tshem tawm, thiab hauv lub sijhawm luv luv.
Lub raum hauv 3D: TISSUE-CLEARING TECHNIQUES THIAB EXPANSION MICROSCOPY
Txawm hais tias kev txhim kho thev naus laus zis, ib qho ntawm cov kev txwv tseem ceeb rau kev ntsuas qhov tob tob yog lub raum cov ntaub so ntswg opacity, uas ua rau lub raum tsis ua haujlwm.raumib qho ntawm feem ntau optically nyuaj lub cev. Qhov no yog vim li cas cov txheej txheem tshem tawm cov ntaub so ntswg tau tsa ntau qhov kev txaus siab [23]. Lub hom phiaj ntawm cov txheej txheem no yog los hloov cov ntaub so ntswg opaque rau hauv pob tshab, khaws cov proteins thiab thaum kawg fluorescent markers, mus rau duab cov ntaub so ntswg tuab. Kev sib xyaw ua ke ntawm kev kho qhov muag nrog lub xeev-ntawm-lub-kos duab microscopy muab qhov muaj peev xwm ntawm kev soj ntsuam morphometric, xws li kev ntsuas ntawm glomerular ntim thiab tus lej, hauv cov ntaub so ntswg tuab lossis txawm nyob hauv lub raum tsis zoo [24]. Lwm daim ntawv thov suav nrog kev tshuaj xyuas ntawm cov tubule sib txawv [25], lawv cov haujlwm [26], thiab ntsuas qhov poob ntawm podocyte [27].
Txhawm rau ua kom pom cov ntaub so ntswg loj thiab, tib lub sijhawm, daws cov ntsiab lus zoo, ib txoj hauv kev tshiab tau qhia tsis ntev los no, uas yog, nthuav dav microscopy (ExM). Lub tswv yim yog kom lub cev nthuav dav tag nrho lub cev 4- rau 5-fold thaum khaws cia tag nrho cov qauv thiab cov ntsiab lus 3D proteome. Siv cov polymer nthuav dav, ncaj qha ua ke hauv cov qauv, cov molecules ze tshaj qhov kev txwv ntawm lub teeb yog isotropically sib cais nyob rau hauv qhov chaw kom ntau dua qhov kev ncua deb thiab yog li ntawd tuaj yeem kho kho qhov muag txawm tias siv lub tshuab fluorescent. Tsis tas li ntawd, ExM muab qhov pom tseeb dua qhov pom tseeb thiab txo qhov tawg ntawm lub teeb, tso cai rau ib tus neeg pom cov ntaub so ntswg loj dua. Siv txoj hauv kev no, qhov chaw ntawm ntau cov proteins nyob rau hauv qhov slit diaphragm thiab GBM tuaj yeem raug txiav tawm txawm tias muaj lub tshuab tsom iav ib txwm muaj [28]. Piv txwv li, nws tuaj yeem nthuav tawm cov txheej txheem ko taw effacement hauv cov nas proteinuric, ob qho tib si hauv 2D thiab 3D, ntawm qhov kev daws teeb meem nanometer tsis tau mus txog ua ntej [29]. Tsis ntev los no ExM tau siv rau kev soj ntsuam histopatho logic kev soj ntsuam ntawm paraffin-embedded specimens twb stained nrog hematoxylin thiab eosin [30]. Cov txheej txheem no tuaj yeem ua tiav cov txheej txheem ko taw effacement ntawm qib nanoscale uas yav dhau los ua tau tsuas yog nrog EM. Kev nce qib hauv volumetric imaging tso cai rau high-resolution 3D tsom xam ntawm tag nrho lub cev nyob rau hauv ib txoj kev uas lub ntsiab kev txwv rau kev sib sib zog nqus imaging yog tam sim no repre xa los ntawm lub peev xwm rau cov tshuaj tiv thaiv mus rau hauv cov ntaub so ntswg.
STUDYING LUB METABOLIC PROFILE: FREQUENCY-DOMAIN FLUORESCENCE-LIFETIME IMAGING MICROSCOPY
CovraumNws yog ib qho metabolic organ nrog cov ntsiab lus siab ntawm mitochondria. Daim ntawv txo NADH thiab lwm yam metabolites yog ib txwm muaj fluorescent thiab autofluorescence no tuaj yeem siv los ua qhov ntsuas ntawm lub xeev redox [20]. Txawm li cas los xij, emission spectra ntawm cov fluorophores endogenous yog sib tshooj, thiab txheeb xyuas lawv cais tau ua tsis tau. Tab sis fluorescence lifetimes (piv txwv li lub sij hawm lwj ntawm fluorescence) tau pom tias txawv heev. Li no fluorescence-lifetime imaging microscopy (FLIM) tso cai rau kev tshawb fawb ntawm metabolic profiles [31]. Tsis ntev los no, siv kev sib xyaw ntawm MPM thiab FLIM, nws tau dhau los ua tus cwj pwm ntawm cov kab mob metabolic tshwj xeeb thiab saib xyuas cov kev hloov pauv hauv vivo thaum lub sijhawm.raumkab mob [32]. FLIM cia siab tias yuav muab kev nkag siab ntxiv rauraummetabolism hauv vivo hauv kev noj qab haus huv thiab kab mob.
PUSHING RESOLUTION BEYOND LIMITS: SUPER-RESOLUTION duab ntawm lub raum
Tus qauv lub teeb microscopy ntsib qhov txwv ntawm qhov kev daws teeb meem kho qhov muag tshaj 200 nm. Cov thev naus laus zis tshiab, hu ua super-resolution imaging, tam sim no tso cai fluorescent microscopy rau cov duab dhau ntawm nws qhov kev txwv tsis pub dhau qhov pib ntawm lub sijhawm ntawm nanoscopy [33]. Cov txheej txheem no ua ke nrog kev daws teeb meem nanoscopic nrog qhov zoo ntawm ntau xim fluorescence labeling. Hauv kev lees paub txog qhov muaj peev xwm cuam tshuam ntawm cov kev tsim kho tshiab no, 2014 Nobel Prize hauv Chemistry tau muab tsub rau Eric Betzig, Stefan Hell, thiab William Moerner 'rau kev txhim kho ntawm super daws teeb meem fluorescence microscopy. Cov tswv yim sib txawv ntawm kev daws teeb meem zoo tshaj plaws tuaj yeem siv los daws cov lus nug sib txawv. Cov txheej txheem no tuaj yeem muab faib ua ob pawg tseem ceeb, nyob ntawm seb qhov kev daws teeb meem super yog siv los ntawm qib ntawm ib qho molecules lossis ntawm pawg [33].
Cov txheej txheem sib xyaw ua ke
Ensemble-based cov txheej txheem rhuav tshem qhov kev txwv tsis pub dhau los ntawm lub sijhawm thiab spatially modulating lub excitation teeb beam. Ntawm lawv, super-resolution stimulated emission depletion (STED) imaging siv ob lub lasers, excitation laser thiab superimposed, red-shifted, depletion laser (STED laser), nrog ib tug donut zoo li, los suppress lub fluorescence emission los ntawm cov fluorophores nyob tawm. qhov chaw ntawm excitation. Qhov kev tawm tsam no tau ua tiav los ntawm kev txhawb nqa emissions thiab tso cai rau khaws tsuas yog lub teeb liab los ntawm qhov chaw focal nrog ib qhov ntev hauv qab qhov kev txwv tsis pub tshaj (Daim duab 2A). STED imaging tau siv los ua duab cov kab diaphragm hauv optically clearedraumcov ntaub so ntswg (Daim duab 2B–c') thiab tso cai rau thaj tsam ntawm qhov chaw faib khoom ntawm podocin thiab nephrin ntawm nanometer scale mus txog yam tsawg kawg 30 lm ntawm qhov tob [34]. Tsis tas li ntawd, qhov ua tau los soj ntsuam thiab ntsuas cov txheej txheem ko taw effacement hauv cov qauv kev sim tuaj yeem ua rau STED imaging muaj txiaj ntsig zoo rau kev tshawb fawb ntawm glomerular filtration barrier [34].
STED microscopy xav tau cov txheej txheem tshwj xeeb rau kev npaj ua qauv, tshwj xeeb fluorophores, thiab kev cob qhia. Vim li no, ob peb xyoos dhau los no tau txais kev saib xyuas ntau ntxiv rau lwm cov txheej txheem daws teeb meem, teeb pom kev zoo microscopy (SIM), uas tuaj yeem txuas ntxiv qhov kev txwv tsis pub dhau los ntawm qhov tseem ceeb ntawm ob qho tib si sab nraud thiab axially. SIM siv lub tshuab ntsuas qhov dav dav nrog cov qauv teeb pom kev zoo, tso cai rau kev ntes cov ntaub ntawv ntau zaus (raws li cov ntsiab lus zoo hauv cov qauv) ntawm qhov qis dua. Los ntawm kev tau txais ntau cov duab nrog cov qauv teeb pom kev sib txawv ntawm cov theem sib txawv thiab kev taw qhia, cov duab siab daws teeb meem tuaj yeem tsim kho dua tshiab (Daim duab 3A). SIM tau sib xws nrog cov qauv fluorophores, tsis tas yuav tsum tau npaj cov qauv tshwj xeeb, thiab tso cai rau pom 3D. Muab tag nrho cov txiaj ntsig no, cov kev tshawb fawb sib txawv tau ua los ntsuas qhov ua tau ntawm kev siv SIM ua cov cuab yeej kuaj mob sai [35], piv txwv li, nrog lub sijhawm luv luv dua li EM. Tsis ntev los no 3D SIM thiab kev ua cov duab ua haujlwm tau siv los ua cov cuab yeej kuaj mob sai ntawm cov txheej txheem ko taw effacement siv cov ntu paraffin niaj hnub los ntawm biopsies ntawm cov neeg mob uas muaj kev hloov pauv tsawg kawg nkaus [36] (Daim duab 3B thiab C). Lub peev xwm no txhawm rau txheeb xyuas qhov kev hloov pauv hauv cov kab mob uas paub tias muaj cov kab mob nanoscale paub tseeb tias SIM yog cov cuab yeej muaj txiaj ntsig rau kev kuaj mob niaj hnub.
Ib leeg-molecule localization-based imaging txoj kev
Cov tswv yim no tso siab rau qhov muaj peev xwm hloov mus rau thiab tawm ntawm ib tus neeg fluorescent molecules uas ze dhau los daws tau. Hauv cov txheej txheem no, cov molecules nyob rau hauv ib cheeb tsam diffraction-limited tuaj yeem qhib tau ntawm lub sijhawm sib txawv kom lawv tuaj yeem ua tus kheej cov duab thiab tom qab ntawd hauv zos nrog nanometer precision los ntawm kev suav nrhiav lawv cov chaw (Daim duab 3D). Ntawm cov txheej txheem no, stochastic optical reconstruction microscopy (STORM) yog thawj zaug hauvraumkev tshawb fawb nyob rau hauv 2013, nrog rau kev tshawb fawb uas qhia txog intricate molecular lub koom haum ntawm GBM ntawm nanometer precision, nrog rau kev taw qhia ntawm molecules nyob rau hauv [37]. Hauv tus qauv nas ntawm qhov tsis tshua muaj tshwm sim hauv lub cev tsis muaj zog, STORM tau qhia txog kev sib koom ua ke thiab kev taw qhia kom raug ntawm laminin 521 hauv GBM tom qab txhaj tshuaj, qhib txoj hauv kev rau kev kho mob siab heev rau cov neeg mob [38]. Lwm txoj kev tshawb fawb tau piav qhia txog lub koom haum ntawm actin cytoskeleton hauv podocytes ntawm kev daws teeb meem molecular [39] (Daim duab 3E–I). Raws li qhov kev tshawb pom tshiab, podocytes muaj cov kab mob sib txuas nrog cov actin bundles uas tsis yog-contractile hauv cov txheej txheem ko taw. Hauv cov qauv kev raug mob podocyte, cov qauv actin hauv cov txheej txheem ko taw disassembled, ua rau lub cev qhuav dej ntawm cov qauv hauv lub cev [39].
VISUALIZING 3D STRUCTURES OF FUNDAMENTAL BIOMOLECULES AT THE ATOMIC LEVEL: CRYO-ELECTRON MICROSCOPY
Txog thaum tsis ntev los no, qhov muaj peev xwm ntawm kev kawm cov qauv ntawm biomolecules tseem ceeb tsuas yog siv rau electron microscopy, nrog rau kev daws teeb meem ntawm ob peb nanometers. Technical hurdles, xws li cov qauv kev puas tsuaj los ntawm khaus electron beam, qis-duab sib piv, thiab molecule txav tom qab muaj kev cuam tshuam nrog electrons los yog teeb meem los khaws cov dej hauv cov qauv lom hauv lub tshuab nqus tsev siv hauv EM chamber, ua rau nws tsis tuaj yeem daws cov molecules hauv qab no. . Txias cov qauv tuaj yeem txo cov dej evaporation thiab electron-induced puas tsuaj [40]. Los ntawm lub tswv yim no, cryo-EM tau qhia hauv xyoo 1950. Ob peb xyoos dhau los, qhov kev taw qhia ntawm ib qho tshiab-electron suav ntsuas thaum kawg thawb kev daws teeb meem dhau qhov txwv dhau los. Hauv 2017, Nobel nqi zog hauv Chemistry tau muab tsub rau Jacques Dubochet, Joachim Frank, thiab Richard Henderson rau 'tsim cryo-electron microscopy rau cov qauv kev txiav txim siab ntawm biomolecules hauv kev daws'. Cov kev nce qib tam sim no tso cai rau kev txiav txim siab ntawm biomolecules hauv kev daws [41].
Hauvraumkev tshawb fawb, cov qauv ntawm cov proteins tseem ceeb tau txiav txim siab ntawm qib atomic [42, 43]. Ntawm lawv, cov qauv ntawm tib neeg polycystin -2, uas nws cov kev hloov pauv yog lub luag haujlwm rau autosomal dominant polycystic.raumkab mob [44], thiab cov qauv ntawm cov hloov pauv hloov pauv muaj peev xwm canonical 6 ion channels, uas nws cov kev hloov pauv ua rau FSGS hauv tib neeg [43], tau txiav txim siab nrog kev daws teeb meem ntawm ob peb angstroms [44].
NYEEM NTXIV MOLECULAR BASIS ntawm KIDNEY DISEASES: INTEGRATIVE KIDNEY MICROSCOPY
Ob peb xyoos dhau los no tau pom muaj kev txaus siab ntxiv rau cov kev tsis muaj daim ntawv lo muaj peev xwm tshwj xeeb thiab ib txhij qhia tag nrho cov spectrum ntawm molecules hauv cov ntaub so ntswg [45]. Ntawm lawv, matrix-as-sisted laser desorption / ionization imaging mass spectrometry (MALDI-IMS) tso cai rau kev ntsuas ntau pua ntawm cov molecules sib txawv tib lub sijhawm ncaj qha rau ntawm cov ntaub so ntswg ntawm qhov kev daws teeb meem siab. Hauv kev sim MALDI-IMS, cov ntaub so ntswg nyias, khov lossis paraffin-embedded, yog coated nrog ib tug matrix uas absorbs lub zog laser thiab txhawb ionization ntawm cov ntaub so ntswg analytes. Kev tsom xam hauv qhov loj spectrometer tsim ib spectrum rau txhua x / y ua haujlwm. Tom qab kev ntsuas MALDI, tib cov ntaub so ntswg tuaj yeem stained nrog cov txheej txheem histochemical thiab immunohistochemical los ua ke cov qauv molecular nrog cov ntsiab lus histological. Hauv kev tshawb fawb lub raum, MALDI-IMS yog siv los kawm txog tshuaj, metabolites, lipids, peptides, thiab cov proteins hauv cov ntaub so ntswg thiab cov kab mob [45].
Ib qho ntawm cov kev siv nquag tshaj plaws ntawm MALDI-IMS yog kev txheeb xyuas cov cim molecular los txheeb xyuas lub raum cell carcinoma [46] thiab tsim kom muaj qhov tseeb ntawm cov kab mob qog noj ntshav thiab cov nqaij mos ntawm cov molecular nrog rau qib histological [47]. MALDI-IMS kuj tau ua tiav siv los kawm txog cov tshuaj toxicity rau lub raum los muab kev tshawb pom ntxov ntawm cov tshuaj lomraumcov txheej txheem ntawm kev puas tsuaj kho los ntawm cov kws tshuaj sib tw [48]. Ntxiv mus, MALDI-IMS yog siv los kawm txog lipids thiab lawv lub luag haujlwm hauv lub raum pathologies, xws li mob ntshav qab zib nephropathy [49], mob raum mob [50], thiab polycystic.raumkab mob [51], thiab tseem yuav nthuav tawm endogenous metabolic profiles rau kev nkag siab tob txog cov kab mob ntsig txog cov txheej txheem [52]. Kev txheeb xyuas ntawm cov molecular profiles kuj tau ua tiav rau cov biomarkers uas tuaj yeem yog tus tshiab muaj peev xwm kuaj mob thiab prognostic biomarkers [53, 54].
Cov ntsiab lus thiab cov kev xav yav tom ntej
Kev txhim kho tas mus li hauv cov txheej txheem kev yees duab tau nce zuj zus daws ntau yam teeb meem tseem ceeb hauvraumkev tshawb fawb thiab tau tso cai rau ib tug dynamic portrayal ntawm cov qauv thiab kev ua hauj lwm nyob rau hauv ib txwm thiab mob raum. Tseeb tiag, qhov kev tshwm sim ntawm cov cuab yeej microscopy tshiab tau ua rau pom kev tshawb fawb tseem ceeb thiab tom qab ntawd hloov peb txoj kev xav ntawm lub raum physiology thiab pathophysiology uas tam sim no tuaj yeem pom hauv 3D thiab txawm ua yeeb yaj kiab, qhov twg lub sijhawm sawv cev rau qhov thib plaub. Nrog rau kev siv thev naus laus zis tsis ntev los no ntawm kev daws teeb meem microscopy thiab kev nce qib ntawm cov qauv duab molecular, cov kws tshawb fawb tuaj yeem pom cov tib neeg bio-molecules ncaj qha rau hauv cov ntaub so ntswg thiab txiav txim siab seb lawv cuam tshuam li cas ntawm cov cellular thiab subcellular.
Muaj peev xwm siv cov cuab yeej no rau pathologicalraumCov qauv, tsom mus rau cov ntsiab lus ntawm cov molecular thiab subcellular hauv kev kho mob biopsies, yuav tsum pab nrog cov kab mob rov ua dua. Yog tias thiab, thaum twg qhov no yuav tsim kev cuam tshuam rau kev kuaj mob raum lossis txawm tias kev tswj hwm tus neeg mob, tam sim no tsis paub meej.
NYIAJ
Cov nyiaj tau txais los ntawm European Research Council nyob rau hauv European Union lub Horizon 2020 kev tshawb fawb thiab kev tsim kho tshiab (kev pom zoo 648274).
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