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I-Thermophiles ziintsholongwane ezikhula kakuhle kumaqondo obushushu aphezulu. Ukuzifunda kunokunika ulwazi oluxabisekileyo malunga nendlela ubomi obuziqhelanisa ngayo neemeko ezinzima. Nangona kunjalo, kunzima ukufezekisa iimeko zokushisa eziphezulu kunye neemicroscopes eziqhelekileyo ezibonakalayo. Izisombululo ezininzi ezenziwe ekhaya ezisekelwe kwindawo yokufudumeza umbane ochasayo ziye zacetywa, kodwa akukho sicombululo esilula sorhwebo. Kweli phepha, sazisa ingqikelelo yokufudumeza nge-laser microscale ngaphezulu kwendawo yokujonga i-microscope ukubonelela ngamaqondo obushushu aphezulu kwizifundo ze-thermophile ngelixa ugcina indawo yomsebenzisi ithambile. Ukufudumeza kweMicroscale kwimodareyitha ye-laser intensity kunokufezekiswa kusetyenziswa i-nanoparticle coated substrate yegolide njenge-biocompatible kunye ne-absorption esebenzayo yokukhanya. Iziphumo ezinokwenzeka ze-microscale fluid convection, ukugcinwa kweeseli, kunye ne-centrifugal thermophoretic motion kuxoxwa ngazo. Indlela ibonakaliswe kwiindidi ezimbini: (i) I-Geobacillus stearothermophilus, ibhaktheriya esebenzayo ye-thermophilic ephindaphinda malunga ne-65 ° C, esiye saqaphela ukuba intshula, ikhula kwaye idada phantsi kokufudumala kwe-microscale; (ii) I-Thiobacillus sp., i-archaea ephezulu kakhulu ye-hyperthermophilic. ku-80°C. Lo msebenzi uvula indlela yoqwalaselo olulula nolukhuselekileyo lwe-thermophilic microorganisms kusetyenziswa izixhobo zemakroskopu zanamhlanje nezifikelelekayo.
Ngaphezulu kweebhiliyoni zeminyaka, ubomi eMhlabeni buye bavela ukuze baqhelane noluhlu olubanzi lweemeko zokusingqongileyo ezithi ngamanye amaxesha zijongwe ngokugqithisileyo ngokwembono yethu yabantu. Ngokukodwa, ezinye ii-microorganisms ze-thermophilic (ibhaktheriya, i-archaea, i-fungi) ebizwa ngokuba yi-thermophiles ikhula kakuhle kwiqondo lokushisa ukusuka kwi-45 ° C ukuya kwi-122 ° C1, 2, 3, 4. I-Thermophiles ihlala kwiindawo ezahlukeneyo zendalo, ezifana neendawo ezinzulu ze-hydrothermal yolwandle, imithombo eshushu. okanye iindawo ezinentaba-mlilo. Uphando lwabo luvelise umdla omkhulu kule minyaka imbalwa idlulileyo ngenxa yezizathu ezibini. Okokuqala, sinokufunda kubo, umzekelo, indlela i-thermophiles 5, 6, i-enzymes 7, i-8 kunye ne-membrane ye-9 ezinzile kubushushu obuphezulu obunjalo, okanye indlela i-thermophiles enokumelana ngayo namazinga aphezulu e-radiation10. Okwesibini, zisisiseko sezicelo ezininzi ezibalulekileyo ze-biotechnological1,11,12 ezifana nokuveliswa kwamafutha13,14,15,16, ukuhlanganiswa kweekhemikhali (dihydro, alcohols, methane, amino acids, njl.)17, biomining18 kunye ne-thermostable biocatalysts7 ,11, 13. Ngokukodwa, i-polymerase chain reaction (PCR)19 eyaziwayo ngoku ibandakanya i-enzyme (Taq polymerase) ekwanti kwibhaktheriya ye-thermophilic i-Thermus aquaticus, enye ye-thermophiles yokuqala ukufunyanwa.
Nangona kunjalo, ukufundwa kwe-thermophiles akuwona umsebenzi olula kwaye akunakuphuculwa kuyo nayiphi na ilebhu yebhayoloji. Ngokukodwa, i-thermophiles ephilayo ayinakubonwa kwi-vitro nayo nayiphi na i-microscope yokukhanya esemgangathweni, kunye namagumbi okufudumeza akhoyo ngokurhweba, adla ngokulinganiswa amaqondo obushushu angaphantsi kwe-40 ° C. Ukusukela ngeminyaka yee-1990s, ngamaqela ambalwa ophando azinikeleyo ekwaziseni ii-high-temperature microscopy (HTM) iinkqubo. Ngo-1994 uGlukh et al. Igumbi lokufudumeza / lokupholisa lakhawulwa ngokusekelwe ekusebenziseni iseli yePeltier elawula ukushisa kwama-capillaries angama-rectangular avaliweyo ukugcina i-anaerobicity 20. Isixhobo sinokufudunyezwa ukuya kuthi ga kwi-100 ° C ngesantya se-2 ° C / s, sivumela ababhali ukuba bafunde ukuhamba kwebhaktheriya ye-hyperthermophilic Thermotoga maritima21. Ngowe-1999 Horn et al. Kuye kwaphuhliswa isixhobo esifana kakhulu, sisasekelwe ekusetyenzisweni kwee-capillaries ezifudumalayo ezifanelekileyo kwi-microscopy yorhwebo ukufunda ulwahlulo/uqhagamshelwano lweseli. Emva kwexesha elide lokungasebenzi okuhlobene, ukukhangela ii-HTM ezisebenzayo zaqala kwakhona kwi-2012, ngokukodwa ngokumalunga noluhlu lwamaphepha liqela le-Wirth elisebenzisa isixhobo esakhiwe nguHorn et al. Kwiminyaka elishumi elinesihlanu edlulileyo, ukuhamba kwenani elikhulu le-archaea, kuquka i-hyperthermophiles, yafundwa kumaqondo okushisa ukuya kwi-100 ° C usebenzisa i-capillaries evuthayo23,24. Baphinde bahlengahlengisa i-microscope yokuqala ukufezekisa ukufudumeza ngokukhawuleza (imizuzu embalwa endaweni yemizuzu engama-35 ukufikelela kwiqondo lobushushu elibekiweyo) kunye nokufezekisa i-gradient yeqondo lokushisa elingaphezulu kwe-2 cm phakathi nendawo. Esi sixhobo sokubunjwa kweqondo lobushushu (TGFD) sisetyenziselwe ukufunda ukushukuma kwee-thermophiles ezininzi ngaphakathi kweqondo lobushushu kwimigama ehambelana nebhayoloji eyi-24, 25.
Ukufudumeza ii-capillaries ezivaliweyo akukuphela kwendlela yokujonga i-thermophiles ephilayo. Kwi-2012, Kuwabara et al. Amagumbi ePyrex alahlwayo enziwe ekhaya atywinwe ngokuncamathelisa ukumelana nobushushu (Super X2; Cemedine, Japan) kwasetyenziswa. Iisampulu zifakwe kwi-plate yokufudumeza ecacileyo efumanekayo yorhwebo (i-Micro Heat Plate, i-Kitazato Corporation, eJapan) ekwazi ukufudumeza ukuya kwi-110 ° C, kodwa kungekhona ekuqaleni i-bioimaging. Ababhali baqaphele ukwahlula okusebenzayo kwebhaktheriya ye-anaerobic thermophilic (i-Thermosipho globiformans, ixesha eliphindwe kabini le-24 min) kwi-65 ° C. Ngo-2020, uPulshen et al. Ukufudumeza okusebenzayo kwezitya zetsimbi zorhwebo (i-Attofluor TM, i-Thermofisher) ibonakaliswe ngokusebenzisa izinto ezimbini zokufudumeza zasekhaya: isiciko kunye nesiteji (ubumbeko oluphefumlelweyo lomatshini we-PCR). Olu nxulumano lubangela ubushushu obufanayo bolwelo kwaye luthintele ukuphuphuma komphunga kunye ne-condensation ezantsi kwesiciko. Ukusetyenziswa kwe-O-ring kugwema ukutshintshiselwa kwegesi kunye nokusingqongileyo. Le HTM, ebizwa ngokuba yiSulfoscope, isetyenziswe ukwenza umfanekiso weSulfolobus acidocaldarius kuma-75°C27.
Unyino olwaziwayo lwazo zonke ezi nkqubo yaba sisithintelo ekusetyenzisweni kwenjongo zomoya, nakuphi na ukuntywiliselwa kwe-oyile kungafanelanga ubushushu obuphezulu obunjalo kunye nokucinga nge->1-mm yeesampulu ezibonisa elubala. Unyino olwaziwayo lwazo zonke ezi nkqubo yaba sisithintelo ekusetyenzisweni kwenjongo zomoya, nakuphi na ukuntywiliselwa kwe-oyile kungafanelanga ubushushu obuphezulu obunjalo kunye nokucinga nge->1-mm yeesampulu ezibonisa elubala. Inkcazelo ngokuthe gabalala malunga nenkqubo yekhompyutha. для такой высокой температуры и для визуализации через прозрачные образцы толщиной > 1 мм. Ukusilela okwaziwayo kuzo zonke ezi nkqubo yaba kukuthintelwa kokusetyenziswa kweenjongo zomoya, kuba nakuphi na ukuntywiliselwa kwe-oyile kwakungafanelanga ubushushu obuphezulu obunjalo kunye nokubonwa ngeesampulu ezicacileyo> 1 mm ubukhulu.所有这些系统的一个公认限制是限制使用空气物镜, 任何油浸都不适合这样的高温和通过> 1 毫米厚的透明样品成像. Umda ovunyiweyo wazo zonke ezi nkqubo kukuthintelwa kokusetyenziswa kwesibuko esinomoya, njengoko nakuphi na ukuntywiliselwa kwe-oyile kungenakulungelelaniswa kumfanekiso weisampulu ezicacileyo> 1 mm ubukhulu kumaqondo obushushu aphezulu ngolo hlobo. Inkcazo malunga nenkqubo yekhompyutha таких высоких температур и визуализации через прозрачные образцы толщиной >1 мм. I-drawback eyaziwayo yazo zonke ezi nkqubo kukusetyenziswa okulinganiselweyo kweelensi zomoya, nayiphi na into yokuntywiliselwa kwe-oyile ayifanelanga ubushushu obuphezulu kunye nokubonwa ngeesampulu ezicacileyo> 1 mm ubukhulu.Kutshanje, lo mda uphakanyiswe nguCharles-Orzag et al. 28, owaphuhlisa isixhobo engasaboneleli ubushushu emhlabeni inkqubo umdla, kodwa kunokuba ngaphakathi iglasi cover ngokwayo, egqunywe umaleko obhityileyo transparent of a resistor eyenziwe ITO (indium-tin oxide). Isivalo sinokufudunyezwa ukuya kuthi ga kwi-75 °C ngokugqithisa umsinga wombane kumaleko akhanyayo. Nangona kunjalo, umbhali kufuneka afudumeze i-lens kwinjongo, kodwa ingabi ngaphezu kwe-65 ° C, ukuze ingonakalisi.
Le misebenzi ibonisa ukuba uphuhliso olusebenzayo lwemakroskopu yobushushu obuphezulu obuphezulu alukhange lwamkelwe ngokubanzi, luhlala lufuna izixhobo zasekhaya, kwaye luhlala luphunyezwa ngeendleko zokusonjululwa kwendawo, nto leyo engalunganga kakhulu xa kujongwe ukuba i-thermophilic microorganisms azikho zikhulu kunembalwa. iimicrometers. Ukunciphisa umthamo wokufudumeza ngundoqo ekusombululeni iingxaki ezintathu zendalo ze-HTM: ukungasonjululwa kakuhle kwendawo, inertia ephezulu ye-thermal xa inkqubo ishushu, kunye nokufudumeza okuyingozi kwezinto ezijikelezileyo (i-oyile yokuntywiliselwa, i-lens yenjongo… okanye izandla zomsebenzisi) kumaqondo obushushu agqithisileyo. ).
Kweli phepha, sazisa i-HTM yoqwalaselo lwe-thermophile olungasekelwanga ekufudumezeni okuxhathisayo. Endaweni yoko, siye safumana ukufudumeza kwendawo ngaphakathi kwendawo elinganiselweyo yendawo yokujonga yemakroskopu ngokukhanya kwelaser yesubstrate efunxa ukukhanya. Ukuhanjiswa kweqondo lokushisa kwabonwa kusetyenziswa i-quantitative phase microscopy (QPM). Ukusebenza kwale ndlela kuboniswa yi-Geobacillus stearothermophilus, i-motile thermophilic bacterium evelisa malunga ne-65 ° C kwaye inexesha elifutshane lokuphindaphinda (malunga nemizuzu engama-20), kunye ne-Sulfolobus shibatae, i-hyperthermophile ekhula ngokufanelekileyo kwi-80 ° C (archaea) ukubonisa. Izinga lokuphindaphinda okuqhelekileyo kunye nokuqubha kwabonwa njengomsebenzi weqondo lokushisa. Le laser HTM (LA-HTM) ayikhawulelwanga ngobunzima be-coverlip okanye ngohlobo lwenjongo (umoya okanye ukucwiliswa kweoli). Oku kuvumela nayiphi na isisombululo esiphezulu se-lens kwimarike ukuba isetyenziswe. Kananjalo ayikhathazwa kukufudumeza okucothayo ngenxa ye-thermal inertia (ifezekisa ukufudumeza okukhawulezileyo kwisikali se-millisecond) kwaye isebenzisa kuphela amacandelo akhoyo ngokurhweba. Ekuphela kweenkxalabo ezitsha zokhuseleko zinxulumene nobukho bemiqadi yelaser enamandla (ngokuqhelekileyo ukuya kuthi ga kwi-100 mW) ngaphakathi kwesixhobo kwaye kunokwenzeka nasemehlweni, afuna iiglasi ezikhuselayo.
Umgaqo we-LA-HTM kukusebenzisa i-laser ukutshisa isampuli kwindawo ngaphakathi kwendawo yokujonga i-microscope (Umfanekiso 1a). Ukwenza oku, isampuli kufuneka ibe lula. Ukusebenzisa amandla e-laser afanelekileyo (ngaphantsi kwe-100 mW), asizange sithembele ekufakweni kokukhanya ngumbane ophakathi, kodwa ngobuchule bonyusa ukufunxwa kwesampulu ngokugubungela i-substrate nge-nanoparticles yegolide (Umfanekiso 1c). Ukufudumeza iinanoparticles zegolide ngokukhanya kubaluleke kakhulu kwintsimi ye-thermal plasmonics, kunye nokusetyenziswa okulindelweyo kwi-biomedicine, i-nanochemistry okanye ukukhanya kwelanga29,30,31. Kwiminyaka embalwa edlulileyo, siye sasebenzisa le LA-HTM kwizifundo ezininzi ezinxulumene ne-thermal plasma izicelo kwi-physics, chemistry kunye ne-biology. Ubunzima obuphambili ngale ndlela kukubonisa iprofayili yokugqibela yeqondo lokushisa, ekubeni ubushushu obuphakanyisiweyo bukhawulelwe kummandla we-microscale ngaphakathi kwesampuli. Siye sabonisa ukuba imephu yobushushu inokufezekiswa nge-four-wavelength transverse shear interferometer, indlela elula, ene-high-resolution, kunye nendlela enovakalelo kakhulu ye-quantitative phase microscopy esekelwe ekusetyenzisweni kwe-diffraction gratings (eyaziwa ngokuba yi-cross gratings) 33,34,35,36. Ukuthembeka kobu buchule bemakroskopu yobushushu, esekwe kwi-cross grating wavefront microscopy (CGM), kubonakaliswe kumaphepha alishumi elinesibini apapashwe kule minyaka ilishumi idlulileyo37,38,39,40,41,42,43.
Inkqubo yofakelo lwe-laser ehambelanayo yokufudumeza, ukubunjwa kunye ne-microscope yobushushu. b Isampulu yejometri ebandakanya igumbi le-Attofluor TM elinoqweqwe lokugquma olugqunywe ngeenanoparticles zegolide. c Jonga ngokusondeleyo isampuli (ungalinganisi). d imele iprofayile yomqadi welaser efanayo kunye (e) nokuhanjiswa kobushushu okufaniswayo okulandelayo kwisampulu yesampulu yeenanoparticles zegolide. f yiprofayili yomqadi welaser annular ilungele ukuvelisa ubushushu obufanayo njengoko kubonisiwe kulinganiso lwesiphumo sokusasazwa kobushushu obuboniswe ku (g). Isikali somgca: 30 µm.
Ngokukodwa, kutshanje sifumene ukufudumeza kweeseli ezincancisayo nge-LA-HTM kunye ne-CGM kwaye silandele iimpendulo zothuso lobushushu beselula kuluhlu lwe-37-42 ° C, ebonisa ukusebenza kobu buchule kumfanekiso weseli ephilayo enye. Nangona kunjalo, ukusetyenziswa kwe-LA-HTM kwisifundo se-microorganisms kumaqondo aphezulu akucacile, njengoko kufuna ukuqaphela ngakumbi xa kuthelekiswa neeseli ze-mammalian: okokuqala, ukufudumeza umzantsi ophakathi ngamadigri amashumi (kunokuba amadigri ambalwa) ukuya kwiqondo lobushushu elithe nkqo elomeleleyo. inokudala i-convection ye-fluid 44 ethi, ukuba ayinamathele ngokuqinileyo kwi-substrate, inokubangela ukunyakaza okungafunekiyo kunye nokuxuba ibhaktheriya. Le convection inokupheliswa ngokunciphisa ubukhulu bomgangatho wolwelo. Ukwenzela le njongo, kuzo zonke iimvavanyo ezithiwe thaca apha ngezantsi, ukunqunyanyiswa kwebhaktiriya kwabekwa phakathi kwezigqubuthelo ezimbini ezimalunga ne-15 µm ubukhulu zifakwe ngaphakathi kwekomityi yentsimbi (Attofluor TM, Thermofisher, Fig. 1b,c). Ngokomgaqo, i-convection inokuphetshwa ukuba ubukhulu bolwelo buncinci kunobukhulu be-laser yokufudumala. Okwesibini, ukusebenza kwijometri enjalo elinganiselweyo kunokufunxa izinto eziphilayo ze-aerobic (jonga umfanekiso we-S2). Le ngxaki inokuphetshwa ngokusebenzisa i-substrate ekwazi ukungena kwi-oksijeni (okanye nayiphi na enye igesi ebalulekileyo), ngokushiya ama-bubbles emoyeni abanjwe ngaphakathi kwe-coverlip, okanye ngokubhoboza imingxuma kwi-coverlip top (jonga umfanekiso we-S1) 45. Kule sifundo, sakhetha isisombululo sokugqibela (Amanani 1b kunye ne-S1). Ekugqibeleni, ukufudumeza kwe-laser akuboneleli ngokusasazwa kobushushu obufanayo. Nangona ukuqina okufanayo kwe-laser beam (Umfanekiso 1d), ukuhanjiswa kweqondo lokushisa akufani, kodwa kunokuba kufana nokusabalalisa kweGaussian ngenxa yokusabalalisa kwe-thermal (Umfanekiso 1e). Xa injongo kukuseka amaqondo obushushu achanekileyo kwinkalo yokujonga ukufunda iinkqubo zebhayoloji, iiprofayili ezingalinganiyo azifanelekanga kwaye zinokukhokelela ekuhambeni kwe-thermophoretic yeebhaktheriya ukuba azibambeleli kwi-substrate (jonga umzobo S3, S4)39. Ukuza kuthi ga ngoku, sasebenzisa imodyuli yokukhanya yendawo (SLM) ukubumba umqadi we-laser we-infrared ngokwemilo yeringi (Fig. 1f) kwinqwelomoya yesampulu ukufezekisa ukuhanjiswa kweqondo lobushushu ngokugqibeleleyo ngaphakathi kwendawo yejometri enikiweyo, nangona ukusabalalisa kwe-thermal (umzobo 1d) 39, 42, 46. Beka i-coverlip phezulu phezu kwesitya sesinyithi (Umfanekiso 1b) ukuphepha ukuvela kwe-evaporation ye-medium kwaye ugcine ubuncinane iintsuku ezimbalwa. Ngenxa yokuba esi sigqubuthelo siphezulu asitywinwanga, i-medium eyongezelelweyo inokongezwa ngokulula nangaliphi na ixesha ukuba kuyimfuneko.
Ukubonisa indlela esebenza ngayo i-LA-HTM kwaye ibonise ukusebenza kwayo kuphando lwe-thermophilic, sifunde ibhaktiriya ye-aerobic Geobacillus stearothermophilus, enobushushu obuphezulu bokukhula obumalunga ne-60-65°C. Ibhaktheriya nayo ine-flagella kunye nokukwazi ukuqubha, inika esinye isalathisi somsebenzi oqhelekileyo weselula.
Iisampulu (umzobo 1b) zifakwe ngaphambili kwi-60 ° C ngeyure enye kwaye emva koko zifakwe kwi-LA-HTM yesibambi sesampula. Le pre-incubation iyakhethwa, kodwa iseluncedo, ngenxa yezizathu ezibini: Okokuqala, xa i-laser ivuliwe, ibangela ukuba iiseli zikhule ngokukhawuleza kwaye zahlule (jonga i-movie M1 kwi-Supplementary Materials). Ngaphandle kokufaka kwangaphambili, ukukhula kwebhaktiriya ngokuqhelekileyo kulibaziseka malunga nemizuzu engama-40 ngexesha ngalinye indawo entsha yokujonga ishushu kwisampulu. Okwesibini, iyure ye-1 yangaphambi kwe-incubation ikhuthaze ukunamathela kwebhaktheriya kwi-coverlip, ukuthintela iiseli ukuba zikhuphe ngaphandle kwebala lokujonga ngenxa ye-thermophoresis xa i-laser ivuliwe (jonga ifilimu i-M2 kwi-Supplementary Materials). I-Thermophoresis yintshukumo yeencinci okanye iimolekyuli kunye ne-gradient yeqondo lokushisa, ngokuqhelekileyo ukusuka kushushu ukuya kubanda, kwaye iibhaktheriya azikho ngaphandle kwe-43,47. Esi siphumo esingathandekiyo sipheliswa phezu kwendawo enikiweyo ngokusebenzisa i-SLM ukubumba i-laser beam kunye nokufezekisa ukusabalalisa kweqondo lokushisa.
Kwikhiwane. Umzobo we-2 ubonisa ukuhanjiswa kweqondo lokushisa elilinganiswa yi-CGM efunyenwe ngokufakela i-glass substrate ehlanganiswe ne-nanoparticles yegolide kunye ne-laser beam annular (Fig. 1f). Ukwabiwa kobushushu obuthe tyaba kuye kwajongwa phezu kwendawo yonke egqunywe ngumqa welaser. Lo mmandla wawumiselwe kuma-65°C, elona qondo lobushushu liphezulu lokukhula. Ngaphandle kwalo mmandla, ijika lobushushu ngokwendalo liwela ku \(1/r\) (apho \(r\) lulungelelwaniso lweradial).
imephu yoBubushushu yemilinganiselo yeCGM efunyenwe ngokusebenzisa i-laser beam annular ukuze i-irradiate umaleko we-nanoparticles yegolide ukufumana iphrofayili yobushushu obucaba kwindawo ejikelezayo. b Isotherm yemephu yobushushu (a). I-contour ye-laser beam imelwe isangqa esinamachaphaza angwevu. Uvavanyo luphindwe kabini (jonga i-Supplementary Materials, Figure S4).
Ukusebenza kweeseli zebhaktheriya kwajongwa iiyure eziliqela usebenzisa i-LA-HTM. Kwikhiwane. I-3 ibonisa ikhefu lexesha lemifanekiso emine ethathwe kwi-3 iyure ye-20 ye-movie yemizuzu (i-Movie M3, iNkcukacha eyoNgezelelweyo). Iibhaktheriya zabonwa ukuba zanda ngokusebenzayo ngaphakathi kwendawo yesetyhula echazwe yilaser apho iqondo lobushushu laliphezulu, lisondela kwi-65 ° C. Ngokwahlukileyo, ukukhula kweeseli kwancitshiswa kakhulu xa iqondo lobushushu liwela ngaphantsi kwe-50 ° C kwi-10 s.
Imifanekiso enzulu ye-Optical deep ye-G. stearothermophilus bacteria ekhula emva kokufudunyezwa kwelaser ngamaxesha ahlukeneyo, (a) t = 0 min, (b) 1 h 10 min, (c) 2 h 20 min, (d) 3 h 20 min, ngaphandle I-200 Icatshulwe kwifilimu yomzuzu omnye (ifilimu ye-M3 enikezelwe kwiiNkcukacha ezoNgezelelweyo) ebekwe phezulu kwimephu yobushushu ehambelanayo. Ilaser ivula ngexesha \(t=0\). Ii-Isotherms zongezwe kumfanekiso wobunzulu.
Ukuqhubela phambili ukulinganisa ukukhula kweeseli kunye nokuxhomekeka kwayo kwiqondo lokushisa, silinganise ukunyuka kwe-biomass yeekholoni ezahlukeneyo zeebhaktheriya ezizimeleyo ekuqaleni kwi-Movie M3 yemboniselo (Fig. 4). Ibhaktheriya yomzali ekhethiweyo ekuqaleni kweyunithi yokwenza i-mini colony forming unit (mCFU) iboniswe kuMfanekiso S6. Imilinganiselo yobunzima obomileyo ithathwe ngekhamera ye-CGM ye-48 eyayisetyenziselwa ukwenza imephu yokusabalalisa ukushisa. Ikhono le-CGM lokulinganisa ubunzima obomileyo kunye nobushushu bunamandla e-LA-HTM. Njengoko bekulindelekile, ukushisa okuphezulu kubangele ukukhula kwebhaktheriya ngokukhawuleza (Umfanekiso 4a). Njengoko kubonisiwe kwisicwangciso semi-log kumfanekiso we-4b, ukukhula kuwo onke amaqondo obushushu kulandela ukukhula okubonakalayo, apho idatha isebenzisa umsebenzi we-exponential \(m={m}_{0}{10}^{t/\ tau}) + {{\mbox{cst}}}\), apho \(\ tau {{{{\rm{log}}}}}}}}2\) - ixesha lesizukulwana (okanye ixesha eliphindwe kabini), \( g =1/ \tau\) - izinga lokukhula (inani lezahlulo ngexesha leyunithi). Kwikhiwane. I-4c ibonisa isantya sokukhula kunye nexesha lesizukulwana njengomsebenzi weqondo lobushushu. Ii-mCFUs ezikhula ngokukhawuleza ziphawulwa kukukhula kokukhula emva kweeyure ezimbini, ukuziphatha okulindelweyo ngenxa yoxinaniso lwebhaktiriya ephezulu (efana nenqanaba lokumisa kwiinkcubeko zolwelo zakudala). Imilo ngokubanzi \(g\left(T\right)\) (Fig. 4c) ihambelana negophe elilindelekileyo lesigaba sesibini se-G. stearothermophilus enezinga eliphezulu lokukhula malunga ne-60-65 °C. Tshatisa idatha usebenzisa imodeli yekhadinali (Figure S5)49 apho \(\ left({{G}_{0}{;\;T}}_{{\min }};{T}_{{opt} } ;{T}_{{\max}}}\ ekunene)\) = (0.70 ± 0.2; 40 ± 4; 65 ± 1.6; 67 ± 3) °C, evumelana kakuhle nezinye imilinganiselo ekhankanywe kuncwadi49. Nangona iiparamitha ezixhomekeke kwiqondo lobushushu ziphinda zikwazi ukuphinda ziphindeke, umlinganiselo wokukhula ophezulu we \({G}_{0}\) unokwahluka ukusuka kumfuniselo omnye ukuya kwenye (jonga amanani S7-S9 kunye nemovie M4). Ngokwahlukileyo kwiiparamitha zokufudumala kweqondo lokushisa, okumele kube yinto yonke, izinga eliphezulu lokukhula lixhomekeke kwiipropati ze-medium (ubukho bezondlo, i-oxygen concentration) ngaphakathi kwejometri ye-microscale ephawulweyo.
Ukukhula kweMicrobial kumaqondo obushushu ahlukeneyo. I-mCFU: IiYunithi zokwenziwa kweKoloni encinci. Idatha efunyenwe kwividiyo yebhaktheriya enye ekhula kwiqondo lokushisa (i-movie M3). b Kunye no-(a), isikali semi-logarithmic. c Izinga lokukhula\(\tau\) kunye nexesha lesizukulwana\(g\) elibalwe ukusuka kumgca wokuhlehla ngomgca (b). Imivalo yempazamo ethe tyaba: uluhlu lobushushu apho i-mCFUs yandiswe kwindawo yokujonga ngexesha lokukhula. Iibar zemposiso ezithe nkqo: imposiso esemgangathweni yobuyiselo lomgca.
Ukongeza ekukhuleni okuqhelekileyo, ezinye iibhaktheriya ngamanye amaxesha zazidada kwimbono ngexesha lokufudumeza ngelaser, nto leyo yindlela elindelekileyo yokuziphatha kwebhaktheriya eneflegi. Ifilimu i-M5 ngolwazi olongezelelweyo ibonisa imisebenzi enjalo yokubhukuda. Kolu vavanyo, imitha yelaser efanayo isetyenziselwe ukwenza iqondo lobushushu, njengoko kubonisiwe kwiMizobo 1d, e kunye ne-S3. Umzobo we-5 ubonisa ulandelelwano lwemifanekiso emibini ekhethiweyo kwimuvi ye-M5 ebonisa ukuba ibhaktheriya enye ibonisa intshukumo eya kwicala ngelixa zonke ezinye iibhaktheriya zihlala zingashukumi.
Amaxesha amabini (a) kunye (b) abonisa ukuqubha kwebhaktiriya ezimbini ezahlukeneyo eziphawulwe ngezangqa ezinamachokoza. Imifanekiso yatsalwa kumboniso bhanyabhanya we-M5 (ubonelelwe njengemathiriyeli eyongezelelweyo).
Kwimeko yeG. stearothermophilus, ukunyakaza okusebenzayo kweebhaktheriya (umzobo 5) kwaqala imizuzwana embalwa emva kokuba i-laser beam ivuliwe. Olu qwalaselo lugxininisa impendulo yesikhashana yale microorganism ye-thermophilic ekwandeni kobushushu, njengoko sele kuphawulwe nguMora et al. 24 . Isihloko se-bacterial motility kunye ne-thermotaxis sinokuphononongwa ngakumbi usebenzisa i-LA-HTM.
Ukuqubha kweMicrobial akufuneki kubhidaniswe kunye nezinye iintlobo zentshukumo yomzimba, ezizezi (i) iBrownian motion, ebonakala ngathi yintshukumo yesiphithiphithi engenacala liqinisekileyo, (ii) i-convection 50 kunye ne-thermophoresis 43, ebandakanya intshukumo eqhelekileyo kunye nobushushu. ukuthambeka.
I-G. stearothermophilus yaziwa ngokukwazi ukuvelisa i-spores exhathisayo (ukwakheka kwe-spore) xa ibonakaliswe kwiimeko ezimbi zokusingqongileyo njengendlela yokukhusela. Xa iimeko zokusingqongileyo zivuma kwakhona, iimbewu ziyantshula, zenze iiseli eziphilayo zize ziphinde zikhule. Nangona le nkqubo ye-sporulation / intshula isaziwa, ayizange ibonwe ngexesha lokwenyani. Ukusebenzisa i-LA-HTM, sichaza apha ukuqwalaselwa kokuqala kweziganeko zokuhluma kwi-G. stearothermophilus.
Kwikhiwane. I-6a ibonisa imifanekiso ye-time-lapse ye-optical deep (OT) efunyenwe ngokusebenzisa isethi ye-CGM yee-spores ze-13. Kulo lonke ixesha lokuqokelela (15 h 6 min, \(t=0\) -ukuqala kokufudumeza kwelaser), i-4 kwi-13 i-spores intshula, ngamaxesha alandelelanayo \ (t=2\) h, \( 3\ ) h \(10 \)', \(9\) h \(40\)' kunye \(11\) h \(30\)'. Nangona inye kuphela yezi ziganeko iboniswe kuMfanekiso 6, iziganeko ezi-4 zokuhluma zinokubonwa kwi-movie ye-M6 kwizinto ezongezelelweyo. Kuyathakazelisa ukuba ukuhluma kubonakala kungenangqondo: akuzona zonke iimbewu ezihlumayo kwaye azihlumi ngexesha elifanayo, nangona utshintsho olufanayo kwiimeko zokusingqongileyo.
i-Time-lapse equkethe imifanekiso ye-8 ye-OT (ukuntywiliselwa kweoli, i-60x, i-1.25 ye-NA injongo) kunye (b) ne-biomass evolution ye-G. stearothermophilus aggregates. c (b) Izotywe kwisikali semi-log ukuqaqambisa ulungelelwaniso lwezinga lokukhula (umgca odawuniweyo).
Kwikhiwane. I-6b,c ibonisa i-biomass yabantu beeseli kwindawo yokujonga njengomsebenzi wexesha kulo lonke ixesha lokuqokelelwa kwedatha. Ukubola okukhawulezayo kobunzima obomileyo obuqatshelwe kwi \(t=5\)h kwifig. 6b, c, ngenxa yokuphuma kwezinye iiseli ukusuka kwindawo yokujonga. Izinga lokukhula kwezi ziganeko zine \(0.77\pm 0.1\) h-1. Eli xabiso liphezulu kunezinga lokukhula elihambelana noMzobo 3. 3 kunye ne-4, apho iiseli zikhula ngokuqhelekileyo. Isizathu sokunyuka kwezinga lokukhula kwe-G. stearothermophilus ukusuka kwi-spores ayicacanga, kodwa le milinganiselo igxininisa umdla we-LA-HTM kwaye isebenze kwinqanaba leseli enye (okanye kwinqanaba elilodwa le-mCFU) ukufunda ngakumbi malunga ne-dynamics yobomi beeseli. .
Ukubonisa ngakumbi ukuguquguquka kwe-LA-HTM kunye nokusebenza kwayo kumaqondo aphezulu, sihlolisise ukukhula kwe-Sulfolobus shibatae, i-hyperthermophilic acidophilic archaea eneqondo lokushisa eliphezulu lokukhula kwe-80 ° C51. Xa kuthelekiswa ne-G. stearothermophilus, ezi archaea nazo zine-morphology eyahluke kakhulu, efana ne-1 micron spheres (cocci) kuneentonga ezinde (bacilli).
Umzobo 7a uquka imifanekiso elandelanayo yobunzulu be-optical ye-S. shibatae mCFU efunyenwe kusetyenziswa i-CGM (jonga ifilimu yefilimu ye-M7 kwiiMathiriyeli ezongezelelweyo). Le mCFU ikhula malunga nama-73°C, ngaphantsi kobona bushushu buphezulu obuyi-80°C, kodwa ngaphakathi koluhlu lobushushu bokukhula okusebenzayo. Siye saqwalasela iziganeko ezininzi ze-fission eyenza i-mCFUs ibonakale njenge-micrograpes ye-archaea emva kweeyure ezimbalwa. Ukusuka kule mifanekiso ye-OT, i-biomass ye-mCFU yalinganiswa ngokuhamba kwexesha kwaye yaboniswa kuMfanekiso 7b. Okubangela umdla kukuba, i-S. shibatae mCFUs ibonise ukukhula komgama kunokukhula okubonakalayo okubonwa nge-G. stearothermophilus mCFUs. Kubekho ingxoxo ende ye-52 malunga nobume bezinga lokukhula kweeseli: ngelixa ezinye izifundo zibika izinga lokukhula kwee-microbes ezihambelana nobukhulu bazo (ukukhula okubonakalayo), ezinye zibonisa izinga eliqhubekayo (ukukhula komgca okanye i-bilinear). Njengoko kuchazwe nguTzur et al.53, ukwahlula phakathi kwe-exponential kunye (bi) ukukhula komgca kufuna ukuchaneka kwe-<6% kwimilinganiselo ye-biomass, engenakufikeleleka kwiindlela ezininzi ze-QPM, nokuba zibandakanya i-interferometry. Njengoko kuchazwe nguTzur et al.53, ukwahlula phakathi kwe-exponential kunye (bi) ukukhula komgca kufuna ukuchaneka kwe-<6% kwimilinganiselo ye-biomass, engenakufikeleleka kwiindlela ezininzi ze-QPM, nokuba zibandakanya i-interferometry. Как объяснили Цур и др.53, различение экспоненциального и (би)линейного роста требует точности <6% в измерениях биомаснионция, биомассия дов QPM, даже с использованием интерферометрии. Njengoko kuchazwe nguZur et al.53, ukwahlula phakathi kwe-exponential kunye (bi) ukukhula komgca kufuna i-<6% ukuchaneka kwimilinganiselo ye-biomass, engenakufikeleleka kwiindlela ezininzi ze-QPM, kwanokusebenzisa i-interferometry.Njengoko kuchazwe nguZur et al. I-53, ukwahlula phakathi kwe-exponential kunye (bi) ukukhula komgca kufuna ngaphantsi kwe-6% ngokuchanekileyo kwimilinganiselo ye-biomass, engenakufikeleleka kwiindlela ezininzi ze-QPM, nangona i-interferometry isetyenziswa. I-CGM ifezekisa oku kuchanekileyo ngokuchaneka kwe-sub-pg kwimilinganiselo ye-biomass36,48.
i-Time-lapse equkethe imifanekiso ye-6 OT (ukuntywiliselwa kweoli, i-60x, i-NA injongo ye-1.25) kunye (b) ne-micro-CFU biomass evolution elinganiswa ne-CGM. Jonga imuvi M7 ngolwazi oluthe vetshe.
Ukukhula komgca ngokugqibeleleyo kwe-S. shibatae bekungalindelekanga kwaye akukaxelwa. Nangona kunjalo, ukukhula okubonakalayo kulindeleke, ubuncinci kuba ekuhambeni kwexesha, izahlulo ezininzi ze-2, 4, 8, 16 ... iiseli kufuneka zenzeke. Siye saqikelela ukuba ukukhula komgca kungabangelwa kukuthintelwa kweeseli ngenxa yokupakishwa kweeseli ezixineneyo, kanye njengokuba ukukhula kweeseli kuncipha kwaye ekugqibeleni kufikelele kwindawo yokulala xa ukuxinana kweeseli kuphezulu kakhulu.
Siqukumbela ngokuxubusha ezi ngongoma zintlanu zilandelayo zomdla ngokutshintshana: ukunciphisa umthamo wokufudumala, ukunciphisa inertia ye-thermal, inzala kwi-nanoparticles yegolide, inzala kwi-microscopy yesigaba sobungakanani, kunye nomgangatho wokushisa onokwenzeka apho i-LA-HTM ingasetyenziswa.
Xa kuthelekiswa nokufudumeza okuxhathisayo, ukufudumeza kwelaser okusetyenziselwa uphuhliso lwe-HTM kunika iingenelo ezininzi, esizibonisayo kwesi sifundo. Ngokukodwa, kumajelo eendaba kwintsimi yokujonga i-microscope, umthamo wokufudumala ugcinwa ngaphakathi kweembalwa (10 μm) 3 imiqulu. Ngale ndlela, kuphela ii-microbes eziqatshelweyo ezisebenzayo, ngelixa ezinye iibhaktheriya zilele kwaye zingasetyenziselwa ukuqhubela phambili ukufunda isampuli - akukho mfuneko yokutshintsha isampuli rhoqo xa kufuneka kuhlolwe ubushushu obutsha. Ukongezelela, ukufudumeza kwe-microscale kuvumela ukuhlolwa ngokuthe ngqo koluhlu olukhulu lwamaqondo okushisa: Umzobo we-4c wafunyanwa kwi-movie yeeyure ezi-3 (i-Movie M3), edla ngokufuna ukulungiswa kunye nokuhlolwa kweesampuli ezininzi - enye yeesampuli eziphantsi kwesifundo. y liqondo lobushushu elimele inani leentsuku kuvavanyo. Ukunciphisa umthamo ovuthayo kwakhona kugcina zonke izinto ezijikelezileyo ze-optical microscope, ngokukodwa i-lens yenjongo, kwiqondo lokushisa, eliye layingxaki enkulu ejongene noluntu ukuza kuthi ga ngoku. I-LA-HTM ingasetyenziselwa nayiphi na i-lens, kubandakanywa iilensi zokucwiliswa kweoli, kwaye iya kuhlala kwindawo yokushisa kwamagumbi kunye nokushisa okugqithiseleyo kwintsimi yokujonga. Umda oyintloko wendlela yokufudumeza i-laser esiyinika ingxelo kolu phononongo kukuba iiseli ezingabambeleliyo okanye ezidadayo zinokuba kude nentsimi yokujonga kwaye kunzima ukuzifunda. Indlela yokusebenza inokuba kukusebenzisa iilensi zokukhulisa okuphantsi ukufikelela kunyuso olukhulu lobushushu obungaphaya kwamakhulu ambalwa eemicrons. Esi silumkiso sihamba kunye nokuncipha kwesisombululo sendawo, kodwa ukuba injongo kukufunda ukuhamba kwe-microorganisms, isisombululo esiphezulu sendawo ayifuni.
Ubungakanani bexesha lokufudumeza (kunye nokupholisa) inkqubo \({{{{\rm{\tau }}}}}}}}}_{{{\mbox{D}}}}\) kuxhomekeke kubungakanani bayo , ngokomthetho \({{({\rm{\tau}}}}}}}}_{{{\mbox{D}}}}}={L}^{2}/D\), apho \ (L\ ) luphawu lobungakanani bomthombo wobushushu (ububanzi bomqadi welaser kuphononongo lwethu ngu \(L\ malunga ne-100\) μm), \(D\) yi-thermal diffusivity yokusingqongileyo (i-avareji yethu icala, iglasi kunye namanzi Diffusion rate\(D\ about 2\fold {10}^{-7}\) m2/s. Ngoko ke, kolu phononongo, iimpendulo zexesha lolandelelwano lwe-50 ms, oko kukuthi, i-quasi-instantaneous). utshintsho lweqondo lobushushu, lunokulindeleka oku kusekwa ngoko nangoko kokunyuka kobushushu akufinyeleli nje ixesha lovavanyo, kodwa kukwavumela ixesha elichanekileyo \(t=0\) kulo naluphi na uphononongo oluguquguqukayo lweziphumo zobushushu.
Indlela yethu ecetywayo iyasebenza kuyo nayiphi na i-substrate yokukhanya (umzekelo, iisampuli zorhwebo ezine-ITO coating). Nangona kunjalo, i-nanoparticles yegolide iyakwazi ukubonelela ngokunyuka okuphezulu kwi-infrared kunye ne-low absorption kuluhlu olubonakalayo, iimpawu zokugqibela ezinomdla wokuqwalasela optical osebenzayo kuluhlu olubonakalayo, ngakumbi xa usebenzisa i-fluorescence. Ukongeza, igolide iyahambelana ne-biocompatible, inert yekhemikhali, ingxinano ye-optical inokulungiswa ukusuka kwi-530 nm ukuya kufutshane ne-infrared, kwaye ukulungiswa kwesampulu kulula kwaye kunoqoqosho29.
I-Transverse grating wavefront microscopy (CGM) ayivumeli kuphela imephu yobushushu kwi-microscale, kodwa kunye nokujongwa kwe-biomass, iyenza ibe luncedo ngakumbi (ukuba akukho mfuneko) ngokudibanisa ne-LA-HTM. Kwiminyaka elishumi edlulileyo, ezinye iindlela ze-microscopy zeqondo lokushisa ziye zaphuhliswa, ngokukodwa kwintsimi ye-bioimaging, kwaye ininzi yazo ifuna ukusetyenziswa kwe-probes ye-fluorescent probes54,55. Nangona kunjalo, ezi ndlela ziye zagxekwa kwaye ezinye iingxelo ziye zalinganisa utshintsho olungenangqiqo lobushushu ngaphakathi kweeseli, mhlawumbi ngenxa yokuba i-fluorescence ixhomekeke kwizinto ezininzi ngaphandle kweqondo lokushisa. Ukongezelela, ezininzi iiprobes ze-fluorescent azizinzile kumaqondo aphezulu. Ke ngoko, i-QPM kwaye ngakumbi i-CGM imele ubuchule obufanelekileyo be-microscopy yobushushu bokufunda ubomi kumaqondo obushushu aphezulu usebenzisa i-optical microscopy.
Izifundo ze-S. shibatae, ezihlala ngokugqibeleleyo kwi-80 ° C, zibonisa ukuba i-LA-HTM ingasetyenziselwa ukufunda i-hyperthermophiles, kungekhona nje i-thermophiles elula. Ngokomgaqo, akukho mda kuluhlu lwamaqondo okushisa anokuthi afikeleleke ngokusebenzisa i-LA-HTM, kwaye amaqondo okushisa angaphezu kwe-100 ° C anokufikelela kuxinzelelo lwe-atmospheric ngaphandle kokubilisa, njengoko kuboniswe liqela lethu le-38 kwizicelo ze-hydrothermal chemistry kwi-atmospheric. uxinzelelo A. I-laser isetyenziselwa ukufudumeza i-nanoparticles yegolide engama-40 ngendlela efanayo. Ngaloo ndlela, i-LA-HTM inamandla okusetyenziswa ukujonga i-hyperthermophiles engazange ibonwe ngaphambili kunye ne-standard high resolution optical microscopy phantsi kweemeko eziqhelekileyo (okt phantsi koxinzelelo lwendalo).
Zonke iimvavanyo zenziwe kusetyenziswa i-microscope yasekhaya, kubandakanywa ukukhanya kwe-Köhler (kunye ne-LED, i-M625L3, i-Thorlabs, i-700 mW), i-specimen holder ene-xy movement ye-manual, iinjongo (i-Olympus, i-60x, i-0.7 NA, umoya, i-LUCPlanFLN60X okanye i-60x, i-Oil. , UPLFLN60XOI), ikhamera ye-CGM (QLSI cross grating, 39 µm pitch, 0.87 mm ukusuka kwi-sensor yekhamera ye-Andor Zyla) ukubonelela ngokuqina kunye nokucinga kwangaphambili, kunye nekhamera ye-sCMOS (i-ORCA Flash 4.0 V3, imowudi ye-16-bit, ukusuka e-Hamamatsu) ukurekhoda irekhodi idatha eboniswe kuMfanekiso 5 (ukubhukuda kwebhaktheriya). I-dichroic beam splitter yi-749 nm BrightLine edge (Semrock, FF749-SDi01). Isihluzi esiphambi kwekhamera sisihluzo sokudlula esifutshane se-694 (FF02-694/SP-25, Semrock). I-laser yesafire ye-Titanium (i-Laser Verdi G10, i-532 nm, i-10 W, i-tsunami ye-laser cavity, i-Spectra-Physics kwi-Fig. 2-5, ithathelwe indawo yi-Millenia laser, i-Spectraphysics 10 W, impompo ye-Mira laser cavity, i-Coherent, ye-Fig. -5). I-6 kunye ne-7) imiselwe kubude be-wavelength \({{({\rm{\lambda}}}}}}}}=800\) nm, ehambelana ne-plasmon resonance spectrum ye-nanoparticles zegolide. 1152 pixels) zathengwa kwi-Meadowlark Optics I-holograms yabalwa ngokusebenzisa i-algorithm ye-Gerchberg-Saxton njengoko ichazwe kwikhonkco.
I-Cross grating wavefront microscopy (CGM) bubuchule be-optical microscopy esekwe ekudibaniseni igrayiti ene-dimensional diffraction (ekwaziwa ngokuba yi-cross grating) kumgama we-millimeter enye ukusuka kwisivamvo sekhamera eqhelekileyo. Owona mzekelo uqhelekileyo weCGM esiwusebenzisileyo kolu phononongo ubizwa ngokuba yi-four-wavelength transverse shift interferometer (QLSI), apho i-cross-grating ibandakanya i-intensitety / phase checkerboard pattern eyaziswa kwaye inelungelo lobunikazi nguPrimot et al. kwi-200034. Imigca yegrayithi ethe nkqo kunye nethe tyaba yenza izithunzi ezinjenge-grid kwi-sensor, ukugqwethwa kwayo kunokucutshungulwa ngokwamanani ngexesha langempela ukufumana ukuguqulwa kwe-wavefront optical (okanye iprofayili yesigaba esilinganayo) sokukhanya kwesiganeko. Xa isetyenziswe kwi-microscope, ikhamera ye-CGM ingabonisa umehluko wendlela ye-optical yento efanekisiweyo, eyaziwa ngokuba yi-optical deep (OT), enovakalelo ngokomyalelo we-nanometers36. Kuyo nayiphi na imilinganiselo ye-CGM, ukuze kupheliswe naziphi na iziphene kumacandelo e-optical okanye imiqadi, umfanekiso osisiseko we-OT kufuneka uthathwe kwaye ususwe kuyo nayiphi na imifanekiso elandelayo.
I-microscopy yobushushu yenziwa ngokusebenzisa ikhamera yeCGM njengoko kuchazwe kwireferensi. 32. Ngamafutshane, ukufudumeza ulwelo kuguqula isalathisi salo, ukudala i-lens ye-thermal effect ephazamisa i-beam yesiganeko. Oku kugqwethwa kwe-wavefront kulinganiswa yi-CGM kwaye kusetyenzwe kusetyenziswa i-algorithm ye-deconvolution ukuze kufumaneke ukuhanjiswa kobushushu obuthathu kwindawo yolwelo. Ukuba i-nanoparticles yegolide isasazwa ngokulinganayo kuyo yonke isampuli, imephu yeqondo lokushisa inokwenziwa kwiindawo ezingenabhaktheriya ukuvelisa imifanekiso engcono, into esiyenzayo ngamanye amaxesha. Umfanekiso wesalathiso weCGM ufunyenwe ngaphandle kokufudumeza (ngelaser off) kwaye emva koko wabanjwa kwindawo enye kumfanekiso ngelaser.
Umlinganiselo wobunzima obomileyo uphunyezwa kusetyenziswa ikhamera yeCGM efanayo esetyenziselwa ukulinganisa ubushushu. Imifanekiso ye-CGM yereferensi ifunyenwe ngokuhambisa ngokukhawuleza isampuli kwi-x kunye ne-y ngexesha lokuvezwa njengendlela yokulinganisa nayiphi na inhomogeneity kwi-OT ngenxa yobukho beebhaktheriya. Ukusuka kwimifanekiso ye-OT yebhaktheriya, i-biomass yabo yafunyanwa kusetyenziswa i-ensemble yemifanekiso kwiindawo ezikhethiweyo kusetyenziswa i-algorithm ye-Matlab ye-segmentation yasekhaya (jonga icandelo "Ikhowudi yamanani"), ngokulandela inkqubo echazwe kwi-ref. 48. Ngamafutshane, sisebenzisa unxulumano \(m={\alpha}^{-1}\iint {{\mbox{OT}}}\left(x,y\right){{\mbox{d}} } x{{\mbox{d}}}}y\), apho \({{\mbox{OT}}}\khohlo(x,y\ekunene)\) ngumfanekiso obunzulu obubonakalayo, \(m\) ubunzima obomileyo kwaye \({{{{\rm{\ alpha }}}}}}\) yinto engatshintshiyo. Sakhetha \({{{\rm{\alpha))))))=0.18\) µm3/pg, nto leyo iqhelekile kwiiseli eziphilayo.
Isiliphu sokugquma esingama-25 mm ububanzi kunye ne-150 µm ubukhulu esiqatywe ngeenanoparticles zegolide safakwa kwigumbi le-AttofluorTM (Thermofisher) iinanoparticles zegolide zijonge phezulu. I-Geobacillus stearothermophilus yalungiswa ngobusuku kwi-LB medium (200 rpm, 60 ° C) phambi kosuku ngalunye lwemifuniselo. Ithontsi ye-5 µl yokunqunyanyiswa kwe-G. stearothermophilus ene-optical density (OD) ye-0.3 ukuya kwi-0.5 ibekwe kwisiliphu sokugquma kunye neenanoparticles zegolide. Emva koko, isiliphu sekhava engqukuva esiyi-18 mm ububanzi kunye nomngxuma oyi-5 mm ububanzi phakathi embindini wehliswa kwithontsi, kwaye i-5 μl yokunqunyanyiswa kwebhaktheriya kunye nokuxinana kwamehlo okufanayo kwasetyenziswa ngokuphindaphindiweyo embindini womngxuma. Amaqula akwizigqubuthelo ezigqunyiweyo alungiswa ngokuhambelana nenkqubo echazwe kwiref. 45 (jonga uLwazi oloNgezelelweyo ngolwazi oluthe vetshe). Emva koko yongeza i-1 ml ye-LB medium kwi-coverlip ukukhusela umaleko wolwelo ukuba wome. Isiliphu sokugqibela sokugquma sibekwe phezu kwesiciko esivaliweyo se-Attofluor™ chamber ukunqanda ukuvela kwamanzi abe ngumphunga ngexesha lokufukamela. Kwimifuniselo yokuhluma, sasebenzisa i-spores, eyathi, emva kovavanyo oluqhelekileyo, ngamanye amaxesha igubungele i-coverlip ephezulu. Kwasetyenziswa indlela efanayo ukufumana iSulfolobus shibatae. Iintsuku ezintathu (i-200 rpm, i-75 ° C) yokulima kwangaphambili kwe-Thiobacillus serrata yenziwa phakathi kwe-182 (DSMZ).
Iisampulu ze-nanoparticles zegolide zalungiswa nge-micellar block copolymer lithography. Le nkqubo ichazwe ngokweenkcukacha kwiSahluko. 60. Ngokufutshane, ii-micelles ezifakela i-ion zegolide zenziwe ngokuxuba i-copolymer kunye ne-HAuCl4 kwi-toluene. Iikhava ezicociweyo zaye zantywiliselwa kwisisombululo kwaye ziphathwa nge-UV irradiation phambi kwe-agent yokunciphisa ukufumana imbewu yegolide. Okokugqibela, imbewu yegolide yakhuliswa ngokudibana nekhava kunye nesisombululo esimanzi se-KAuCl4 kunye ne-ethanolamine imizuzu eli-16, nto leyo ekhokelele kulungiselelo lwe-quasi-periodic kunye nolungelelwano olufana kakhulu lwe-nanoparticles yegolide engeyo-spherical kwi-infrared ekufutshane.
Ukuguqula i-interferograms kwimifanekiso ye-OT, sasebenzisa i-algorithm yasekhaya, njengoko kucacisiwe kwikhonkco. 33 kwaye iyafumaneka njengephakeji yeMatlab kwindawo yokugcina yoluntu elandelayo: https://github.com/baffou/CGMprocess. Iphakheji ingabala ubunzima kunye nemifanekiso ye-OT esekelwe kwii-interferograms ezirekhodiweyo (kubandakanywa nemifanekiso yereferensi) kunye nemigama yekhamera.
Ukubala ipateni yesigaba esetyenzisiweyo kwi-SLM ukufumana iprofayili yobushushu obunikiweyo, sisebenzise i-algorithm39,42 eyenziwe yasekhaya eyenziwe ngaphambili efumaneka kwindawo yokugcina yoluntu: https://github.com/baffou/SLM_temperatureShaping. Igalelo yintsimi yeqondo lokushisa elifunwayo, elinokuthi libekwe ngokwedijithali okanye ngomfanekiso we-monochrome bmp.
Ukwahlula iiseli kunye nokulinganisa ubunzima bazo obomileyo, sisebenzise i-algorithm yeMatlab epapashwe kule ndawo ilandelayo yokugcina uluntu: https://github.com/baffou/CGM_magicWandSegmentation. Kumfanekiso ngamnye, umsebenzisi kufuneka acofe kwibhaktheriya okanye i-mCFU yomdla, ulungelelanise uvakalelo lwe-wand, kwaye uqinisekise ukhetho.
Ukufumana ulwazi oluthe kratya malunga noyilo lwesifundo, bona iNgxelo yoPhando lweNdalo eqhagamshelwe kweli nqaku.
Idatha exhasa iziphumo zolu phononongo iyafumaneka kubabhali abachaphazelekayo ngesicelo esinengqiqo.
Ikhowudi yomthombo esetyenzisiweyo kolu phononongo icacisiwe kwicandelo leeNdlela, kwaye iinguqulelo zokulungisa zinokukhutshelwa kwi-https://github.com/baffou/ kwezi zixhobo zilandelayo: SLM_temperatureShaping, CGMprocess, kunye neCGM_magicWandSegmentation.
Mehta, R., Singhal, P., Singh, H., Damle, D. & Sharma, AK Insight kwi-thermophiles kunye nezicelo zabo ezibanzi. Mehta, R., Singhal, P., Singh, H., Damle, D. & Sharma, AK Insight kwi-thermophiles kunye nezicelo zabo ezibanzi.Mehta, R., Singhal, P., Singh, H., Damle, D. and Sharma, AK Overview of thermophiles kunye nokusetyenziswa kwazo okubanzi. Mehta, R., Singhal, P., Singh, H., Damle, D. & Sharma, AK 深入了解嗜热菌及其广谱应用. Mehta, R., Singhal, P., Singh, H., Damle, D. & Sharma, AK.Mehta R., Singhal P., Singh H., Damle D. kunye noSharma AK Ukuqonda okunzulu kwe-thermophiles kunye noluhlu olubanzi lwezicelo.3 Biotechnology 6, 81 (2016).
Ixesha lokuposa: Sep-26-2022