insimbi engenasici 321 8 * 1.2 ityhubhu edibeneyo yokutshintsha ubushushu

Imibhobho yeCapillary

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Ubonisa ikharawusi yezilayidi ezithathu ngaxeshanye.Sebenzisa amaqhosha angaphambili nalandelayo ukutyhutyha izilayidi ezithathu ngexesha, okanye sebenzisa amaqhosha esilayidi ekupheleni ukuya kwizilayidi ezithathu ngexesha.
I-ultra-compact (54 × 58 × 8.5 mm) kunye ne-wide-aperture (1 × 7 mm) i-spectrometer yemibala esithoba yaphuhliswa, "icandwe kubini" ngoluhlu lwezipili ze-dichroic ezilishumi, ezazisetyenziselwa ukucinga ngokukhawuleza kwe-spectral.Isiganeko sokukhanya okukhanyayo kunye necandelo elinqamlezayo elincinane kunobungakanani bokuvula lihlulwe libe ngumcu oqhubekayo we-20 nm ububanzi kunye nombala othoba umbala kunye ne-wavelengths ephakathi kwe-530, 550, 570, 590, 610, 630, 650, 670 kunye ne-690 nm.Imifanekiso yemijelo yemibala esithoba ngaxeshanye ilinganiswa ngokufanelekileyo yinzwa yomfanekiso.Ngokungafaniyo ne-dichroic mirror arrays eqhelekileyo, i-dichroic mirror array ephuhlisiwe ine-configuration ye-piece-piece eyodwa ekhethekileyo, enganyusi nje inani lemibala enokulinganiswa ngaxeshanye, kodwa iphucula isisombululo somfanekiso kumjelo wombala ngamnye.I-spectrometer ephuhlisiwe yemibala esithoba isetyenziselwa i-electrophoresis ye-capillary emine.Uhlalutyo lwaxeshanye lobungakanani bedayi ezisibhozo ezifudukayo ngaxeshanye kwi-capillary nganye zisebenzisa i-laser-induced fluorescence enemibala esithoba.Ekubeni i-spectrometer yemibala esithoba ayiyona nje i-ultra-incinci kwaye ingabizi kuphela, kodwa inokukhanya okuphezulu okukhanyayo kunye nesisombululo esaneleyo se-spectral yezicelo ezininzi ze-imaging, inokusetyenziswa ngokubanzi kwiinkalo ezahlukeneyo.
I-Hyperspectral kunye ne-multispectral imaging ibe yinxalenye ebalulekileyo ye-astronomy2, i-remote sensing ye-Earth observation3,4, ulawulo lokutya kunye nomgangatho wamanzi5,6, ukugcinwa kobugcisa kunye ne-archeology7, i-forensics8, ukuhlinzwa9, uhlalutyo lwe-biomedical kunye ne-diagnostics10,11 njl Ummandla 1 Iteknoloji eyimfuneko ,12,13.Iindlela zokulinganisa i-spectrum yokukhanya okukhutshiweyo kwindawo nganye yokukhutshwa kwindawo yokujonga zahlulwe zibe (1) ukuskena kwamanqaku (“umtshayelo”)14,15, (2) ukuskena ngomgca (“panicle”)16,17,18 , (3) ubude buskena amaza19,20,21 kunye (4) nemifanekiso22,23,24,25.Kwimeko yazo zonke ezi ndlela, isisombululo sendawo, isisombululo se-spectral kunye nesisombululo sexeshana sinobudlelwane borhwebo9,10,12,26.Ukongeza, ukuphuma kokukhanya kunempembelelo enkulu kubuntununtunu, oko kukuthi umlinganiselo wesignali ukuya kwingxolo kwi-imaging spectral26.Ukukhanya okuguquguqukayo, oko kukuthi, ukusebenza kakuhle kokusetyenziswa kokukhanya, kuthelekiseka ngokuthe ngqo kumlinganiselo womlinganiselo woko wokulinganisa wokukhanya kwindawo nganye ekhanyayo ngexesha leyunithi ukuya kwisixa esipheleleyo sokukhanya komlinganiselo woluhlu lwamaza.Udidi (4) yindlela efanelekileyo xa ubunzulu okanye i-spectrum yokukhanya ekhutshwa kwindawo nganye yokukhupha itshintsha ngexesha okanye xa indawo yokukhupha itshintsha ngexesha ngenxa yokuba i-spectrum yokukhanya ekhutshwa zizo zonke iindawo ezikhuphayo ilinganiswa ngaxeshanye.24.
Uninzi lweendlela ezingentla zidityaniswe ne-spectrometers ezinkulu, ezintsonkothileyo kunye / okanye ezibizayo zisebenzisa i-18 gratings okanye i-14, 16, 22, 23 iiprism kwiiklasi (1), (2) kunye (4) okanye 20, 21 iidiski zokucoca, izihluzi zolwelo. .Izihluzi zeCrystalline eziguqulekayo (LCTF)25 okanye i-acousto-optic izihluzo ezifuthwayo (AOTF)19 zodidi (3).Ngokwahlukileyo, udidi (4) ii-spectrometers ezininzi ze-mirror zincinci kwaye azibizi kakhulu ngenxa yokucwangciswa kwazo okulula27,28,29,30.Ukongeza, banokukhanya okukhanyayo okuphezulu ngenxa yokuba ukukhanya okwabelwana ngaso sisibuko ngasinye se-dichroic (oko kukuthi, ukukhanya okugqithisiweyo kunye nokubonakaliswa kokukhanya kwesiganeko kwisibuko ngasinye se-dichroic) kusetyenziswa ngokupheleleyo kwaye ngokuqhubekayo.Nangona kunjalo, inani leebhanti zobude bobude (okt imibala) ekufuneka zilinganiswe ngaxeshanye zilinganiselwe malunga nezine.
I-spectral imaging esekelwe kwi-fluorescence yokukhangela isetyenziswa ngokuqhelekileyo uhlalutyo lwe-multiplex kwi-biomedical ukuxilongwa kunye nokuxilongwa kwe-10, i-13.Kwi-multiplexing, ekubeni i-analytes ezininzi (umzekelo, i-DNA ethile okanye iiprotheni) zibhalwe ngeedayi ezahlukeneyo ze-fluorescent, i-analyte nganye ekhoyo kwindawo nganye yokukhutshwa kwindawo yokujonga ibalwa ngokusebenzisa uhlalutyo lwe-multicomponent.I-32 yophula i-spectrum ye-fluorescence efunyenweyo kwindawo nganye ephumayo.Ebudeni bale nkqubo, iidayi ezahlukeneyo, nganye ekhupha i-fluorescence eyahlukileyo, iyakwazi ukuhlangana, oko kukuthi, ukuhlala kwindawo kunye nexesha.Okwangoku, inani eliphezulu ledayi ezinokuvuyiswa ngumqa we-laser enye yi-833.Lo mda uphezulu awumiselwanga kwisisombululo sespectral (okt, inani lemibala), kodwa ngobubanzi be-fluorescence spectrum (≥50 nm) kunye nobungakanani bedayi ye-Stokes shift (≤200 nm) kwi-FRET (usebenzisa iFRET)10 .Nangona kunjalo, inani lemibala kufuneka libe likhulu okanye lilingane nenani ledayi ukuphelisa ukugqithwa kwe-spectral yeedayi ezixutywe31,32.Ngoko ke, kuyimfuneko ukwandisa inani lemibala elinganisiweyo ngaxeshanye ukuya kwisibhozo okanye ngaphezulu.
Kutshanje, i-ultra-compact heptachroic spectrometer (esebenzisa uluhlu lwezibuko ze-heptychroic kunye ne-sensor yomfanekiso ukulinganisa ii-fluorescent fluxes ezine) zenziwe.I-spectrometer yi-odolo ezimbini ukuya kwezintathu zobukhulu ezincinci kunee-spectrometer eziqhelekileyo usebenzisa i-gratings okanye iiprisms34,35.Nangona kunjalo, kunzima ukubeka ngaphezu kwesixhenxe izibuko ze-dichroic kwi-spectrometer kwaye ngaxeshanye ukulinganisa imibala engaphezulu kwesixhenxe36,37.Ngokunyuka kwenani lezibuko ze-dichroic, umahluko omkhulu kubude beendlela ze-optical dichroic light fluxes zanda, kwaye kuba nzima ukubonisa zonke izibane zokukhanya kwinqwelomoya enye.Obona bude bubude bendlela yokubona yokukhanya okuguquguqukayo nabo buyanda, ngoko ke ububanzi bendawo yokuvula i-spectrometer (okt obona bubanzi buphezulu bokukhanya okuhlalutywayo yi-spectrometer) buyancipha.
Ukuphendula kwezi ngxaki zingentla, i-ultra-compact enemibala esithoba ye-spectrometer ene-"dichroic" ye-decachromatic mirror array kunye ne-sensor yomfanekiso we-imaging spectral engoko nangoko [udidi (4)] yaphuhliswa.Xa kuthelekiswa ne-spectrometers yangaphambili, i-spectrometer ephuhlisiweyo inomahluko omncinci kubude bendlela yokukhanya kunye nobude obuncinci bendlela yokukhanya.Isetyenziswe kwi-electrophoresis emine ye-capillary ukuze ibone i-laser-induced nine-color fluorescence kunye nokulinganisa ukufuduka kwangaxeshanye kweedayi ezisibhozo kwi-capillary nganye.Ekubeni i-spectrometer ephuhlisiweyo ayiyona nje i-ultra-incinci kwaye ingabizi kuphela, kodwa inokukhanya okuphezulu okukhanyayo kunye nesisombululo esaneleyo se-spectral yezicelo ezininzi ze-imaging, inokusetyenziswa ngokubanzi kwiinkalo ezahlukeneyo.
I-spectrometer yesiqhelo yemibala esithoba iboniswe kwifig.1a.Uyilo lwayo lulandela i-spectrometer yangaphambili ye-ultra-encinci enemibala esixhenxe 31. Iqukethe izibuko ezisithoba ze-dichroic ezicwangciswe ngokuthe tye kwi-angle ye-45 ° ukuya ngasekunene, kunye ne-sensor yomfanekiso (S) ibekwe ngaphezu kwezibuko ezilithoba ze-dichroic.Ukukhanya okungena ukusuka ngaphantsi (C0) kwahlulahlulwe ngoluhlu lwezipili ze-dichroic ezilithoba zibe yi-9 light flows ukuya phezulu (C1, C2, C3, C4, C5, C6, C7, C8 and C9).Yonke imijelo yemibala esithoba yondliwa ngokuthe ngqo kwinzwa yomfanekiso kwaye ifunyenwe ngaxeshanye.Kule sifundo, i-C1, i-C2, i-C3, i-C4, i-C5, i-C6, i-C7, i-C8, kunye ne-C9 i-wavelength kwaye imelwe yi-magenta, i-violet, i-blue, i-cyan, eluhlaza, i-yellow, i-orange, i-red-orange, kunye ebomvu, ngokulandelelanayo.Nangona ezi zibizo zemibala zisetyenziswa kolu xwebhu, njengoko kubonisiwe kuMfanekiso 3, kuba zahlukile kwimibala yokwenyani ebonwa liliso lomntu.
Imizobo ecwangcisiweyo yeespectrometers eziqhelekileyo kunye nezintsha ezinemibala esithoba.(a) I-spectrometer eqhelekileyo enemibala esithoba kunye noluhlu lwezipili eziyi-dichroic ezilithoba.(b) I-spectrometer entsha enemibala esithoba kunye noluhlu lwesipili se-dichroic esinemigangatho emibini.Isiganeko sokukhanya okuguquguqukayo kwe-C0 sahlulwe kwii-fluxes zokukhanya ezilithoba ezinemibala ye-C1-C9 kwaye ifunyenwe yinzwa yomfanekiso S.
I-spectrometer entsha ephuhlisiwe yemibala esithoba ine-mirror-mibini yesibuko se-dichroic grating kunye ne-sensor yomfanekiso, njengoko kuboniswe kwi-Fig. 1b.Kwinqanaba elisezantsi, izibuko ezihlanu ze-dichroic zithambekele kwi-45 ° ukuya ngasekunene, zilungelelaniswe ngasekunene ukusuka kumbindi woluhlu lwee-decamers.Kwinqanaba eliphezulu, izibuko ezihlanu ezongezelelweyo ze-dichroic zifakwe kwi-45 ° ukuya ngakwesobunxele kwaye zibekwe ukusuka embindini ukuya ngasekhohlo.Isibuko se-dichroic esisekhohlo somaleko ongezantsi kunye nesipili se-dichroic esisekunene somaleko ongasentla siyadibana.Isiganeko sokukhanya okukhanyayo (C0) sahlulwe ukusuka ngaphantsi ukuya kwii-chromatic fluxes ezine eziphumayo (C1-C4) ngezibuko ezihlanu ze-dichroic ngasekunene kunye nezintlanu eziphumayo ze-chromatic fluxes (C5-C4) ngezibuko ezinhlanu ze-dichroic ngakwesobunxele C9).Njengeespectrometers eziqhelekileyo ezinemibala esithoba, yonke imijelo yemibala esithoba ifakwe ngokuthe ngqo kwinzwa yomfanekiso (S) kwaye ifunyenwe ngaxeshanye.Ukuthelekisa i-Figure 1a kunye ne-1b, umntu unokubona ukuba kwimeko ye-spectrometer entsha yemibala esithoba, zombini umehluko omkhulu kunye nobude obude bendlela ye-optical yemibala esithoba iyancipha.
Ulwakhiwo oluneenkcukacha lwe-ultra-encinci ye-dichroic mirror array 29 mm (ububanzi) × 31 mm (ubunzulu) × 6 mm (ubude) iboniswe kuMfanekiso 2. Uluhlu lwesipili se-decimal dichroic luqulathe izipili zedichroic ezintlanu ngasekunene. (M1-M5) kunye nezibuko ezintlanu ze-dichroic ngakwesobunxele (i-M6-M9 kunye nenye i-M5), isibuko ngasinye se-dichroic sigxininiswe kwi-bracket ye-aluminium ephezulu.Zonke izibuko ze-dichroic ziyagungxulwa ukuhlawulela ukufuduswa okuhambelanayo ngenxa ye-refraction of flow through the mirrors.Ngaphantsi kwe-M1, i-band-pass filter (BP) ilungisiwe.I-M1 kunye ne-BP imilinganiselo yi-10mm (icala elide) x 1.9mm (icala elifutshane) x 0.5mm (ubukhulu).Imilinganiselo yezibuko ezisele ze-dichroic ziyi-15 mm × 1.9 mm × 0.5 mm.I-matrix pitch phakathi kwe-M1 kunye ne-M2 yi-1.7 mm, ngelixa i-matrix pitch yezinye izibuko ze-dichroic yi-1.6 mm.Kwikhiwane.I-2c idibanisa isiganeko sokukhanya ukukhanya kwe-C0 kunye nesithoba esinembala esikhanyayo se-C1-C9, esahlulwe yi-matrix ye-de-chamber yezibuko.
Ukwakhiwa kwe-matrix yesipili se-dichroic enemigangatho emibini.(a) Imbono yembono kunye (b) nembono enqamlezileyo yoluhlu lwesipili se-dichroic esinemigangatho emibini (imilinganiselo 29 mm x 31 mm x 6 mm).Iqukethe izibuko ezintlanu ze-dichroic (M1-M5) ezibekwe kwinqanaba elingaphantsi, izibuko ezihlanu ze-dichroic (M6-M9 kunye nenye i-M5) ebekwe kwinqanaba eliphezulu, kunye ne-bandpass filter (BP) engaphantsi kwe-M1.(c) Umbono we-cross-sectional kwicala elithe nkqo, kunye ne-C0 kunye ne-C1-C9.
Ububanzi be-aperture kwicala elithe tye, eliboniswe ngobubanzi C0 kwi-Fig. – 7 mm.Oko kukuthi, i-spectrometer entsha yemibala esithoba inomlinganiselo omkhulu wokuvula we-1 mm × 7 mm.Indlela ye-Optical ye-C4 iyona ende kunazo zonke phakathi kwe-C1-C9, kwaye indlela ye-optical ye-C4 ngaphakathi kwe-mirror mirror array, ngenxa ye-ultra-small size (29 mm × 31 mm × 6 mm), yi-12 mm.Ngexesha elifanayo, ubude bendlela ye-C5 bufutshane phakathi kweC1-C9, kunye nobude bendlela ye-C5 yi-5.7mm.Ngoko ke, umahluko omkhulu kubude bendlela ye-optical yi-6.3 mm.Ubude bendlela ye-optical engentla bulungiswa kubude bendlela ye-optical transmission ye-M1-M9 kunye ne-BP (ukusuka kwi-quartz).
Iimpawu ze-spectral ze-М1−М9 kunye ne-VR zibalwa ukuze i-fluxes С1, С2, С3, С4, С5, С6, С7, С8 kunye ne-С9 ikuluhlu lwamaza 520-540, 540-560, 560-5800, 560-5800, -600 , 600-620, 620-640, 640-660, 660-680, kunye ne-680-700 nm, ngokulandelanayo.
Ifoto ye-matrix eyenziwe yezibuko ze-decachromatic iboniswe kwi-Fig. 3a.I-M1-M9 kunye ne-BP inamathele kwi-slope ye-45 ° kunye ne-horizontal plane yenkxaso ye-aluminium, ngokulandelanayo, ngelixa i-M1 kunye ne-BP zifihliwe ngasemva komzobo.
Ukuveliswa koluhlu lwezibuko ze-decan kunye nokubonakaliswa kwayo.(a) Uluhlu lwezipili ezenziwe ngedecachromatic.(b) Umfanekiso oyi-1 mm × 7 mm wemibala esithoba ocandekileyo oboniswe kwiphepha elibekwe phambi koluhlu lwezipili zedecachromatic nezikhanyiswe ngasemva ngokukhanya okumhlophe.(c) Uluhlu lwezipili ezidekokhromatic ezikhanyiswe ngokukhanya okumhlophe ukusuka ngasemva.(d) Umjelo ocandayo onemibala esithoba ophuma kuluhlu lwesipili se-decane, ebonwa ngokubeka inkonkxa ye-acrylic ezele umsi phambi kwesipili se-decane ku-c kunye nokwenza mnyama igumbi.
I-spectra yothumelo olulinganisiweyo lwe-M1-M9 C0 kwi-angle ye-45 ° kunye ne-spectrum yokudluliselwa kwe-BP C0 kwi-angle ye-engeli ye-0 ° iboniswe kwiiFig.4a.I-spectra yokudluliselwa kwe-C1-C9 ngokumalunga ne-C0 iboniswe kwiMifanekiso.4b.Ezi mbonakalo zabalwa kwi-spectra kwiFig.4a ngokuhambelana nendlela ye-optical C1-C9 kwi-Fig. 4a.1b kunye no-2c.Umzekelo, TS(C4) = TS (BP) × [1 − TS (M1)] × TS (M2) × TS (M3) × TS (M4) × [1 − TS (M5)], TS(C9) = TS (BP) × TS (M1) × [1 − TS (M6)] × TS (M7) × TS (M8) × TS (M9) × [1 − TS (M5)], apho TS(X) kunye [ 1 − TS(X)] lusasazo kunye ne-reflection spectra ka-X, ngokulandelelanayo.Njengoko kuboniswe kwi-Figure 4b, i-bandwidth (i-bandwidth ≥50%) ye-C1, i-C2, i-C3, i-C4, i-C5, i-C6, i-C7, i-C8 kunye ne-C9 yi-521-540, 541-562, 563-580, 581-602, 603 -623, 624-641, 642-657, 659-680 kunye ne-682-699 nm.Ezi ziphumo zihambelana noluhlu oluphuhlisiweyo.Ukongezelela, ukusetyenziswa kakuhle kokukhanya kweC0 kuphezulu, oko kukuthi, i-avareji ephezulu ye-C1-C9 yokudlulisa ukukhanya yi-92%.
Imbonakalo yokuhanjiswa kwesibuko se-dichroic kunye nokwahlulwa kwemibala esithoba.(a) Ukulinganisa ukuhanjiswa kwe-spectra ye-M1-M9 kwi-45 ° izehlo kunye ne-BP kwi-0 ° izehlo.(b) I-spectra yokudluliselwa kwe-C1–C9 ngokunxulumene ne-C0 ibalwe ukusuka ku-(a).
Kwikhiwane.I-3c, uluhlu lwezibuko ze-dichroic lubekwe ngokuthe nkqo, ukuze icala lalo lasekunene kwi-Fig.Uluhlu lwezibuko ze-decachromatic eziboniswe kwi-Figure 3a zifakwe kwi-adapter ye-54 mm (ubude) × 58 mm (ubunzulu) × 8.5 mm (ubukhulu).Kwikhiwane.3d, ukongeza kwimeko eboniswe kwifig.I-3c, itanki ye-acrylic ezele ngumsi yabekwa phambi koluhlu lwezibuko ze-decochromatic, kunye nezibane zegumbi zicinyiwe.Ngenxa yoko, imijelo esithoba ye-dichroic ibonakala kwitanki, ephuma kuluhlu lwezibuko ze-decachromatic.Umlambo ngamnye oqhekezayo unecandelo lomnqamlezo oluxande kunye nemilinganiselo ye-1 × 7 mm, ehambelana nobukhulu bokuvula i-spectrometer entsha yemibala esithoba.Kwi-Figure 3b, iphepha libekwe phambi koluhlu lwezibuko ze-dichroic kwi-Figure 3c, kunye nomfanekiso we-1 x 7 mm wemijelo esithoba ye-dichroic eqikelelwe kwiphepha ibonwa ukusuka kwicala lokuhamba kwephepha.imisinga.Imibala esithoba iphuma kwifig.I-3b kunye no-d yi-C4, i-C3, i-C2, i-C1, i-C5, i-C6, i-C7, i-C8 kunye ne-C9 ukusuka phezulu ukuya ezantsi, enokuthi ibonakale kumanani 1 kunye no-2. 1b kunye no-2c.Zijongwa ngemibala ehambelana nobude bazo.Ngenxa yokukhanya okuphantsi kokukhanya okumhlophe kwe-LED (jonga i-Supplementary Fig. S3) kunye novakalelo lwekhamera yombala esetyenziselwa ukubamba i-C9 (682-699 nm) kwi-Fig. Okunye ukuhamba okuqhekezayo kubuthathaka.Ngokufanayo, i-C9 yayibonakala ngokukhawuleza kwiliso lenyama.Okwangoku, i-C2 (umlambo wesibini ukusuka phezulu) ibonakala iluhlaza kwi-Figure 3, kodwa ibonakala iphuzi ngakumbi kwiso lenyama.
Ukutshintsha ukusuka kwi-Figure 3c ukuya kwi-d kuboniswe kwiVidiyo eyoNgezelelweyo 1. Ngokukhawuleza emva kokuba ukukhanya okumhlophe okuvela kwi-LED kudlule kwisibuko sesibuko se-decachromatic, kwahlula ngaxeshanye kwimibala esithoba.Ekugqibeleni, umsi okwivathi waphela ngokuthe ngcembe ukusuka phezulu ukuya ezantsi, kangangokuba imida enemibala elithoba nayo yanyamalala ukusuka phezulu ukuya ezantsi.Ngokwahlukileyo, kwiVidiyo eyoNgezelelweyo yesi-2, xa ubude bokukhanya kwesiganeko sokukhanya kuluhlu lwezibuko ze-decachromatic zatshintshwa ukusuka kwixesha elide ukuya kwifutshane ngokomyalelo we-690, 671, 650, 632, 610, 589, 568, 550 kunye ne-532 nm. ., Kuphela yimijelo ehambelanayo yokwahlula kwemilambo esithoba yokwahlula ngokulandelelana kweC9, C8, C7, C6, C5, C4, C3, C2, kunye neC1 iboniswa.I-acrylic reservoir ithathelwa indawo yi-quartz pool, kwaye iiflakes ze-shunted flow nganye zingabonwa ngokucacileyo ukusuka kwicala elihlayo ukuya phezulu.Ukongezelela, i-sub-video ye-3 ihlelwe ngendlela yokuba inxalenye yokutshintsha kwe-wavelength ye-sub-video ye-2 iphinda iphindwe.Le yeyona ndlela ibalaseleyo yokubonakaliswa kweempawu ze-decochromatic array yezibuko.
Ezi ziphumo zingentla zibonisa ukuba i-decachromatic mirror array eyenziweyo okanye i-spectrometer entsha yemibala esithoba isebenza njengoko bekucetyiwe.I-spectrometer entsha yemibala esithoba yenziwa ngokunyusa uluhlu lwezipili ze-decachromatic ezineadaptha ngqo kwibhodi yoluvo lomfanekiso.
I-flux ekhanyayo kunye ne-wavelength range ukusuka kwi-400 ukuya kwi-750 nm, ekhutshwe ngamanqaku amane emitha ye-radiation φ50 μm, ebekwe kwi-1 mm yezithuba kwi-perpendicular kwi-plane ye-Fig. iilensi ezine φ1 mm kunye nobude obugxile kwi-1.4 mm kunye ne-pitch ye-1 mm.Imijelo emine edibeneyo (emine C0) iyenzeka kwi-DP ye-spectrometer entsha yemibala esithoba, ibekwe kwizithuba ze-1 mm.Uluhlu lwezibuko ze-dichroic zahlula umlambo ngamnye (C0) kwimijelo yemibala esithoba (C1-C9).Iziphumo ze-36 zemilambo (iisethi ezine ze-C1-C9) zifakwe ngokuthe ngqo kwi-CMOS (S) yomfanekiso wenzwa edityaniswe ngokuthe ngqo kuluhlu lwezibuko ze-dichroic.Ngenxa yoko, njengoko kuboniswe kwi-Fig. 5a, ngenxa yendlela encinci ye-optical path difference kunye ne-short maximum optical path, imifanekiso yazo zonke ii-streams ze-36 zafunyanwa ngaxeshanye kwaye ngokucacileyo kunye nobukhulu obufanayo.Ngokombono osezantsi (jonga i-Supplementary Figure S4), ubukhulu bomfanekiso wamaqela amane i-C1, i-C2 kunye ne-C3 iphantsi.Imifanekiso engamashumi amathathu anesithandathu yayiyi-0.57 ± 0.05 mm ngobukhulu (ithetha ± SD).Ngaloo ndlela, ulwandiso lomfanekiso luyi-avareji ye-11.4.Isithuba esithe nkqo phakathi kwemifanekiso yi-avareji ye-1 mm (isithuba esifanayo ne-lens array) kunye ne-horizontal space i-avareji yi-1.6 mm (isithuba esifanayo nesipili se-dichroic).Ngenxa yokuba ubungakanani bomfanekiso buncinci kunomgama phakathi kwemifanekiso, umfanekiso ngamnye unokulinganiswa ngokuzimeleyo (nge-crosstalk ephantsi).Okwangoku, imifanekiso yamashumi amabini anesibhozo emilambo ebhalwe yi-spectrometer yemibala esixhenxe eqhelekileyo esetyenziswe kwisifundo sethu sangaphambili iboniswe kwi-Fig. izibuko kuMfanekiso 1a.Ayiyiyo yonke imifanekiso ebukhali, ubungakanani bomfanekiso buyanda ukusuka kwiC1 ukuya kwiC7.Imifanekiso engamashumi amabini anesibhozo yi-0.70 ± 0.19 mm ngobukhulu.Ngaloo ndlela, kunzima ukugcina isisombululo esiphezulu kuyo yonke imifanekiso.I-coefficient of variation (CV) yobungakanani bomfanekiso 28 kuMfanekiso 5b yayiyi-28%, ngelixa i-CV yobukhulu bomfanekiso 36 kuMfanekiso 5a yehle yaya kutsho kwi-9%.Ezi ziphumo zingentla zibonisa ukuba i-spectrometer entsha yemibala esithoba ayinyusi nje kuphela inani lemibala elinganisiweyo ngaxeshanye ukusuka kwisixhenxe ukuya kwisithoba, kodwa inomfanekiso ophezulu wesisombululo kumbala ngamnye.
Ukuthelekiswa komgangatho womfanekiso oqhekezayo owenziwe ngama-spectrometers aqhelekileyo kunye namatsha.(a) Amaqela amane emibala esithoba yemifanekiso eyahlukanisiweyo (C1-C9) eveliswe yi-spectrometer emitsha enemibala esithoba.(b) Iiseti ezine zemibala esixhenxe yemifanekiso eyahlukanisiweyo (C1-C7) yenziwe ngespectrometer yesiqhelo enemibala esixhenxe.I-Fluxes (C0) ene-wavelengths ukusuka kwi-400 ukuya kwi-750 nm ukusuka kwiindawo ezine zokukhupha i-collimated kunye nesiganeko kwi-spectrometer nganye, ngokulandelanayo.
Iimpawu ze-spectral ze-spectrometer yemibala esithoba ziye zavavanywa ngokuvavanywa kwaye iziphumo zovavanyo ziboniswa kuMfanekiso 6. Qaphela ukuba i-Figure 6a ibonisa iziphumo ezifanayo kunye noMzobo 5a, oko kukuthi kubude be-4 C0 400-750 nm, yonke imifanekiso ye-36 ifunyenwe. (Amaqela ama-4 C1–C9).Ngokuchasene noko, njengoko kubonisiwe kumfanekiso 6b–j, xa iC0 nganye inobude obuthile obuyi-530, 550, 570, 590, 610, 630, 650, 670, okanye 690 nm, kukho phantse imifanekiso emine ehambelanayo (emine). amaqela achongiwe C1, C2, C3, C4, C5, C6, C7, C8 okanye C9).Nangona kunjalo, eminye imifanekiso esecaleni kwemifanekiso emine ehambelanayo ibonakala ibuthathaka kakhulu ngenxa yokuba i-spectra yokudlulisa i-C1-C9 eboniswe kwi-Fig.Ezi ziphumo zihambelana ne-C1-C9 yokuhanjiswa kwe-spectra eboniswe kwiMifanekiso.I-4b kunye neevidiyo ezongezelelweyo 2 kunye ne-3. Ngamanye amazwi, i-spectrometer yombala elithoba isebenza njengoko kulindelekile ngokusekelwe kwiziphumo eziboniswe kwifig.4b.Ngoko ke, kugqitywa ukuba ukusabalalisa kokuqina komfanekiso C1-C9 yi-spectrum yeC0 nganye.
Iimpawu ze-Spectral ze-spectrometer yemibala esithoba.I-spectrometer entsha yemibala esithoba ivelisa iiseti ezine zemibala esithoba yemifanekiso eyahlukanisiweyo (C1-C9) xa ukukhanya kwesiganeko (i-C0 emine) inobude obungamaza (a) 400-750 nm (njengoko kubonisiwe kuMfanekiso 5a), (b) 530 nm.nm, (c) 550 nm, (d) 570 nm, (e) 590 nm, (f) 610 nm, (g) 630 nm, (h) 650 nm, (i) 670 nm, (j) 690 nm, ngokulandelelana.
I-spectrometer ephuhlisiwe yemibala esithoba yayisetyenziselwa i-electrophoresis ye-capillary ezine (ukufumana iinkcukacha, jonga i-Supplementary Materials)31,34,35.I-matrix ene-capillary ine-capillaries ezine (i-diameter yangaphandle eyi-360 μm kunye ne-diameter yangaphakathi eyi-50 μm) ebekwe kwi-1 mm yezithuba kwindawo ye-laser irradiation.Iisampulu eziqulathe amaqhekeza eDNA abhalwe ngeedayi eziyi-8, ezizezi FL-6C (dayi 1), JOE-6C (dayi 2), dR6G (dayi 3), TMR-6C (dayi 4), CXR-6C (dayi 5), TOM- I-6C (idayi 6), i-LIZ (idayi 7), kunye ne-WEN (idayi 8) ngokunyuka kwe-wavelength ye-fluorescent, yahlulwe kwi-capillaries nganye kwezine (emva koku kubhekiselwa kuyo njenge-Cap1, i-Cap2, i-Cap3, ne-Cap4).I-laser-induced fluorescence esuka kwi-Cap1-Cap4 yadityaniswa noluhlu lweelensi ezine kwaye ngaxeshanye zarekhodwa nge-spectrometer enemibala elithoba.I-intensity dynamics ye-nine-color (C1-C9) fluorescence ngexesha le-electrophoresis, oko kukuthi, i-electrophoregram yemibala esithoba ye-capillary nganye, iboniswe kwi-Fig. 7a.I-electrophoregram elinganayo yemibala esithoba ifumaneka kwi-Cap1-Cap4.Njengoko kubonisiwe ngeentolo ze-Cap1 kuMfanekiso 7a, iincopho ezisibhozo kwi-electrophoregram yemibala esithoba ibonisa ukukhutshwa kwe-fluorescence enye kwi-Dye1-Dye8, ngokulandelanayo.
Ubungakanani obungaxeshanye bedayi ezisibhozo kusetyenziswa ispectrometer yemibala esithoba ye-capillary electrophoresis.(a) Imibala esithoba (C1-C9) i-electrophoregram ye-capillary nganye.Iincopho ezisibhozo ezibonakaliswe ngeentolo i-Cap1 ibonisa ukukhutshwa kwe-fluorescence nganye kwiidayi ezisibhozo (i-Dye1-Dye8).Imibala yeentolo ihambelana nemibala (b) kunye no-(c).(b) I-Fluorescence spectra yeedayi ezisibhozo (i-Dye1-Dye8) nge-capillary nganye.c Ii-Electropherograms zedayi ezisibhozo (Dye1-Dye8) ngecapillary nganye.Incopho ye-Dye7 ebhalwe ngamaqhekeza e-DNA ibonakaliswe ngeentolo, kwaye ubude bayo besiseko seCap4 bubonisiwe.
Ukuhanjiswa kokuqina kwe-C1-C9 kwiincopho ezisibhozo kuboniswe kwiiFig.7b, ngokulandelelana.Ngenxa yokuba zombini i-C1-C9 kunye ne-Dye1-Dye8 i-wavelength order, ulwabiwo olusibhozo kwi-Fig. 7b lubonisa i-fluorescence spectra ye-Dye1-Dye8 ngokulandelelana ukusuka ekhohlo ukuya ekunene.Kolu phononongo, iDye1, iDye2, iDye3, iDye4, iDye5, iDye6, iDye7, kunye neDye8 zivela kwi-magenta, violet, blue, cyan, green, yellow, orenji, nebomvu ngokulandelelanayo.Qaphela ukuba imibala yeentolo kwi-Fig. 7a ihambelana nemibala yedayi kwi-Fig. 7b.Amandla e-C1-C9 e-fluorescence kwi-spectrum nganye kwi-Figure 7b yenziwe yesiqhelo ukuze isixa sabo silingane nesinye.Iimbonakalo ezisibhozo ezilinganayo ze-fluorescence zafunyanwa kwi-Cap1-Cap4.Umntu unokujonga ngokucacileyo ukuhlangana okubonakalayo kwe-fluorescence phakathi kwedayi ye-1-dayi yesi-8.
Njengoko kuboniswe kwi-Figure 7c, kwi-capillary nganye, i-electrophoregram yemibala esithoba kwi-Figure 7a yaguqulelwa kwi-electropherogram yedayyi ezisibhozo ngokuhlalutya kwamacandelo amaninzi ngokusekelwe kwi-spectra ye-fluorescence esibhozo kwi-Figure 7b (jonga i-Supplementary Materials ngeenkcukacha).Ekubeni ukudityaniswa kwembonakalo ye-fluorescence kuMfanekiso 7a awuboniswanga kuMfanekiso 7c, iDye1-Dye8 inokuchongwa kwaye ibalwe ngokwahlukeneyo ngexesha ngalinye, nokuba izixa ezahlukeneyo zeDye1-Dye8 fluoresce ngaxeshanye.Oku akunakwenziwa ngobhaqo lwemibala esixhenxe yesintu31, kodwa inokuphunyezwa ngokufunyaniswa kwemibala esithoba.Njengoko kubonisiwe ngeentolo ze-Cap1 kumfanekiso we-7c, kuphela i-fluorescent emission singlets i-Dye3 (ebhlowu), i-Dye8 (ebomvu), i-Dye5 (eluhlaza), i-Dye4 (i-cyan), i-Dye2 (imfusa), i-Dye1 (i-magenta), kunye ne-Dye6 (ephuzi). ) zijongwa ngokolandelelwano olulindelekileyo.Ukukhutshwa kwe-fluorescent yedayi 7 (i-orenji), ngaphezu kweyona nto iphakamileyo eboniswe lutolo lwe-orenji, ezinye iinqununu ezininzi zabonwa.Esi siphumo sibangelwa kukuba iisampuli ziqulethe imilinganiselo yobungakanani, i-Dye7 ibhalwe ngamaqhekeza e-DNA anobude obuhlukeneyo.Njengoko kubonisiwe kuMfanekiso 7c, kwiCap4 obu bude busisiseko ngama-20, 40, 60, 80, 100, 114, 120, 140, 160, 180, 200, 214 kunye ne-220 ubude besiseko.
Iimpawu eziphambili ze-spectrometer yemibala esithoba, ephuhliswe ngokusebenzisa i-matrix yezibuko ezimbini ze-dichroic, zincinci kunye noyilo olulula.Ekubeni uluhlu lwezibuko ze-decachromatic ngaphakathi kwe-adapter eboniswe kumkhiwane.I-3c ifakwe ngokuthe ngqo kwibhodi ye-sensor yomfanekiso (jonga i-Fig. S1 kunye ne-S2), i-spectrometer yemibala esithoba inemilinganiselo efanayo ne-adapter, oko kukuthi 54 × 58 × 8.5 mm.(ubukhulu).Obu bukhulu buncinci buyi-odolo ezimbini ukuya kwezintathu zobukhulu ezincinci kunezibukeli eziqhelekileyo ezisebenzisa iigrayiti okanye iiprism.Ukongeza, kuba i-spectrometer yemibala esithoba icwangciswe ngohlobo lokuba ukukhanya kubethe umphezulu wenzwa yomfanekiso ngokuthe ngqo, indawo inokwabelwa ngokulula i-spectrometer yemibala esithoba kwiinkqubo ezinjenge-microscopes, ii-flow cytometers, okanye abahlalutyi.I-Capillary grating electrophoresis analyzer ye-miniaturization enkulu ngakumbi yenkqubo.Ngelo xesha, ubukhulu bezibuko ezilishumi ze-dichroic kunye nezihlungi ze-bandpass ezisetyenziswe kwi-spectrometer yemibala esithoba yi-10 × 1.9 × 0.5 mm kuphela okanye 15 × 1.9 × 0.5 mm.Ke, ngaphezu kwe-100 izibuko ezincinci ze-dichroic kunye nezihluzo ze-bandpass, ngokulandelelana, zinokusikwa kwisibuko se-dichroic kunye ne-60 mm2 bandpass filter, ngokulandelanayo.Ngoko ke, uluhlu lwezibuko ze-decachromatic lungenziwa ngexabiso eliphantsi.
Olunye uphawu lwe-spectrometer yemibala esithoba yimpawu zayo ezintle kakhulu.Ngokukodwa, ivumela ukufunyanwa kwemifanekiso ye-spectral ye-snapshots, oko kukuthi, ukufunyanwa kwemifanekiso kunye nolwazi lwe-spectral.Kumfanekiso ngamnye, i-spectrum eqhubekayo ifunyenwe kunye ne-wavelength range ukusuka kwi-520 ukuya kwi-700 nm kunye nesisombululo se-20 nm.Ngamanye amazwi, ukuqaqamba kokukhanya kwemibala elithoba kuchongiwe kumfanekiso ngamnye, okt amabhanti alithoba angama-20 nm ahlula ngokulinganayo uluhlu lwamaza ukusuka kuma-520 ukuya kuma-700 nm.Ngokutshintsha iimpawu ze-spectral zesibuko se-dichroic kunye nesihlungi se-bandpass, uluhlu lwamaza e-nine bands kunye nobubanzi bebhendi nganye lunokulungelelaniswa.Ukufunyanwa kwemibala esithoba kungasetyenziselwa kuphela imilinganiselo ye-fluorescence nge-imaging spectral (njengoko kuchazwe kule ngxelo), kodwa nakwezinye ezininzi izicelo eziqhelekileyo zisebenzisa i-spectral imaging.Nangona i-imaging ye-hyperspectral ingakwazi ukubona amakhulu emibala, kufunyenwe ukuba nangona kuncitshiswa kakhulu kwinani lemibala ebonakalayo, izinto ezininzi kwibala lokujonga zingachongwa ngokuchaneka okwaneleyo kwizicelo ezininzi38,39,40.Ngenxa yokuba isisombululo sendawo, isisombululo se-spectral, kunye nesisombululo sexeshana sine-tradeoff kwi-imaging ye-spectral, ukunciphisa inani lemibala kunokuphucula ukulungiswa kwendawo kunye nesisombululo sexeshana.Isenokusebenzisa ii-spectrometers ezilula ezifana nale iphuhliswe kolu phononongo kwaye inciphise ngakumbi inani lokubala.
Kolu phononongo, iidayi ezisibhozo ziye zabalwa ngaxeshanye ngokwahlulwa kwembonakalo ye-fluorescence spectra ethe kratya ngokusekwe ekubhaqweni kwemibala elithoba.Ukuya kuthi ga kwiidayi ezisithoba zingabalwa ngaxeshanye, zihlala ngexesha kunye nendawo.Inzuzo ekhethekileyo ye-spectrometer yemibala esithoba yi-flux yayo ephezulu ekhanyayo kunye ne-aperture enkulu (1 × 7 mm).Uluhlu lwesipili se-decane lugqithise kakhulu kwi-92% yokukhanya ukusuka kwindawo evulekileyo kuluhlu ngalunye lwamaza alithoba.Ukusebenza kokusetyenziswa kokukhanya kwesiganeko kwi-wavelength range ukusuka kwi-520 ukuya kwi-700 nm phantse i-100%.Kuluhlu olubanzi lwamaza obude, akukho diffraction grating inokubonelela ngobuchule obuphezulu bokusebenzisa.Nokuba i-diffraction esebenzayo yegrayiti ye-diffraction idlula i-90% kumgama we-wavelength othile, njengoko umahluko phakathi kobude bezaza kunye ne-wavelength ethile isanda, ukusebenza kakuhle kokuphazamiseka kobunye ubude bezakuza kuncipha41.Ububanzi be-aperture perpendicular ukuya kwicala lendiza kwi-Fig. 2c inokwandiswa ukusuka kwi-7 mm ukuya kububanzi be-sensor yomfanekiso, njengakwimeko ye-sensor yomfanekiso esetyenziswe kule sifundo, ngokuguqula kancinane uluhlu lwe-decamer.
I-spectrometer yemibala esithoba ingasetyenziselwa kuphela i-capillary electrophoresis, njengoko kuboniswe kolu cwaningo, kodwa nakwezinye iinjongo ezahlukeneyo.Ngokomzekelo, njengoko kubonisiwe kumzobo ongezantsi, i-spectrometer yemibala esithoba ingasetyenziswa kwi-microscope ye-fluorescence.I-plane yesampuli iboniswa kwi-sensor yomfanekiso we-spectrometer yemibala esithoba ngokusebenzisa injongo ye-10x.Umgama obonakalayo phakathi kwenjongo ye-lens kunye ne-sensor yomfanekiso ngu-200 mm, ngelixa umgama we-optical phakathi kwendawo yesiganeko se-spectrometer yemibala esithoba kunye ne-sensor yomfanekiso ngu-12 mm kuphela.Ngoko ke, umfanekiso wanqunyulwa malunga nobukhulu be-aperture (1 × 7 mm) kwindiza yeziganeko kwaye yahlulahlulwe kwimifanekiso yemibala esithoba.Okokuthi, umfanekiso we-spectral we-snapshot yemibala esithoba ingathathwa kwindawo ye-0.1 × 0.7 mm kwi-plane yesampuli.Ukongezelela, kunokwenzeka ukufumana umfanekiso we-spectral wemibala esithoba yendawo enkulu kwisampulu yesampulu ngokuskena isampuli ngokumalunga nenjongo kwicala elithe tye kwi-Fig.
Amacandelo e-decachromatic mirror array, okuyi-M1-M9 kunye ne-BP, ayenziwe ngokwezifiso ngu-Asahi Spectra Co., Ltd. usebenzisa iindlela eziqhelekileyo zemvula.Izinto ze-dielectric ze-Multilayer zisetyenziswe ngabanye kwiipleyiti ze-quartz ezilishumi ze-60 × 60 mm ngobukhulu kunye ne-0.5 mm ubukhulu, zihlangabezana nezi mfuno zilandelayo: M1: IA = 45 °, R ≥ 90% kwi-520-590 nm, i-Tave ≥ 90% kwi-610- 610 nm.700 nm, M2: IA = 45 °, R ≥ 90% kwi-520-530 nm, Tave ≥ 90% kwi-550-600 nm, M3: IA = 45 °, R ≥ 90% kwi-540-550 nm, Tave 0 % kwi-570-600 nm, M4: IA = 45 °, R ≥ 90% kwi-560-570 nm, Tave ≥ 90% kwi-590-600 nm, M5: IA = 45 °, R ≥ 98% kwi-580-600 nm , R ≥ 98% kwi-680-700 nm, M6: IA = 45 °, Tave ≥ 90% kwi-600-610 nm, R ≥ 90% kwi-630-700 nm, M7: IA = 45 °, R ≥ 90% 620-630 nm, Taw ≥ 90% kwi-650-700 nm, M8: IA = 45 °, R ≥ 90% kwi-640-650 nm, Taw ≥ 90% kwi-670-700 nm, M9: IA = 45 ° ≥ 90% kwi-650-670 nm, i-Tave ≥ 90% kwi-690-700 nm, i-BP: IA = 0 °, T ≤ 0.01% kwi-505 nm, i-Tave ≥ 95% kwi-530-690 nm kwi-530 nm T0≥ kwi -690 nm kunye ne-T ≤ 1% kwi-725-750 nm, apho i-IA, i-T, i-Tave, kunye ne-R i-angle ye-icidence, i-transmittance, i-avareji yokudlulisa, kunye nokukhanya okungabonakaliyo.
Ukukhanya okumhlophe (C0) kunye noluhlu lwe-wavelength ye-400-750 nm ekhutshwe ngumthombo wokukhanya kwe-LED (AS 3000, AS ONE CORPORATION) yahlanganiswa kwaye yenzeke ngokuthe nkqo kwi-DP yoluhlu lwezipili ze-dichroic.I-spectrum yokukhanya okumhlophe kwee-LEDs iboniswe kwi-Supplementary Figure S3.Beka itanki ye-acrylic (imilinganiselo ye-150 × 150 × 30 mm) ngokuthe ngqo phambi koluhlu lwesibuko se-decamera, ngokuchasene ne-PSU.Umsi oveliswe xa umkhenkce owomileyo untywiliselwa emanzini emva koko wagalelwa kwitanki ye-acrylic ukujonga imijelo enemibala esithoba yeC1-C9 ephuma kuluhlu lwezipili ze-decachromatic.
Ngenye indlela, ukukhanya okumhlophe okudibeneyo (C0) kudlula kwisihluzo ngaphambi kokungena kwi-DP.Izihluzo ekuqaleni bezizihluzo ezingathathi hlangothi zoxinaniso ezinoxinano lwe-0.6.Emva koko sebenzisa isihluzo semoto (FW212C, FW212C, Thorlabs).Ekugqibeleni, vula i-ND yokucoca kwakhona.I-bandwidths ye-nine bandpass filters ihambelana ne-C9, i-C8, i-C7, i-C6, i-C5, i-C4, i-C3, i-C2 kunye ne-C1, ngokulandelanayo.Iseli ye-quartz enemilinganiselo yangaphakathi ye-40 (ubude obude) x 42.5 (ubude) x 10 mm (ububanzi) ibekwe phambi koluhlu lwezibuko ze-decochromatic, ezichasene neBP.Umsi ke utyiswa ngetyhubhu kwiseli ye-quartz ukuze kugcinwe ukuxinwa komsi kwiseli ye-quartz ukuze ubone ngeso lengqondo imijelo enemibala esithoba ye-C1-C9 ephuma kuluhlu lwesibuko se-decachromatic.
Ividiyo yemibala esithoba yokukhanya komlambo ophuma kuluhlu lwezibuko ze-decanic yabanjwa kwimowudi yokuphelelwa lixesha kwi-iPhone XS.Thatha imifanekiso yescene kwi-1 fps kwaye uqokelele imifanekiso ukwenza ividiyo kwi-30 fps (kwividiyo ekhethiweyo 1) okanye i-24 fps (kwiividiyo ezikhethiweyo ze-2 kunye ne-3).
Beka ipleyiti yentsimbi etyebileyo engama-50 µm (enemingxuma emine ye-50 µm yedayamitha nge-1 mm intervals) kwipleyiti ye-diffusion.Ukukhanya okunobude be-400-750 nm kufakwe kwi-diffuser plate, efunyenwe ngokudlula ukukhanya kwisibane se-halogen ngokusebenzisa isihluzo esifutshane sokuhambisa kunye ne-cutoff wavelength ye-700 nm.I-spectrum yokukhanya iboniswe kwi-Supplementary Figure S4.Kungenjalo, ukukhanya kudlula kwesinye sezihluzi ze-10 nm bandpass ezizinze kwi-530, 550, 570, 590, 610, 630, 650, 670 kunye ne-690 nm kwaye ibethe i-diffuser plate.Ngenxa yoko, iindawo ezine zokusasazeka kwemitha ezinobubanzi obuyi-φ50 μm kunye nobude obuhlukeneyo bobude benziwa kwipleyiti yentsimbi echasene nepleyiti ye-diffuser.
Uluhlu olune-capillary ezine iilensi ezine lufakwe kwi-spectrometer yemibala esithoba njengoko kuboniswe kwiMifanekiso 1 kunye ne-2. I-C1 kunye ne-C2.Ii-capillaries ezine kunye neelensi ezine zazifana nezifundo zangaphambili31,34.I-laser beam ene-wavelength ye-505 nm kunye namandla e-15 mW i-irradiated ngaxeshanye kwaye ngokulinganayo ukusuka kwicala ukuya kwiindawo zokukhupha ezine-capillaries.I-fluorescence ekhutshwa kwindawo nganye yokukhutshwa ihlanganiswe yi-lens ehambelanayo kwaye ihlulwe ibe yimijelo yemibala esithoba ngoluhlu lwezibuko ze-decachromatic.Iziphumo ze-36 ezibangelwa yimijelo zaye zafakwa ngokuthe ngqo kwi-sensor ye-CMOS yomfanekiso (C11440-52U, i-Hamamatsu Photonics K · K.), kunye nemifanekiso yabo yabhalwa ngaxeshanye.
I-ABI PRISM® BigDye® Primer Cycle Sequencing Ready Reaction Kit (I-Applied Biosystems), 4 µl GeneScan™ 600 LIZ™ idayi yaxutywa kwi-capillary nganye ngokuxuba i-1 µl PowerPlex® 6C Matrix Standard (Promega Corporation), i-1 µl yobungakanani bomgangatho womxube.v2.0 (Thermo Fisher Scientific) kunye ne-14 µl yamanzi.I-PowerPlex® 6C Matrix Standard iquka amaqhekeza amathandathu e-DNA abhalwe ngeedayi ezintandathu: FL-6C, JOE-6C, TMR-6C, CXR-6C, TOM-6C, kunye ne-WEN, ngokomyalelo wobude bobude.Ubude besiseko sezi ziqwenga ze-DNA azichazwanga, kodwa ukulandelelana kobude besiseko se-DNA fragments ebhalwe nge-WEN, CXR-6C, TMR-6C, JOE-6C, FL-6C kunye ne-TOM-6C iyaziwa.Umxube kwi-ABI PRISM® BigDye® Primer Cycle Sequencing Ready Reaction Kit iqulethe iqhekeza le-DNA elibhalwe ngedayi ye-dR6G.Ubude beziseko zamaqhekeza eDNA abuchazwanga.I-GeneScan™ 600 LIZ™ Ubungakanani beDayi uMgangatho we-v2.0 ubandakanya amaqhekeza e-DNA angama-36 e-LIZ.Ubude besiseko bezi ziqwenga zeDNA ngama-20, 40, 60, 80, 100, 114, 120, 140, 160, 180, 200, 214, 220, 240, 250, 260, 280, 40, 30, 30, 30 360, 380, 400, 414, 420, 440, 460, 480, 500, 514, 520, 540, 560, 580 kunye ne-600 isiseko.Iisampulu zahlanjululwa kwi-94 ° C kwimizuzu emi-3, emva koko zipholile kwi-ice imizuzu emi-5.Iisampuli zafakwa kwi-capillary nganye kwi-26 V / cm kwi-9 s kwaye yahlulwe kwi-capillary nganye ezaliswe isisombululo se-polymer ye-POP-7™ (i-Thermo Fisher Scientific) enobude obusebenzayo obuyi-36 cm kunye ne-voltage ye-181 V / cm kunye i-engile ye-60 °.UKUSUKA.
Yonke idatha efunyenweyo okanye ehlalutyiweyo ekuhambeni kwesi sifundo ifakwe kweli nqaku elipapashwe kunye nolwazi olongezelelweyo.Enye idatha efanelekileyo kolu phononongo iyafumaneka kubabhali abachaphazelekayo ngesicelo esifanelekileyo.
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