Kom meej meej Imaging thiab Quantifying lub raum nyob rau hauv 3D
Feb 27, 2022
Tau ntau xyoo, kev ntsuas ntawmraummicroanatomy siv 2- seem seem tau muab rau peb paub meej txog raum morphology nyob rau hauv physiological thiab pathological tej yam kev mob. Txawm li cas los xij, kev txhim kho sai ntawm cov txheej txheem tshem tawm cov ntaub so ntswg nyob rau hauv xyoo tas los no, nrog rau kev txhim kho tshiab ntawm 3- cov qauv kev ntsuas qhov ntev tau muab cov kev nkag siab tshiab rau hauvraumqauv thiab kev ua haujlwm. Tsab ntawv tshuaj xyuas no piav qhia txog ntau yam kev nkag siab tshiab rau hauvraumkev loj hlob thiab kab mob tau txais tsis ntev los no siv cov txheej txheem tshiab no. Piv txwv li, hauv kev txhim khoraumCov txheej txheem no tau muab kev nkag siab tshiab ntawm ureteric branching morphogenesis, nephron progenitor cell proliferation thiab kev cog lus, kev cuam tshuam ntawm ureteric tip cells thiab nephron progenitor cells, thiab tsim kom muaj nephron segmentation. Nyob rau hauv tag nrho cov neeg laus naslub raum, cov ntaub so ntswg clearing ua ke nrog lub teeb daim ntawv microscopy tuaj yeem duab thiab ntsuas tag nrho cov glomeruli, ib qho kev tawg loj hauv thaj teb. Cov txheej txheem zoo sib xws tau muab cov kev nkag siab tshiab rau hauv cov qauv ntawm lub raum vasculature thiab innervation, tubulointerstitial remodeling, podocyte poob thiab hypertrophy, cyst tsim, evolution ntawm cellular crescents, thiab cov qauv ntawm glomerular filtration barrier. Muaj ntau yam kev nce qib hauv kev nkag siab ntawmraumbiology thiab pathology tuaj yeem xav tau raws li kev tshem tawm ntxiv thiab cov duab kos duab tau tsim thiab pom zoo los ntawm cov neeg tshawb xyuas ntau dua.
Ntsiab lus:3D duab; glomerulus; kev loj hlob ntawm lub raum; nephropathology; tshem tawm cov ntaub so ntswg; tubules; lub raum
CISTANCHE yuav txhim kho lub raum / raum FUCTION
Tag nrho tus naj npawb nephron hauv txhua tus tib neegraumthaj tsam li ntawm kwv yees li 200,000 mus rau ntau dua 2 lab, 1 qhov nruab nrab kwv yees li 1 lab. Cov nephrons no zoo li lwm yam khoom ntawm covraumnrog rau kev sau cov ducts, cov hlab ntsha, interstitia, thiab cov hlab ntsha fibers yog teem nrog exquisite microanatomical precision. 2 Qhov no tus qauv zoo meej underpins ib txwmlub raum ua haujlwmthiab kev noj qab haus huv, uas tuaj yeem cuam tshuam loj heev thaum cov ntaub so ntswg hloov pauv vim glomerular lossis tubular hypertrophy lossis shrinkage, nephron poob, congenital abnormalities, hlab ntsha rarefaction, thiab interstitial fibrosis. Ntau yam ntawm cov kev yees duab tau ua rau peb nkag siab zooraummicroanatomy thaum lub sij hawm kev loj hlob thiab nyob rau hauv cov neeg laus noj qab nyob zoo thiab muaj mobraum. Feem ntau, qhov kev nkag siab no tau los ntawm kev txheeb xyuas ntawm 2- seem (2D) seem seb lawv puas yog lub teeb lossis hluav taws xob microscopic lub cev los yog confocal optical seem. Qhov ntau ntawm qhov microanatomy no feem ntau yog nyob ntawm kev soj ntsuam stereological ntawm cov seem uas tau muab kwv yees ntawm cov lej, qhov ntev, thaj chaw saum npoo, thiab ntim ntawm glomeruli, tubules, hlab ntsha, interstitia, thiab lawv cov khoom ntawm tes. 3,4 Txog thaum tsis ntev los no, 3D pom ntawmraummicrostructure feem ntau txwv rau confocal microscopy, uas ua rau kho qhov muag thiab 3D reconstruction mus txog qhov tob ntawm 50 mus rau 80 m m. Hmoov tsis zoo, cov khoom refractive ntawm cov protein thiab lipid Cheebtsam hauv cov ntaub so ntswg tawg lub teeb thiab txo cov duab zoo li qhov tob zuj zus. Nyob rau hauv xyoo tas los no, ntau txoj kev tshem tawm tau raug tsim los tshem tawm cov lipid thiab pigment cov ntsiab lus ntawm cov ntaub so ntswg thiab phim cov khoom refractive ntawm cov ntaub so ntswg thiab mounting xov xwm los ua kom cov ntaub so ntswg pob tshab. 5 Txoj kev tshem tawm muaj qhov nyuaj los ntawm kev tsim cov ntaub so ntswg hauv ntau cov kev daws teeb meem nyob rau lub sijhawm ntev mus rau cov txheej txheem txuas ntxiv nrog cov khoom siv hluav taws xob. Peb coj cov neeg nyeem mus rau qhov kev tshuaj xyuas tsis ntev los no 5,6 rau cov ncauj lus kom ntxaws txog cov txheej txheem tam sim no, thiab cov ntaub ntawv tau hais hauv no rau cov txheej txheem tshwj xeeb thiab siv cov xwm txheej.
Cov qauv subcellular thiab tag nrho cov pejxeem ntawm cov hlwb tuaj yeem daws tau nyob rau hauv tshem tawm tag nrho cov kabmob lossis cov ntu tuab. Ua kom pom tseeb cov yam ntxwv hauv cov ntaub so ntswg tshem tawm yuav tsum tau siv lub tshuab tsom iav tshwj xeeb thiab cov duab ua kom ntes thiab rov tsim cov ntaub ntawv ntim los ntawm cov qauv uas muaj peev xwm nyob ntawm ntau pua microns mus rau centimeters. Point scanning thiab spinning disk confocal microscopes tau siv tau zoo rau cov duab nta hauv kev tshem tawmraumcov ntaub so ntswg tab sis raug kev txom nyem los ntawm kev daws teeb meem hauv Z axis thiab kev txo qis hauv fluorescence ntawm qhov tob vim yog ib qho axis ntawm illumination thiab duab. Lub teeb daim ntawv microscopy cov tswv yim yog tshwj xeeb tshaj yog haum rau imaging cleared cov ntaub so ntswg raws li lawv pab kom tau ceev nrooj ntawm 3D ntim loj thiab muaj peev xwm muaj xws li multiangle illumination thiab imaging. Optical projection tomography yog lwm txoj hauv kev uas suav nrog ntau lub duab duab thiab digital reconstruction los tsim cov ntaub ntawv uas X, Y, thiab Z axes ntawm txhua lub voxel yog tib qhov kev daws teeb meem. Tsis hais txog ntawm txoj hauv kev tshwj xeeb, cov ntaub so ntswg tshem tawm thiab tag nrho cov duab mount muab lub sijhawm tshiab los tshawb txog cov kab mob tsis muaj kab mob hauv cov kab mob microenvironments thiab cov ntaub so ntswg ntau dua. Hauv kev tshuaj xyuas no, peb qhia txog qee qhov kev nkag siab tshiab rau hauvraumkev loj hlob, neeg lausraumqauv, thiab pathology uas tau tshwm sim los ntawm cov ntaub so ntswg clearing tom qab 3D quantitative tsom xam.
Kev nkag siab tshiab txog kev txhim kho lub raumNtau xyoo ntawm gene qhia profileing thiab kev tshawb fawb knockout tau ua rau kev nkag siab ntxaws txog cov hom cell, kev tswj hwm kev sib txuas ntawm cov noob caj noob ces, thiab cov cim qhia txoj hauv kev uas tswj cov tsiaj txhu.raumkev loj hlob. Qhov kev paub no qhia txog kev kuaj mob ntawm tus kab mob congenital thiab enabled zus tau tej covraumcell hom los ntawm pluripotent qia hlwb. 7 Txawm li cas los xij, qhov loj, opacity, thiab complexity ntawm kev txhim khoraumnthuav tawm qhov teeb meem tseem ceeb rau kev ntsuas qhov morphogenesis ntawm lub cev thiab 3D kev faib tawm ntawm cov hlwb thiab cov protein nyob hauv nws. Kev siv cov ntaub so ntswg clearing thiab multiscale duab nyob rau hauv lubraumtau ua kom pom kev pom thiab kev tsom xam ntau ntawm ureteric branching morphogenesis, progenitor cell populations, thiab nephron endowment nyob rau hauv lub cev tsis zoo (Daim duab 1). 8 Cov txheej txheem no txhawb nqa lub peev xwm tshiab rau qhov tseeb phenotyping thiab qhib txoj hauv kev los tshuaj xyuas cov cellular thiab molecular tsav tsheb ntawmraumkev loj hlob thiab kab mob congenital.
Kev yees duab thiab txheeb xyuas cov ceg morphogenesis hauv 3D.Kev tsim ntawm tsob ntoo ureteric yog ib qho kev txhais ntawmraumtxoj kev loj hlob thiab tau kawm dav siv nas caj ces thiab flattenedraumexplant kab lis kev cai. Txawm li cas los xij, kev nkag siab tias tib neeg cov noob ua li cas pab txhawb kev loj hlob, daim ntawv, thiab qauv ntawm cov kab mob epithelial duct no hauv vivo xav tau lub peev xwm los ua duab thiab txheeb xyuas qhov kev sib txuas no thoob plaws ntau qhov ntau thiab tsawg ntawm cov qauv. Luv et al. 8 tau hais txog cov teeb meem no siv tag nrho mount immunofluorescence, hnyav-raws li tshem tawm (benzyl cawv benzyl benzoate) thiab ntsuas tag nrho lub cev nrog optical projection tomography. Siv txoj hauv kev no los ntes 3D snapshots txij thaum ntxov mus rau nruab nrab-raumtxoj kev loj hlob, pab neeg no tau tsim kev cai tsom xam software kom tau txais kev nthuav dav tsis tau pom dua txog tsob ntoo ureteric nrog rau tus lej xov tooj, ceg kaum thiab ntev, ceg ntim, thiab tus naj npawb ntawm tiam ntawm cov ceg ntoo los ntawm cov ntoo ureteric tsis zoo (Daim duab 1c thiab d). 8 Thaum xub thawj siv los ua tus cwj pwm thiab txheeb xyuas cov yam ntxwv tshiab ntawm lub raum ceg, 9,10 kev siv zog txuas ntxiv mus nrhiav kev txhais cov ntsiab lus thiab cov tswj hwm tseem ceeb uas tswj hwm cov ceg qauv hauv vivo. 1
Txuas cov progenitor dynamics rau lub cev morphogenesis nrog multiscale imaging.Molecular kev sib cuam tshuam ntawm ureterictip cells thiab nephron progenitor cells ua lub luag haujlwm tseem ceeb hauv kev tsim kom muaj kev sib ntxiv ntawm nephrons uas pab txhawb.lub raum ua haujlwmnyob rau hauv neeg laus lub neej. Uninduced nephron progenitors tsim yam uas txhawb nqaraumKev loj hlob los ntawm ureteric branching thaum spatial txwv cues nyob rau hauv lub ureteric tip ob leeg tuav nephron progenitor xeev thiab induce ib tug subset ntawm cov progenitors kom sib txawv rau hauv ib tug nephron thaum ntxov. Cov kev sib cuam tshuam no tsav lub voj voog ntawm cov ceg thiab nephron induction uas tsis nyob ib puag ncig thaum yug, thaum cov nephron progenitors tseem tshuav sib txawv. Ua ntej cov ntaub so ntswg clearing txoj kev, peb to taub txog cov dynamics ntawmraummorphogenesis thiab cov txheeb ze ntau ntawm cov neeg nyob hauv cov neeg laus nyob thoob plaws lub sijhawm raug txwv hnyav. Qhov no tau hloov pauv nrog kev txhim kho ntawm ntau qhov kev ntsuas kev ntsuas uas siv cov txheej txheem immunofluorescence txuas ntxiv, tshem tawm cov ntaub so ntswg, thiab cov duab thaij duab kom muaj nuj nqis.raumtxoj kev loj hlob ntawm cellular (confocal) thiab tag nrho cov khoom nruab nrog cev (optical projection tomography, lub teeb ntawv). 9,10 Nws tau pom meej tias cov nqi ntawmraumKev loj hlob sib txawv heev nrog pib sai sai thiab kev loj hlob tus nqi poob qis hauv cov theem uas cuam tshuam nrog kev txo qis ntawm cov cell progenitor hauv nephrogenic niche. 10 Peb-dimensional confocal imaging ntawm cleared tag nrho tib neeg fetallub raum(lub lim tiam 11) los yog cov qauv ntawm covraumcortex (lub lis piam 11–23) lees paub tias qhov kev txo qis hauv niche loj yog khaws cia thoob plaws hom. 12 Kev sib piv ntxiv ntawm tib neeg thiab nas nephrogenic niche hauv cov ntaub so ntswg tshem tawm coj mus rau lub sijhawm tshiab raws li kev nrhiav neeg ua qauv ntawm kev tsim nephron, uas cov hlwb progenitor tau nce ntxiv mus rau nephron thaum ntxov thiab txais yuav ib ntu tus kheej raws li qhov kev txiav txim uas lawv tuaj txog. . 13 Hauv lwm txoj kev tshawb fawb tsom rau kev cog lus nephron progenitor, hydrogel-raws li cov ntaub so ntswg tshem tawm txoj kev (passive Clear Lipid-exchanged Acryl-amide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue-hYdrogel [CLARITY] 14) tau siv rau nws. muaj peev xwm khaws cov fluorescence ntawm endogenous tdTomato reporter. Kev soj ntsuam ntawm tdTomato-labeling nyob rau hauv cov ntaub so ntswg tshem tawm tau pom tias cov hlwb nyob rau hauv thaum ntxov nephron tuaj yeem rov qab mus rau lub xeev progenitor, qhov twg cov txiaj ntsig ua ntej tau pom tias kev cog lus yog unidirectional. 15 Yog li, kev tshem tawm cov ntaub so ntswg tau txhim kho peb txoj kev nkag siab ntawm cov txheej txheem kev loj hlob ntawm cov nplai thiab hom.
Precision phenotyping. Kev nce qib hauv cov ntaub so ntswg tshem tawm thiab kev tsom xam kom muaj nuj nqis tau ua rau muaj peev xwm tshiab rau precision phenotyping. Cov kev hloov pauv ruaj khov hauv cov ceg thiab cov nephron tus lej tau raug txheeb xyuas hauv cov qauv nas nrog cov phenotypes hloov maj mam xws li Ret þ /? thiab Six2 þ/? nas (Daim duab 2), uas yav tas los muab faib ua "ib txwm." 10,16 High- daws teeb meem confocal imaging ntawm cleared Wnt11 ?/?lub raumtau txheeb xyuas qhov tshiab cellular phenotype qhov twg nephron progenitors tshwm sim tsis zoo vim qhov tsis muaj peev xwm tsim cov cellular ruaj khov nrog cov ureteric tip thiab ntxov ntxov sib txawv. 17 Cov hau kev no kuj tau siv los txheeb xyuas seb cov neeg tswj hwm tshiab ntawm nephron progenitor cell xeev li cas DNA methyltransferase 1, microRNAs Lin28 thiab Let7, thiab TSC1 cuam tshuam.raumkev loj hlob thiab nephron tooj. 18-20 Precision phenotyping yuav dhau los ua ib qho cuab yeej tseem ceeb hauv peb txoj kev siv zog kom nkag siab txog cov txiaj ntsig ntxiv ntawm cov caj ces thiab ib puag ncig uas ua rau muaj tus lej nephron tsawg thiab muaj kev pheej hmoo rau mob ntev.mob raum.
Mathematical modeling ntawmraummorphogenesis. Cov ntaub ntawv raug tsim los ntawm cleared tsimraumyog ua rau lub nthwv dej ntawm cov duab-raws li kev ua lej ua qauv uas sim ua kom pom tias daim ntawv macroscopic thiab kev ua haujlwm ntawm cov ntaub so ntswg tshwm sim los ntawm kev sib cuam tshuam ntawm molecular ntawm progenitor cell pejxeem. 11,21–23 Ntxiv rau daim ntawv thov ntawm tag nrho cov khoom hauv nruab nrog cev hauv cov ntaub so ntswg tshem tawm yuav yog qhov tseem ceeb kom nkag siab txog cov molecular thiab cellular tsav ntawm 3D cov ntaub so ntswg qauv thiab yuav tuav tus yuam sij rau kev txhim kho cov qauv thiab qhov tseeb ntawm qia cell qauv ntawm lubraum.
3D morphology thiab pathology ntawm cov neeg laus lub raumLub intricate morphology ntawm tus neeg lausraumtxwv tsis pub siv 2D morphological cov cuab yeej raws li lawv yuav qhia tsis tiav los yog kev nkag siab yuam kev ntawm 3D qauv. Ntawm no, peb tham txog qee qhov tseem ceeb ntawm kev nce qib hauv xyoo tas los no uas tam sim no tso cai rau 3D tsom xam ntawm cov qauv hauv cov neeg laus.raum, xws li los ntawm qhov tsis zoolub raumtag nrho txoj kev mus rau cov khoom ntawm glomerular filtration barrier (Daim duab 3). 2
CISTANCHE yuav txhim kho lub raum / raum tsis ua haujlwm
Nephron tus naj npawb thiab qhov loj me.Tib neegraumqhia tau hais tias muaj ntau theem ntawm intra- thiab intersubject variability nyob rau hauv nephron tooj thiab ntim, uas tau piav nyob rau hauv autopsy kev tshawb fawb los ntawm cov kev kawm tsis overtkab mob raum1, 30, 31 thiab tau txuas nrog kev txhim kho kev cuam tshuam. Tau ntau xyoo lawm, cov cuab yeej kub txheej txheem los ntsuas tus lej nephron hauv kev sim thiab tib neeg cov qauv tau yog tus qauv disector / fractionator, tus qauv tsim qauv stereological. 3,4 Txawm hais tias txoj hauv kev no yog qhov tseeb thiab meej, nws kuj yog siv tes ua, siv sijhawm ntev, thiab yuav tsum muaj kev cob qhia tseem ceeb. Vim li no, lwm txoj hauv kev tau thov los tsim cov txheej txheem ua tau zoo uas tuaj yeem muab cov ntsiab lus zoo kawg nkaus: 32 piv txwv li, kev sib txuas ntawm cov txheej txheem kho qhov muag, suav nrog hydrophobic (yav tas los hu ua hnyav-raws li), hydrophilic lossis hydrogel-based, nrog rau qib siab. lub teeb microscopy (xws li, confocal, multiphoton, los yog lub teeb ntawv).
Rau peb txoj kev paub, thawj daim ntawv tshaj tawm uas ua ke kho qhov muag pom thiab lub teeb daim ntawv microscopy rau kev tsom xam ntawm nephron tooj thiab qhov loj me tau luam tawm los ntawm Klingberg li al. 24 nyob rau hauv ib daim ntawv tseem ceeb uas qhia txog ntau yam kev nce qib tseem ceeb. Ua ntej, daim ntawv tshaj tawm no qhia txog ethyl cinnamate, cov kuab tshuaj tsis zoo uas tuaj yeem hloov cov tshuaj lom neeg xws li benzyl cawv-benzyl benzoate, uas txog thaum ntawd tau siv tsis tu ncua hauv hydrophobic clearing raws tu qauv. Qhov thib ob, cov neeg tshawb xyuas tau ua nephron suav hauv cov nas tsis zoocev in tus qauv aggressive and progressive immune mediatedmob raum, txheeb xyuas qhov poob ntawm nephron thaum lub sijhawm tus kab mob. Tsis tas li ntawd, cov kauj ruam tseem ceeb rau kev siv computer automation ntawm cov duab segmentation algorithms tau qhia, muab txoj hauv kev rau kev txhim kho tseem ceeb. Tsis ntev los no, Østergaard et al. 25 tau nthuav tawm ib qho zoo sib xws hydrophobic clearing txoj kev ua ke nrog lub teeb daim ntawv microscopy thiab automated duab segmentation uas muab tag nrho cov nephron tooj thiab ib tug glomerular ntim nyob rau hauv tus nas qauv ntawm ntshav qab zib nephropathy. Tsis tas li ntawd, txoj kev tshawb no tau pom cov ntawv sau ua haujlwm zoo nkauj ntawm cov tubuli ze ze ntawm hauv vivo albumin txhaj kom pom cov glomerulartubular juncture thiab cov txheej txheem yooj yim rau kev sib txheeb histopathology hauv tib cov qauv kho qhov muag. Daim ntawv tshaj tawm no qhia txog qhov hloov pauv ntawm cov txheej txheem no, uas tau dhau los ua cov kav dej sib txuas rau cov ntaub so ntswg dav dav.
Lub raum vasculature thiab innervation.Tom qab lem ntawm branching vasculature los yog paj hlwb nyob rau hauv cov neeg lausraumyuav luag tsis yooj yim sua nyob rau hauv 2D pa histology raws li qhov luaj li cas ntawm lub cev thiab cov complexity ntawm lub structural faib yuav tsum tau nws kim heev thiab cov kws txawj serial sectioning. Tsis ntev los no, Huang et al. 33 tau tshaj tawm cov cationic ze ntawm infrared fluorescent dye (MHI148-PEI) rau cov hlab ntsha tshwj xeeb, uas tuaj yeem ua ke nrog kev hloov kho thiab nrawm nrawm nrawm raws li ethyl cinnamate kom txo lub sijhawm ua cov ntaub so ntswg. Txoj hauv kev no qhib txoj hauv kev tshiab rau kev tshawb fawb vascular hauv lubraum,tshwj xeeb tshaj yog thaum muaj kev daws teeb meem siab hauv 3D yog xav tau. Ib yam li ntawd, Hasegawa et al. 26 siv Clear Unobstructed Brain/Cody Imaging Cocktails and Computational analysis (CUBIC) ua ke nrog rau lub teeb pom kev zoo uas siv los txheeb xyuas qhov kev faib tawm ntawm kev sib haum xeeb innervation hauv lub raum tsis zoo nas. Sympathetic qab haus huv feem ntau yog faib nyob ib ncig ntawm cov hlab ntsha. Tom qab 10 hnub ntawm ischemia lossis reperfusion raug mob, sympathetic innervation ceev tau txo qis, cuam tshuam nrog txo cov qib norepinephrine hauvcov ntaub so ntswg. Cov kev hloov pauv no ib nrab txuas ntxiv mus 28 hnub tom qab qhov kev raug mob thawj zaug, qhia tias kev hloov pauv tsis tu ncua tuaj yeem pom thaum muaj kev mob ntev.mob raum.
Tubulointerstitial remodeling.Lub raum tubular system yog qhov nyuaj tshwj xeeb los txheeb xyuas hauv 3D. Lub convoluted thiab complex morphology poses ntau yam kev cov nyom rau cov pa txoj kev raws li nyob rau hauv serial sectioning thiab standard light microscopy. Txawm li cas los xij, optical clearing tso cai rau kev tsom xam ntawm cov tubuli tsis zoo thiab lawv cov microenvironment ib puag ncig. Saritas et al. 34 tau ua ib qho kev sib xyaw ua ke ntawm hydrogel-based thiab hydrophobic optical clearing ua raws li lub teeb pom kev zoo siab thiab semiautomatic 3D tsom xam kom pom qhov kev hloov pauv ntawm cov tubular distal vim kev noj zaub mov tsis zoo. Cov kws tshawb fawb tau txheeb xyuas cov tubular hyperplasia thiab kuaj pom qhov nce hauv cov hlwb hauv cov tubulointerstitium. Txoj kev tshawb no tau siv ntau yam ntawm ob qho tib si optical clearing thiab
Daim duab 3|Peb-dimensional (3D) morphometry nyob rau hauv cov neeg laus lub raum. (a) suav nephrons nyob rau hauv tag nrho ob lub raum siv CD31 labeling ntawm endothelial hlwb. Lub vaj huam sib luag qhia txog kev tsim kho 3D (sab laug), ib qho kev kho qhov muag hauv ob sab (2D) pom (nruab nrab), thiab cov chaw tshwj xeeb uas qhia ib leeg glomeruli hauv kev sib piv ntawm lub teeb daim ntawv microscopy (LSFM) thiab confocal thiab 2- photon (2P) microscopy. Ua raws li kev tso cai los ntawm American Society of Nephrology, los ntawm Phau Tsom Faj ntawm American Society of Nephrology, Kev Ntsuas Kev Ntsuas Tag Nrho ntawm Tag Nrho Glomerular Number thiab Capillary Tuft Loj hauv Nephritic Raum Siv Lightsheet Microscopy, Klingberg A, Hasenberg A, Ludwig-Portugal I, thiab al. ., ntim 28, qhov teeb meem 2, 2017; kev tso cai xa tawm los ntawm Copyright Clearance Center, Inc. 24 (b) Sizing glomeruli nrog spatial sau npe—xim-coding sawv cev rau glomerular loj, qhov liab yog cov khoom loj tshaj plaws thiab liab dawb qhov tsawg tshaj plaws. Hloov kho nrog kev tso cai los ntawm American Society of Nephrology, los ntawm Kidney360, Automated Image Analyzes of Glomerular Hypertrophy in a Mouse Model of Diabetic Nephropathy, Østergaard MV, Sembach FE, Skytte JL, et al., volume 6, issue 6 , 2020; Kev tso cai xa tawm los ntawm Lub Chaw Tiv Thaiv Txoj Cai Lij Choj, Inc. 25 (c) Taug qab vasculature thiab innervation, featuring 3D reconstruction ntawm lub raum tsis zoo nrog immunolabeling ntawm sympathetic qab haus huv (ntsuab), vasculature (magenta), thiab merged (sab laug). Hloov los ntawm Raum International, ntim 96, qhov teeb meem 1, Hasegawa S, Susaki EA, Tanaka T, et al., Kev tsom xam peb-dimensional (CUBIC-lub raum) pom qhov txawv txav rau lub raum sympathetic qab haus huv tom qab ischemia/reperfusion raug mob, nplooj 129–138, Copyright ª 2019, nrog kev tso cai los ntawm International Society of Nephrology. 26 (d) Podocyte morphometry nyob rau hauv ib tug neeg glomeruli siv immunolabeling rau podocyte-specific marker p57 (ntsuab) thiab DNA marker 4 0 , 6-diamidino-2-phenylindole (DAPI). Cov vaj huam sib luag qhia (sab laug) kev tsim kho 3D thiab (txoj cai) cov chaw ua haujlwm. Kho nrog kev tso cai los ntawm American Society of Nephrology, los ntawm Phau Tsom Faj ntawm American Society of Nephrology, Validation of a Three-Dimensional Method for Counting and Sizing Podocytes in Whole Glomeruli, Puelles VG, van der Wolde JW, Schulze KE, et al., ntim 27, qhov teeb meem 10, 2016; kev tso cai nthuav tawm los ntawm Lub Chaw Tiv Thaiv Txoj Cai Lij Choj, Inc. 27 (e) Cellular dynamics siv kab tracing, qhia podocytes (sab laug), parietal epithelial cells (PECs) tsim cov cellular crescents hauv 3D (middle-sab laug), 2D saib ntawm PECs invading sab glomerular nrog vascular raug mob (nruab nrab-txoj cai), thiab 2D pom ntawm lub qhov hluav taws xob tubular (saum sab xis). Hloov los ntawm Raum International, ntim 96, qhov teeb meem 2, Puelles VG, Fleck D, Ortz L, thiab al., Novel 3D tsom xam siv cov ntaub so ntswg tshem tawm cov ntaub ntawv kev hloov pauv ntawm murine sai sai glomerulonephritis, nplooj 505–516, Copyright ª 2019, International Society of Nephrology, raws li daim ntawv tso cai CC BY-NC-ND (http://creativecommons.org/licenses/by-nc-nd/4.0/). 28 (f) Kev pom ntawm lub qhov diaphragm ntawm kev nthuav dav ntawm sab laug mus rau sab xis. Hloov los ntawm lub raum International, ntim 99, qhov teeb meem 4, Unnersjö-Jess D, Butt L, Höhne M, et al., Kev ceev thiab yooj yim tshem tawm thiab o raws tu qauv rau 3D in-situ duab ntawm lub raum hla cov nplai, nplooj 1010–1020 , Copyright ª 2021, International Society of Nephrology, raws li daim ntawv tso cai CC BY-NC-ND (http://creativecommons.org/licenses/by-nc-nd/4.0/). 29 Txhawm rau ua kom zoo saib cov duab no, thov mus saib hauv online version ntawm tsab xov xwm no ntawmwww.kidney-international.org.
Lub teeb microscopy los ua tubular tsom xam nyob rau hauv lub raum tsis zoo siv hydrogel-embedding thiab lub teeb daim ntawv microscopy, nrog rau ib leeg-cell kom muaj nuj nqis nyob rau hauv lub raum slices siv hydrophobic clearing thiab confocal microscopy, raws li txhua qhov sib xyaw ua ke tau zoo dua rau cov lus nug tshwj xeeb. Kev sib xyaw ntawm qhov muag pom thiab pom lub teeb pom kev zoo tau siv los ntawm Tahaei li al. 35 leej twg tus yam ntxwv ntawm kev sib deev dimorphism nyob rau hauv qhov ntev ntawm lub distal convoluted tubule, uas tej zaum yuav txiav txim siab nws lub peev xwm los hloov mus rau physiological kev nyuaj siab, thiab Schuh li al. 36 uas tau qhia txog qhov sib txawv ntawm axial nyob rau hauv proximal tubule ligand uptake thiab endolysosomal muaj nuj nqi, qhia tias S1 ntu yog tshwj xeeb heev rau reabsorb proteins ntawm receptor-mediated endocytosis. Hauv kev tshawb fawb tsis ntev los no, Blanc et al. 2 soj ntsuam tus qauv ntawm cyst tsim, qhia tias cysts tsim tsuas yog nyob rau hauv tej yam nephron ntu, uas txiav txim lawv cov duab thiab nyob rau hauv sib txuas mus rau lub cev tsis muaj zog tubules. Zuag qhia tag nrho, tam sim no peb muaj ntau yam piv txwv ntawm kev siv ncaj qha ntawm kev kho qhov muag rau kev tsom xam ntawm tubulointerstitial compartment.
Podocyte poob thiab hypertrophy.Lub glomerulus muab ib qho piv txwv tshwj xeeb rau kev tsom xam ntawm cov haujlwm tsis zoo hauv lub cev. Tshwj xeeb, txoj kev tshawb fawb ntawm podocyte depletion tau txais txiaj ntsig ncaj qha los ntawm cov kev siv thev naus laus zis no vim tias cov txheej txheem kub tsim raws li cov txheej txheem xav tau kev nqis peev tseem ceeb ntawm lub sijhawm thiab cov peev txheej. 37 Thawj qhov cuab yeej rau 3D podocyte morphometric tsom xam ntawm cov nas tsis zoo glomeruli ua ke ntawm kev tiv thaiv kab mobraumslices siv ib tug hloov raws tu qauv ntawm indirect immunofluores cence, hydrophobic optical clearing, thiab confocal microscopy, uas pab kom muaj nuj nqis ntawm tag nrho cov podocyte tooj nyob rau hauv tag nrho ib tug neeg glomeruli ntawm paub ntim. Peb ntseeg tias txoj hauv kev no yog qhov zoo tshaj plaws rau kev kawm txog cov txheej txheem uas cuam tshuam rau cov txheej txheem tshwj xeeb (piv txwv li, podocyte poob ua rau focal thiab segmental glomerulosclerosis). 27 Cov txheej txheem tib yam tom qab ntawd tau siv los ua tus cwj pwm lub luag haujlwm ntawm podocyte hypertrophy raws li kev them nyiaj rov qab tom qab podocyte poob, ib qho txheej txheem uas tau kho los ntawm lub hom phiaj mammalian ntawm rapamycin signaling pathway. 38 Thaum cov kev tshawb fawb 2 no tau tsom mus rau kev tsom xam ntawm glomerular podocytes, cov raj xa dej no raws li kev siv tshuaj tiv thaiv kab mob tuaj yeem siv los ua tus cwj pwm hloov pauv ntawm txhua hom cell ntawm kev daws teeb meem siab.
Lub evolution ntawm cellular crescents.Lwm yam cuab yeej tseem ceeb uas yuav tsum tau ua kom tiav rau hauv lub toolbox rau 3Draummorphometry yog kev tshawb nrhiav caj ces. Feem ntau cov kev kho qhov muag pom zoo yuav ua rau muaj qee yam ntawm fluorescence quenching (uas txawv ntawm me me mus rau qhov hnyav). Tsis ntev los no peb tau nthuav tawm cov txheej txheem uas sib txuas cov kab ke taug qab, kho qhov muag pom, thiab multiphoton microscopy rau kev txheeb xyuas tag nrho ntawm kev poob ntawm podocyte thiab parietal epithelial cell activation. 28 Nyob rau hauv txoj kev tshawb no, podocyte poob tau raug txheeb xyuas tias yog ib qho ntawm cov kev sim crescentic nephritis siv metabolic labeling ntawm podocytes nrog txhim kho ntsuab fluorescent protein. Cov evolution ntawm cellular crescents tau pom thiab ntsuas raws li kev loj hlob thiab kev tsiv teb tsaws ntawm parietal epithelial hlwb, uas kuj tau cim los ntawm kev txhim kho ntsuab fluorescent protein thiab taug qab thaum lub sij hawm ntawm kev sim ua qauv ntawm crescentic nephritis. Kev soj ntsuam ntawm qhov tsis zoo glomeruli tso cai rau kev txheeb xyuas ntawm focal podocyte poob thiab tsim cov kab mob, uas tau nce mus rau tubular occlusions los ntawm parietal epithelial hlwb, ua rau tsim ntawm atubular glomeruli. Txoj kev tshawb no pave txoj hauv kev rau yav tom ntej kev tshawb fawb ntawm complex kev tiv thaiv kab mob-epithelial kev sib cuam tshuam hauv qhov qublub raum.
CISTANCHE yuav txhim kho lub raum / raum mob ntshav qab zib
Glomerular filtration barrier.Ib qho tseem ceeb tshuaj ntsuam xyuas cov cuab yeej rau niaj hnub kev ntsuam xyuas ntawm cov kab mob glomerular yog qhov kev tsom xam ntawm lub raum biopsies los ntawm electron microscopy. Ib qho kev xaiv rau electron microscopy tuaj yeem pom nyob rau hauv thaj chaw ntawm kev nthuav dav microscopy, uas los ntawm kev txhais lub hom phiaj los ua kom kho qhov muag los ntawm kev nce cov ntaub so ntswg. Tau ntau xyoo, Unnersjo-Jess et al. 39,40 tau muab ntau txoj cai rau kev pom ntawm cov qauv nanoscale hauvraum.Tsis ntev los no, cov neeg tshawb xyuas no tau tshaj tawm cov txheej txheem ceev thiab yooj yim rau kev pom 3D ntawmraummorphology, combining optical clearing thiab cov ntaub so ntswg nthuav cov ntsiab lus los daws cov qauv nanoscale siv cov pa confocal microscopy. 29 Cov txheej txheem no tau siv los ua kom pom thiab ntsuas ntau yam kab mob hauv nas thiab tib neegraumbiopsies nrog rau cov txheej txheem ko taw effacement, kev hloov pauv ntawm glomerular hauv qab daus daim nyias nyias, slit diaphragm ntev, IgG deposits, thiab classical pathology nta (xws li, glomerulo-sclerosis, tubulointerstitial fibrosis, thiab tubular atrophy). Hauv cov ntsiab lus, txoj kev no tso cai rau ntau qhov kev pom pom thiab ua kom muaj nuj nqis ntawm lub raum pathology siv txoj hauv kev yooj yim thiab siv tau cov duab tsom xam cov cuab yeej. Lub scalability thiab imple-mentation ntawm no mus kom ze nyob rau hauv kev kho mob xyaum tej zaum yuav txiav txim los ntawm qhov yuav tsum tau ntawm advanced lub teeb microscopy khoom thiab kev loj hlob ntawm automated duab tsom xam cov cuab yeej.
Xaus
Tam sim no spectrum ntawm cov ntaub so ntswg clearing txoj kev muaj xws li cov tswv yim yooj yim uas yuav siv tau nyob rau hauv feem ntau lub chaw soj nstuam los txhim kho 3D imaging ntawm ib tug uas muaj ntau microscopes. Raws li cov hauv kev tau txais los ntawm cov txheej txheem tshiab no tau tshwm sim, peb cia siab tias yuav muaj kev siv dav dav ntawm cov tshuab kuaj kab mob tshwj xeeb kom ua tau zoo cov duab tshem tawm cov ntaub so ntswg ntawm qhov kev daws teeb meem siab thiab kev txhim kho cov kev tshawb fawb thiab kev kho mob ua haujlwm los teb cov lus nug tshwj xeeb. Lub teb ntawm neuroscience tau ua qhov chaw sim rau kev tshem tawm cov ntaub so ntswg thiab kev siv. Kev txheeb xyuas cov duab tsis siv neeg, cov txheej txheem tshiab los kawm txog kev sib txuas ntawm tes, thiab cov ntaub so ntswg-dav cov noob thiab cov protein qhia atlas tau tsim nyob rau hauv cov ntsiab lus no. Nws yog qhov zoo li qhov zoo sib xws yuav ua rau muaj kev nkag siab tshiab rau hauv cellular anatomy thiab interdependency ntawm cell cell nyob rau hauv kev loj hlob thiab cov neeg laus.raum.
Cov kev txwv tsis pub siv rau cov kev nce qib no suav nrog tus nqi ntawm cov khoom siv kho mob, thiab teeb meem hauv kev tuav, khaws cia, thiab tshuaj xyuas cov ntaub ntawv loj. Lwm qhov kev txwv yog tshwm sim los ntawm qhov tsis muaj kev rov tsim dua tshiab ntawm staining nrog cov ntaub so ntswg sib txawv cuam tshuam los ntawm kev kho cov mob thiab cov tshuaj tiv thaiv daim ntawv lo. Raws li qhov tshwm sim, yuav tsum muaj kev tshuaj ntsuam 3D tshiab los soj ntsuam thiab muab piv rau cov qauv tsim. Txawm tias muaj kev vam meej hauv kev ntsuam xyuasraumkev loj hlob thiab cov neeg lausraumcov qauv thiab kab mob tseem ceeb hauv qhov kev tshuaj xyuas no, peb ntseeg tias qhov zoo tshaj plaws ntawm cov ntaub so ntswg tshem tawm thiab 3D duab tseem tsis tau tuaj thiab yog qhov yuav yog qhov chaw zoo siab rau kev loj hlob.raumkev tshawb fawb rau xyoo tom ntej.
