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Ama-Thermophiles ama-microorganisms akhula kahle emazingeni okushisa aphezulu. Ukuzifunda kunganikeza ulwazi olubalulekile mayelana nendlela impilo evumelana ngayo nezimo ezimbi kakhulu. Kodwa-ke, kunzima ukufeza izimo zokushisa eziphezulu nge-microscopes evamile. Kuphakanyiswe izixazululo eziningana ezenziwe ekhaya ezisekelwe ekushiseni kukagesi okumelana nendawo, kodwa asikho isisombululo esilula sokuhweba. Kuleli phepha, sethula umqondo wokushisisa nge-laser microscale endaweni yokubuka ye-microscope ukuze kuhlinzekwe amazinga okushisa aphezulu ezifundweni ze-thermophile kuyilapho kugcinwa indawo yomsebenzisi ithambile. Ukushisisa i-Microscale ngokuqina kwe-laser okumaphakathi kungafinyelelwa kusetshenziswa i-nanoparticle yegolide ehlanganiswe ingxenye engaphansi njengesibambisi sokukhanya esivumelana ne-biocompatible. Imiphumela engaba khona ye-microscale fluid convection, ukugcinwa kwamaseli, kanye nokunyakaza kwe-centrifugal thermophoretic kuyaxoxwa. Le ndlela iye yaboniswa ezinhlotsheni ezimbili: (i) I-Geobacillus stearothermophilus, i-bacterium esebenzayo ye-thermophilic ephindaphinda cishe ku-65°C, esiye sayibona ukuze imile, ikhule futhi ibhukude ngaphansi kokushisisa okuncane; (ii) Thiobacillus sp., i-archaea ephezulu kakhulu ye-hyperthermophilic. ku-80°C. Lo msebenzi uvula indlela yokubhekwa okulula nokuphephile kwamagciwane amancane ashisayo kusetshenziswa amathuluzi e-microscopy esimanje futhi athengekayo.
Eminyakeni eyizigidigidi, ukuphila eMhlabeni kuye kwavela ukuze kujwayelane nezimo ezihlukahlukene zemvelo ngezinye izikhathi ezibhekwa njengezeqisayo ngokombono wethu womuntu. Ikakhulukazi, amanye ama-microorganisms e-thermophilic (amabhaktheriya, i-archaea, isikhunta) abizwa ngokuthi ama-thermophiles akhula kahle emazingeni okushisa asuka ku-45 ° C kuya ku-122 ° C1, 2, 3, 4. Ama-thermophiles ahlala ezindaweni ezihlukahlukene zemvelo, njengama-deep hydrothermal olwandle, iziphethu ezishisayo. noma izindawo ezinezintaba-mlilo. Ucwaningo lwabo selukhiqize intshisekelo enkulu emashumini ambalwa eminyaka adlule okungenani ngezizathu ezimbili. Okokuqala, singafunda kubo, isibonelo, ukuthi i-thermophiles 5, 6, enzymes 7, 8 kanye ne-membrane 9 zizinzile kanjani emazingeni okushisa aphezulu, noma ukuthi ama-thermophiles angamelana kanjani namazinga aphezulu e-radiation10. Okwesibili, ziyisisekelo sezinhlelo zokusebenza eziningi ezibalulekile ze-biotechnological1,11,12 njengokukhiqizwa kukaphethiloli13,14,15,16, ukuhlanganiswa kwamakhemikhali (i-dihydro, utshwala, i-methane, ama-amino acid, njll.)17, i-biomining18 kanye ne-thermostable biocatalysts7 ,11, 13. Ikakhulukazi, i-polymerase chain reaction (PCR)19 eyaziwa njengamanje ihlanganisa i-enzyme (Taq polymerase) ehlukanisiwe ne-thermophilic bacterium i-Thermus aquaticus, enye ye-thermophiles yokuqala ukutholwa.
Nokho, ukufunda ama-thermophiles akuwona umsebenzi olula futhi akunakuthuthukiswa kunoma iyiphi ilabhorethri yezinto eziphilayo. Ikakhulukazi, ama-thermophiles aphilayo awakwazi ukubonwa ku-vitro nganoma yisiphi isibonakhulu esivamile sokukhanya, ngisho namagumbi okufudumeza atholakala nokudayiswa, ngokuvamile alinganiselwe amazinga okushisa aphansi aze afike ku-40°C. Kusukela ngeminyaka yawo-1990s, ambalwa kuphela amaqembu ocwaningo azinikezele ekwethulweni kwezinhlelo ze-high-temperature microscopy (HTM). Ngo-1994 uGlukh et al. Igumbi lokushisisa/ukupholisa laqanjwa ngokusekelwe ekusetshenzisweni kweseli ye-Peltier elawula izinga lokushisa lama-capillary angama-rectangular avaliwe ukuze kugcinwe i-anaerobicity 20. Idivayisi ingashiswa kufikela ku-100 °C ngesilinganiso esingu-2 °C/s, okuvumela ababhali ukuthi bafunde ukuhamba kwebhaktheriya ye-hyperthermophilic Thermotoga maritima21. Ngo-1999 Horn et al. Kuye kwasungulwa idivayisi efana kakhulu, esekelwe ekusetshenzisweni kwama-capillary ashisayo afanele i-microscopy yokuhweba ukuze kufundwe ukuhlukaniswa/ukuxhumana kwamaseli. Ngemuva kwesikhathi eside sokungasebenzi okuhlobene, ukuseshwa kwama-HTM asebenzayo kwaqala kabusha ngo-2012, ikakhulukazi mayelana nochungechunge lwamaphepha eqembu le-Wirth elisebenzisa idivayisi eyasungulwa ngu-Horn et al. Eminyakeni eyishumi nanhlanu edlule, ukuhamba kwenani elikhulu le-archaea, kuhlanganise ne-hyperthermophiles, kwafundwa emazingeni okushisa afika ku-100 ° C kusetshenziswa ama-capillaries ashisayo23,24. Baphinde bashintsha isibonakhulu soqobo ukuze bathole ukushisisa ngokushesha (imizuzu embalwa esikhundleni semizuzu engama-35 ukuze bafinyelele izinga lokushisa elimisiwe) futhi bafinyelele izinga lokushisa elilinganayo elingaphezu kuka-2 cm phakathi nendawo. Le divayisi yokubunjwa kwe-gradient shape (TGFD) iye yasetshenziselwa ukutadisha ukuhamba kwama-thermophiles amaningi ngaphakathi kwama-gradients okushisa ebangeni elifanele le-biologically 24, 25.
Ukushisa ama-capillary avaliwe akuyona ukuphela kwendlela yokubuka ama-thermophiles aphilayo. Ngo-2012, Kuwabara et al. Amakamelo e-Pyrex alahlwayo enziwe ekhaya ayevalwe nge-adhesive engashisi (Super X2; Cemedine, Japan) asetshenziswa. Amasampula abekwe epuleti lokushisisa elitholakala ngokudayiswa (i-Micro Heat Plate, i-Kitazato Corporation, e-Japan) elikwazi ukushisisa kufika ku-110°C, kodwa ekuqaleni kwakungahloselwe ukuthwebula izithombe. Ababhali babone ukwahlukaniswa okusebenzayo kwebhaktheriya ye-anaerobic thermophilic (i-Thermosipho globiformans, isikhathi esiphindwe kabili 24 min) ku-65°C. Ngo-2020, uPulshen et al. Ukushisisa kahle kwezitsha zensimbi ezithengiswayo (i-Attofluor TM, i-Thermofisher) kwaboniswa kusetshenziswa izinto ezimbili zokushisisa zasekhaya: isivalo kanye nesiteji (ukucushwa okuphefumlelwe umshini we-PCR). Lokhu kuhlotshaniswa kuholela ekushiseni okufanayo koketshezi futhi kuvimbela ukuhwamuka nokufiphala phansi kwesivalo. Ukusetshenziswa kwe-O-ring kugwema ukushintshaniswa kwegesi nemvelo. Le HTM, ebizwa ngokuthi i-Sulfoscope, yasetshenziswa ukwenza isithombe se-Sulfolobus acidocaldarius ku-75°C27.
Umkhawulo oqashelwayo wazo zonke lezi zinhlelo kwakuwumkhawulo ekusetshenzisweni kwezinhloso zomoya, noma yikuphi ukucwiliswa kukawoyela okungafaneleki ezingeni lokushisa elinjalo kanye nokuthwebula izithombe ngamasampula angaphezu kuka-1-mm abonisa ngale. Umkhawulo oqashelwayo wazo zonke lezi zinhlelo kwakuwumkhawulo ekusetshenzisweni kwezinhloso zomoya, noma yikuphi ukucwiliswa kukawoyela okungafaneleki ezingeni lokushisa elinjalo kanye nokuthwebula izithombe ngamasampula angaphezu kuka-1-mm abonisa ngale. Ukuthola imininingwane eyengeziwe mayelana nokusetshenziswa kwe-akhawunti yakho для такой высокой температуры и для визуализации через прозрачные образцы толщиной > 1 мм. Ukushiyeka okwabonwayo kuzo zonke lezi zinhlelo kwaba umkhawulo ekusetshenzisweni kwezinhloso zomoya, njengoba noma yikuphi ukucwiliswa kukawoyela kwakungafanele izinga lokushisa eliphezulu kangaka kanye nokuboniswa ngeso lengqondo ngamasampula asobala> ugqinsi oluyi-1 mm.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. Umkhawulo owaziwayo wazo zonke lezi zinhlelo uwumkhawulo wokusebenzisa isibuko esifakwe emoyeni, njengoba noma yikuphi ukucwiliswa kwamafutha akulungele ukuthwebula amasampula abonisa ngale > 1 mm ubukhulu emazingeni okushisa aphezulu kangaka. Общепризнанным недостатком всех этих систем является ограниченное использование воздушных объективов, любое иммерсидон таких высоких температур и визуализации через прозрачные образцы толщиной >1 мм. Umphumela obonwayo wazo zonke lezi zinhlelo ukusetshenziswa okulinganiselwe kwamalensi omoya, noma yikuphi ukucwiliswa kukawoyela akuwafanele amazinga okushisa anjalo kanye nokuboniswa ngeso lengqondo ngamasampula akhanyelayo >1 mm ubukhulu.Muva nje, lo mkhawulo ususwe nguCharles-Orzag et al. 28, owakha umshini ongasanikezi ukushisa eduze kwesistimu yesithakazelo, kodwa kunalokho ngaphakathi kwengilazi yekhava ngokwayo, embozwe ungqimba oluncane olukhanyayo lwe-resistor eyenziwe nge-ITO (i-indium-tin oxide). Isivalo singashisisa kufikela ku-75 °C ngokudlula amandla kagesi kungqimba olusobala. Kodwa-ke, umbhali kufanele futhi ashise ilensi emgomweni, kodwa hhayi ngaphezu kuka-65 ° C, ukuze angalimazi.
Le misebenzi ibonisa ukuthi ukuthuthukiswa kwe-microscopy ye-optical esebenza kahle yokushisa okuphezulu akukaze kwamukelwe kabanzi, ngokuvamile kudinga imishini yasekhaya, futhi kuvame ukufezwa ngezindleko zokulungiswa kwendawo, okuwububi obukhulu uma kubhekwa ukuthi ama-microorganisms we-thermophilic awekho makhulu kunembalwa. ama-micrometer. Ukwehliswa kwevolumu yokushisisa kuyisihluthulelo sokuxazulula izinkinga ezintathu zemvelo ze-HTM: ukulungiswa kahle kwendawo, izinga lokushisa eliphezulu lapho isistimu ishisa, kanye nokushisisa okuyingozi kwezinto ezizungezile (uwoyela wokucwiliswa, i-lens eqondile… noma izandla zomsebenzisi) emazingeni okushisa adlulele. ).
Kuleli phepha, sethula i-HTM yokubuka i-thermophile engasekelwe ekushiseni okubambezelayo. Kunalokho, sizuze ukushisisa okwenziwe kwasendaweni endaweni elinganiselwe yenkambu yokubuka yesibonakhulu ngemisebe ye-laser ye-substrate emunca ukukhanya. Ukusatshalaliswa kwezinga lokushisa kuye kwabonwa kusetshenziswa i-quantitative phase microscopy (QPM). Ukuphumelela kwale ndlela kuboniswa i-Geobacillus stearothermophilus, igciwane le-thermophilic elinyakazayo elizala cishe ku-65°C futhi linesikhathi esifushane sokuphindaphinda (cishe imizuzu engama-20), kanye ne-Sulfolobus shibatae, i-hyperthermophile ekhula kahle kakhulu ku-80°C (archaea) ukufanekisa. Izinga elijwayelekile lokuphindaphinda nokubhukuda kwabonwa njengomsebenzi wokushisa. Le laser HTM (LA-HTM) ayinqunyelwe ukujiya kwesilip sekhava noma ubunjalo benhloso (ukucwiliswa komoya noma uwoyela). Lokhu kuvumela noma iyiphi ilensi yokucaca okuphezulu emakethe ukuthi isetshenziswe. Futhi ayihlushwa ukushisisa kancane ngenxa ye-thermal inertia (izuza ukushisisa okusheshayo esikalini se-millisecond) futhi isebenzisa izingxenye ezitholakalayo zentengiso kuphela. Okuwukuphela kokukhathazeka okusha kwezokuphepha kuhlobene nokuba khona kwemishayo ye-laser enamandla (imvamisa efika ku-100 mW) ngaphakathi kwedivayisi futhi ngokunokwenzeka nangamehlo, adinga izibuko zokuvikela.
Umgomo we-LA-HTM ukusebenzisa i-laser ukushisa isampula endaweni ngaphakathi kwendawo yokubuka i-microscope (Fig. 1a). Ukuze wenze lokhu, isampula kufanele ibambe ukukhanya. Ukuze sisebenzise amandla e-laser anengqondo (angaphansi kuka-100 mW), asizange sithembele ekumuntshweni kokukhanya yi-liquid medium, kodwa sikhulise ngokuzenzela ukumuncwa kwesampula ngokumboza i-substrate ngama-nanoparticles egolide (Fig. 1c). Ukushisisa ama-nanoparticles egolide ngokukhanya kubaluleke kakhulu emkhakheni we-thermal plasmonics, okulindeleke ukuthi kusetshenziswe ku-biomedicine, nanochemistry noma ekuvuneni ukukhanya kwelanga29,30,31. Eminyakeni embalwa edlule, sisebenzise le-LA-HTM ezifundweni eziningana ezihlobene nezicelo ze-plasma ezishisayo ku-physics, chemistry kanye ne-biology. Ubunzima obukhulu ngale ndlela kusekubonisweni kwephrofayili yokugcina yezinga lokushisa, njengoba izinga lokushisa eliphakeme lilinganiselwe endaweni ye-microscale ngaphakathi kwesampula. Sibonise ukuthi imephu yezinga lokushisa ingafinyelelwa nge-interferometer ye-shear eguquguqukayo enamaza amane, indlela elula, enokulungiswa okuphezulu, futhi ebucayi kakhulu ye-quantitative phase microscopy esekelwe ekusetshenzisweni kwe-diffraction gratings enezinhlangothi ezimbili (eyaziwa nangokuthi ama-cross gratings) 33,34,35,36. Ukuthembeka kwale nqubo ye-microscopy eshisayo, esekelwe ku-crossed grating wavefront microscopy (CGM), kuboniswe emaphepheni ayishumi nambili ashicilelwe kule minyaka eyishumi edlule37,38,39,40,41,42,43.
Uhlelo lokufakwa kokushisisa kwe-laser okuhambisanayo, ukubunjwa kanye nesibonakhulu sezinga lokushisa. b I-geometry yesampula ehlanganisa igumbi le-Attofluor TM eliqukethe isiliphu sekhava esimbozwe ngama-nanoparticles egolide. c Bukisisa isampula (hhayi ukukala). d imele iphrofayili ye-laser ye-laser efanayo kanye (e) nokulingiswa okulandelayo kokusatshalaliswa kwezinga lokushisa endizeni eyisampula yama-nanoparticles egolide. f iphrofayili ye-laser ye-annular efanelekile ukukhiqiza izinga lokushisa elifanayo njengoba kuboniswa ekulingiseni okuwumphumela wokusabalalisa izinga lokushisa okuboniswe ku-(g). Ibha yesikali: 30 µm.
Ikakhulukazi, sisanda kuzuza ukushisisa kwamaseli ezilwane ezincelisayo nge-LA-HTM kanye ne-CGM futhi salandelela izimpendulo zokushaqeka kokushisa kwamaselula ebangeni lika-37-42°C, okubonisa ukusebenza kwale nqubo ekufanekisweni kweseli elilodwa eliphilayo. Kodwa-ke, ukusetshenziswa kwe-LA-HTM ekutadisheni ama-microorganisms emazingeni okushisa aphezulu akuyona into ecacile, njengoba kudinga ukuqapha okwengeziwe uma kuqhathaniswa namangqamuzana ezilwane ezincelisayo: okokuqala, ukushisa phansi okuphakathi ngamashumi amadigri (kunokuba amadigri ambalwa) kuholela. kuya kwigradient eqinile yokushisa eqondile. ingakha i-fluid convection 44 okuthi, uma ingaxhunywanga ngokuqinile ku-substrate, ingabangela ukunyakaza okungathandeki kanye nokuxubana kwamagciwane. Le convection ingaqedwa ngokunciphisa ukujiya kongqimba lwe-liquid. Ngale njongo, kuzo zonke izivivinyo ezivezwe ngezansi, ukumiswa kwebhaktheriya kwabekwa phakathi kwamakhava amabili acishe abe ngu-15 µm awugqinsi afakwe ngaphakathi kwenkomishi yensimbi (AttofluorTM, Thermofisher, Fig. 1b,c). Empeleni, i-convection ingagwenywa uma ukushuba koketshezi kuncane kunosayizi wogongolo we-laser yokushisa. Okwesibili, ukusebenza ku-geometry elinganiselwe kungenza izinto eziphilayo ze-aerobic (bheka Fig. S2). Le nkinga ingagwenywa ngokusebenzisa i-substrate ekwazi ukungena ku-oxygen (noma yimuphi omunye igesi ebalulekile), ngokushiya amabhamuza omoya avaleleke ngaphakathi kwe-coverlip, noma ngokubhoboza izimbobo ku-coverlip top (bheka Fig. S1) 45. Kulolu cwaningo, sikhethe isisombululo sakamuva (Izibalo 1b kanye ne-S1). Okokugcina, ukushisa kwe-laser akuhlinzeki ngokusatshalaliswa kokushisa okufanayo. Ngisho nokuqina okufanayo kwe-laser beam (Fig. 1d), ukusatshalaliswa kwezinga lokushisa akufani, kodwa kunalokho kufana nokusabalalisa kwe-Gaussian ngenxa yokusabalalisa okushisayo (Fig. 1e). Lapho umgomo uwukusungula amazinga okushisa anembile emkhakheni wokubuka wokutadisha izinhlelo zebhayoloji, amaphrofayili angalingani awalungile futhi angaholela ekuhambeni kwe-thermophoretic kwamabhaktheriya uma enganamatheli ku-substrate (bheka Fig. S3, S4)39. Kuze kube sekugcineni, sisebenzise imodulator yokukhanya yendawo (SLM) ukuze silolonge ugongolo lwe-laser ye-infrared ngokuvumelana nokuma kwendandatho (Fig. 1f) endizeni yesampula ukuze sizuze ukusatshalaliswa kwezinga lokushisa okufana ngokuphelele ngaphakathi kwendawo yejometri enikeziwe, naphezu kokusabalalisa okushisayo (Fig. 1d) 39, 42, 46. Beka isiliphu esiphezulu phezu kwesitsha sensimbi (Umfanekiso 1b) ukuze ugweme ukuhwamuka kwendawo futhi ugcine okungenani izinsuku ezimbalwa. Ngenxa yokuthi lesi silip sekhava esiphezulu asivaliwe, i-medium eyengeziwe ingangezwa kalula nganoma yisiphi isikhathi uma kunesidingo.
Ukukhombisa ukuthi i-LA-HTM isebenza kanjani futhi sibonise ukusebenza kwayo ocwaningweni lwe-thermophilic, sifunde ibhaktheriya ye-aerobic i-Geobacillus stearothermophilus, enezinga lokushisa eliphezulu lokukhula okungaba ngu-60-65°C. I-bacterium nayo ine-flagella kanye nekhono lokubhukuda, ihlinzeka ngesinye inkomba yomsebenzi ojwayelekile wamaselula.
Amasampula (Fig. 1b) afakwe ngaphambili ku-60°C ihora elilodwa bese efakwa kusibambi wesampula ye-LA-HTM. Lokhu kufakela kwangaphambi kofukamela kuyinketho, kodwa kusewusizo, ngenxa yezizathu ezimbili: Okokuqala, lapho i-laser ivuliwe, ibangela ukuthi amaseli akhule ngokushesha futhi ahlukane (bona i-movie M1 kokuthi Supplementary Materials). Ngaphandle kokufakwa ngaphambilini, ukukhula kwebhaktheriya kuvame ukubambezeleka cishe ngemizuzu engama-40 isikhathi ngasinye lapho indawo entsha yokubuka ishiselwa kusampula. Okwesibili, ihora elingu-1 langaphambi kokufukamela likhuthaze ukunamathela kwamagciwane ku-coverlip, kuvimbela amaseli ukuthi angakhukhuleki aphume endaweni yokubuka ngenxa ye-thermophoresis lapho i-laser ivuliwe (bheka ifilimu M2 kokuthi Izinto Ezingeziwe). I-Thermophoresis ukuhamba kwezinhlayiya noma ama-molecule eduze kwe-gradient yokushisa, ngokuvamile ukusuka ekushiseni kuya kubanda, futhi amagciwane awahlukile ku-43,47. Lo mphumela ongathandeki uqedwa endaweni ethile ngokusebenzisa i-SLM ukuze ulolonge ugongolo lwe-laser futhi uzuze ukusatshalaliswa kwezinga lokushisa eliyisicaba.
Emkhiwaneni. Umfanekiso wesi-2 ubonisa ukusatshalaliswa kwezinga lokushisa okukalwa nge-CGM etholwe ngokufaka i-radiayidi yengilazi engaphansi embozwe ngama-nanoparticles egolide nge-laser beam annular (Fig. 1f). Ukusatshalaliswa kwezinga lokushisa eliyisicaba kuye kwabonwa phezu kwayo yonke indawo embozwe yi-laser beam. Le ndawo yayibekwe ku-65°C, izinga lokushisa elilungile lokukhula. Ngaphandle kwalesi sifunda, ijika lezinga lokushisa liwela ngokwemvelo kokuthi \(1/r\) (lapho \(r\) kuyisixhumanisi se-radial).
imephu Yezinga lokushisa yezilinganiso ze-CGM etholwe ngokusebenzisa i-laser ye-annular ukuze kukhanye ungqimba lwama-nanoparticles egolide ukuze kutholwe iphrofayela yezinga lokushisa eliyisicaba endaweni eyindilinga. b I-Isotherm yemephu yezinga lokushisa (a). Ikhonta ye-laser beam imelwe indilinga enamachashazi ampunga. Ukuhlolwa kuphindwe kabili (bona Izinto Ezingeziwe, Umfanekiso S4).
Ukusebenza kwamaseli ebhaktheriya kwaqashwa amahora ambalwa kusetshenziswa i-LA-HTM. Emkhiwaneni. 3 ibonisa isikhawu sesikhathi sezithombe ezine ezithathwe ku-movie yamahora angu-3 nemizuzu engu-20 (I-Movie M3, Ulwazi Olwengeziwe). Amagciwane aqashelwa ukuthi anda ngenkuthalo endaweni eyindilinga echazwe yilaser lapho izinga lokushisa lalisezingeni eliphezulu, lifinyelela ku-65°C. Ngokuphambene, ukukhula kwamaseli kwehliswe kakhulu lapho izinga lokushisa lehla ngaphansi kwama-50°C amasekhondi ayi-10.
Izithombe ze-Optical deep ze-G. stearothermophilus bacteria ekhula ngemva kokushisisa nge-laser ngezikhathi ezihlukene, (a) t = 0 min, (b) 1 h 10 min, (c) 2 h 20 min, (d) 3 h 20 min, out of 200 Ikhishwe kufilimu yomzuzu owodwa (ifilimu ye-M3 enikezwe Ulwazi Olwengeziwe) ebekwe phezulu kumephu yokushisa ehambisanayo. I-laser ivula ngesikhathi \(t=0\). Ama-Isotherm engeziwe esithombeni sokuqina.
Ukuze siqhubeke nokulinganisa ukukhula kwamangqamuzana nokuncika kwawo ekushiseni, silinganise ukwanda kwe-biomass yamakholoni ahlukahlukene amagciwane okuqala angawodwa emkhakheni wokubuka we-Movie M3 (Fig. 4). Ibhaktheriya elingumzali elikhethwe ekuqaleni kokwakheka kweyunithi ye-mini colony forming (mCFU) kuboniswa kuMfanekiso S6. Izilinganiso zesisindo esomile zithathwe ngekhamera ye-CGM 48 eyasetshenziswa ukwenza imephu yokusatshalaliswa kwezinga lokushisa. Ikhono le-CGM lokukala isisindo esomile nezinga lokushisa lingamandla e-LA-HTM. Njengoba bekulindelekile, izinga lokushisa eliphezulu labangela ukukhula kwebhaktheriya ngokushesha (Fig. 4a). Njengoba kuboniswe esakhiweni se-semi-log ku-Fig. 4b, ukukhula kuwo wonke amazinga okushisa kulandela ukukhula komchazi, lapho idatha isebenzisa umsebenzi womchazi othi \(m={m}_{0}{10}^{t/\ tau }+) {{ \mbox{cst}}}}\), lapho \(\tau {{{{\rm{log }}}}}}}2\) - isikhathi sokukhiqiza (noma isikhathi esiphindwe kabili), \( g =1/ \tau\) - izinga lokukhula (inombolo yezigaba ngesikhathi seyunithi ngayinye). Emkhiwaneni. I-4c ikhombisa izinga lokukhula elilandelanayo kanye nesikhathi sokukhiqiza njengomsebenzi wezinga lokushisa. Ama-mCFU akhula ngokushesha abonakala ngokugcwala kokukhula ngemva kwamahora amabili, ukuziphatha okulindelekile ngenxa yokuminyana kwamagciwane (okufana nesigaba esimile emasikweni oketshezi asendulo). Umumo ojwayelekile \(g\left(T\right)\) (Fig. 4c) uhambisana nejika lezigaba ezimbili elilindelekile le-G. stearothermophilus enezinga lokukhula elilungile elizungeze u-60-65°C. Qondanisa idatha usebenzisa imodeli yekhadinali (Umfanekiso S5)49 lapho \(\kwesokunxele({{G}_{0}{;\;T}}_{{\min }};{T}_{{opt} } } ;{T}_{{\max}}\kwesokudla)\) = (0.70 ± 0.2; 40 ± 4; 65 ± 1.6; 67 ± 3) °C, evumelana kahle nezinye izindinganiso ezishiwo ezincwadini49. Nakuba amapharamitha ancike kuzinga lokushisa akwazi ukuphinda akhiqizeke, izinga eliphezulu lokukhula \({G}_{0}\) lingahluka kusukela ekuhlolweni okukodwa kuya kwesinye (bona izibalo S7-S9 ne-movie M4). Ngokuphambene nemingcele yokulinganisa izinga lokushisa, okufanele ibe yindawo yonke, izinga eliphezulu lokukhula lincike ezintweni ezimaphakathi (ukutholakala kwezakhi, ukugxila kwe-oxygen) ngaphakathi kwejometri ye-microscale ephawuliwe.
ukukhula kweMicrobial emazingeni okushisa ahlukahlukene. I-mCFU: Amayunithi Okwenza Ikholoni Elincane. Idatha etholwe kuvidiyo yebhaktheriya eyodwa ekhula endaweni yezinga lokushisa (ifilimu M3). b Kufana no-(a), isikali se-semi-logarithmic. c Izinga lokukhula\(\tau\) nesikhathi sokukhiqiza\(g\) esibalwe kusukela ekuhlehleni komugqa (b). Amabha wephutha avundlile: izinga lokushisa lapho ama-mCFU anwetshwa khona endaweni yokubuka phakathi nokukhula. Amabha wephutha aqondile: iphutha elijwayelekile lokuhlehla komugqa.
Ngaphezu kokukhula okuvamile, amanye amagciwane ngezinye izikhathi ayentanta ukuze abonakale ngesikhathi sokushisisa nge-laser, okuwukuziphatha okulindelekile kubhaktheriya ane-flagella. Ifilimu i-M5 ngolwazi olwengeziwe ibonisa imisebenzi yokubhukuda enjalo. Kulesi sivivinyo, kusetshenziswe imisebe ye-laser efanayo ukuze kwakhiwe izinga lokushisa, njengoba kuboniswe ku-Figure 1d, e no-S3. Umfanekiso wesi-5 ubonisa ukulandelana kwezithombe ezimbili ezikhethwe ku-movie ye-M5 ebonisa ukuthi ibhaktheriya elilodwa libonisa ukunyakaza okuqondile kuyilapho wonke amanye amagciwane ehlala enganyakazi.
Amafreyimu wesikhathi amabili (a) kanye (b) abonisa ukubhukuda kwamagciwane amabili ahlukene amakwe ngeziyingi ezinamachashazi. Izithombe zikhishwe ku-movie ye-M5 (ihlinzekwe njengezinto ezingeziwe).
Endabeni ye-G. stearothermophilus, ukunyakaza okusebenzayo kwamabhaktheriya (Fig. 5) kwaqala imizuzwana embalwa ngemva kokuvulwa kwe-laser beam. Lokhu kubuka kugcizelela impendulo yesikhashana yale microorganism ye-thermophilic ekukhuphukeni kwezinga lokushisa, njengoba sekuphawulwe nguMora et al. 24 . Isihloko sokuhamba kwebhaktheriya ngisho ne-thermotaxis singabuye sihlolwe kusetshenziswa i-LA-HTM.
Ukubhukuda kwezilwanyana ezincane akufanele kudidaniswe nezinye izinhlobo zokunyakaza komzimba, okungukuthi (i) ukunyakaza kwe-Brownian, okubonakala kuwukunyakaza okuyisiphithiphithi okungenayo inkomba ethile eqondile, (ii) i-convection 50 kanye ne-thermophoresis 43, ehlanganisa ukunyakaza okuvamile okuhambisana nezinga lokushisa. i-gradient.
I-G. stearothermophilus yaziwa ngekhono layo lokukhiqiza izinhlamvu ezimelana kakhulu (ukwakheka kwezinhlamvu) lapho zichayeka ezimweni zemvelo ezimbi njengesivikelo. Lapho izimo zemvelo zivuma futhi, izinhlamvu ziyamila, zakhe amangqamuzana aphilayo futhi ziphinde zikhule. Nakuba le nqubo ye-sporulation/ukuhluma yaziwa kahle, ayikaze ibonwe ngesikhathi sangempela. Sisebenzisa i-LA-HTM, sibika lapha ukubonwa kokuqala kwemicimbi yokuhluma ku-G. stearothermophilus.
Emkhiwaneni. I-6a ibonisa izithombe ezidlula isikhathi zokujula kokubona (OT) ezitholwe kusetshenziswa isethi ye-CGM yezinhlamvu eziyi-13. Ngaso sonke isikhathi sokuqoqwa (15 h 6 min, \(t=0\) – ukuqala kokushisisa ngelaser), 4 kweziyi-13 izinhlamvu ezimile, ngezikhathi ezilandelanayo \(t=2\) h, \( 3\ ) h \(10 \)', \(9\) h \(40\)' kanye \(11\) h \(30\)'. Yize munye kuphela wale micimbi okhonjiswa kuMdwebo 6, izehlakalo ezi-4 zokuhluma zingabonwa ku-movie ye-M6 kokubalulekile okungeziwe. Kuyathakazelisa ukuthi ukuhluma kubonakala kungenangqondo: akuzona zonke izinhlamvu ezihlumayo futhi azihlumi ngesikhathi esifanayo, naphezu kwezinguquko ezifanayo ezimweni zemvelo.
Ukuphelelwa yisikhathi okuhlanganisa izithombe ezingu-8 ze-OT (ukucwiliswa kwamafutha, 60x, 1.25 NA injongo) kanye (b) nokuvela kwe-biomass ye-G. stearothermophilus aggregates. c (b) Idwetshwe esikalini se-semi-log ukuze kugqanyiswe umugqa wezinga lokukhula (umugqa odeshi).
Emkhiwaneni. 6b,c ibonisa i-biomass yamaseli emkhakheni wokubuka njengomsebenzi wesikhathi phakathi naso sonke isikhathi sokuqoqwa kwedatha. Ukubola okusheshayo kwesisindo esomile esibonwe ku-\(t=5\)h kufig. 6b, c, ngenxa yokuphuma kwamanye amaseli endaweni yokubuka. Izinga lokukhula lale micimbi emine ngu-\(0.77\pm 0.1\) h-1. Leli nani lingaphezulu kwezinga lokukhula elihlotshaniswa noMfanekiso 3. 3 no-4, lapho amaseli akhula ngokujwayelekile. Isizathu sokukhula kwezinga lokukhula kwe-G. stearothermophilus kusuka ezinhlamvu asicacile, kodwa lezi zilinganiso ziqokomisa isithakazelo se-LA-HTM futhi zisebenza ezingeni leseli elilodwa (noma ezingeni elilodwa le-mCFU) ukuze ufunde kabanzi mayelana nokuguquguquka kokuphila kweseli. .
Ukuze siqhubeke sibonise ukuguquguquka kwe-LA-HTM kanye nokusebenza kwayo emazingeni okushisa aphezulu, sihlole ukukhula kwe-Sulfolobus shibatae, i-hyperthermophilic acidophilic archaea enezinga lokushisa eliphezulu lokukhula elingu-80°C51. Uma kuqhathaniswa ne-G. stearothermophilus, lawa ma-archaea nawo ane-morphology ehluke kakhulu, efana ne-micron spheres engu-1 (cocci) kunezinduku ezinde (bacilli).
Umfanekiso 7a uqukethe izithombe zokushona ezilandelanayo zokushona kwe-S. shibatae mCFU ezitholwe kusetshenziswa i-CGM (bona ifilimu yesici se-M7 kokuthi Izinto Ezingeziwe). Le mCFU ikhula cishe ku-73°C, ngaphansi kwezinga lokushisa elilungile lika-80°C, kodwa ngaphakathi kwezinga lokushisa lokukhula okusebenzayo. Sibone izehlakalo eziningi ze-fission ezenza ama-mCFU abukeke njengama-micrograpes of archaea ngemva kwamahora ambalwa. Kusukela kulezi zithombe ze-OT, i-biomass ye-mCFU ikalwa ngokuhamba kwesikhathi futhi yethulwa kuMfanekiso 7b. Kuyathakazelisa ukuthi i-S. shibatae mCFUs ibonise ukukhula komugqa kunokukhula okunamandla okubonwa nge-G. stearothermophilus mCFUs. Kube nengxoxo ende 52 mayelana nemvelo yamazinga okukhula kwamangqamuzana: kuyilapho ezinye izifundo zibika amazinga okukhula kwama-microbes alingana nobukhulu bawo (ukukhula okubonakalayo), ezinye zibonisa izinga eliqhubekayo (ukukhula komugqa noma okubili). Njengoba kuchazwe u-Tzur et al.53, ukuhlukanisa phakathi kokukhula komugqa kanye (bi) nomugqa kudinga ukunemba okungu-<6% kuzilinganiso ze-biomass, okungenakufinyeleleka kumasu amaningi e-QPM, ngisho nokubandakanya i-interferometry. Njengoba kuchazwe u-Tzur et al.53, ukuhlukanisa phakathi kokukhula komugqa kanye (bi) nomugqa kudinga ukunemba okungu-<6% kuzilinganiso ze-biomass, okungenakufinyeleleka kumasu amaningi e-QPM, ngisho nokubandakanya i-interferometry. Как объяснили Цур и др.53, различение экспоненциального и (би)линейного роста требует точности <6% в измерениях биомасссы, чтомассы, биомассия дов QPM, даже с использованием интерферометрии. Njengoba kuchazwe u-Zur et al.53, ukuhlukanisa phakathi kokukhula komugqa kanye (bi) nomugqa kudinga ukunemba okungu-<6% kuzilinganiso ze-biomass, okungatholakali ngezindlela eziningi ze-QPM, ngisho nokusebenzisa i-interferometry.Njengoba kuchazwe uZur et al. 53, ukuhlukanisa phakathi kokukhula komugqa kanye (bi) nomugqa kudinga ukunemba okungaphansi kuka-6% ezilinganisweni ze-biomass, okungafinyeleleki ezindleleni eziningi ze-QPM, ngisho nalapho kusetshenziswa i-interferometry. I-CGM ifinyelela lokhu kunemba ngokunemba kwe-sub-pg ezilinganisweni ze-biomass36,48.
Ukudlula kwesikhathi okuhlanganisa izithombe eziyisi-6 ze-OT (ukucwiliswa kwamafutha, 60x, inhloso ye-NA 1.25) kanye (b) ne-micro-CFU biomass evolution kukalwa nge-CGM. Bheka imuvi M7 ukuze uthole ulwazi olwengeziwe.
Ukukhula komugqa okuphelele kwe-S. shibatae bekungalindelekile futhi akukakabikwa. Nokho, ukukhula okunamandla kulindelekile, okungenani ngoba ngokuhamba kwesikhathi, ukuhlukana okuningi kuka-2, 4, 8, 16 … amaseli kufanele kwenzeke. Sacabanga ukuthi ukukhula komugqa kungase kube ngenxa yokuvinjwa kwamaseli ngenxa yokupakishwa kwamaseli aminyene, njengoba nje ukukhula kweseli kuncipha futhi ekugcineni kufinyelele esimweni sokungalali lapho ukuminyana kwamaseli kuphezulu kakhulu.
Siphetha ngokuxoxa ngamaphuzu amahlanu alandelayo ezithakazelisayo ngokushintshana: ukunciphisa umthamo wokushisa, ukunciphisa inertia eshisayo, isithakazelo kuma-nanoparticles egolide, isithakazelo ku-quantitative phase microscopy, kanye nebanga lokushisa elingenzeka lapho i-LA-HTM ingasetshenziswa khona.
Uma kuqhathaniswa nokushisisa okuphikisayo, ukushisisa ngelaser okusetshenziselwa ukuthuthukiswa kwe-HTM kunikeza izinzuzo ezimbalwa, esizibonisa kulolu cwaningo. Ikakhulukazi, emithonjeni ye-liquid emkhakheni wokubuka kwe-microscope, ivolumu yokushisa igcinwa ngaphakathi kwamavolumu ambalwa (10 μm) angu-3. Ngale ndlela, ama-microbes abonwayo kuphela asebenzayo, kuyilapho amanye amagciwane elele futhi angasetshenziswa ukuze kuqhutshekwe nokufunda isampula - asikho isidingo sokushintsha isampula njalo lapho izinga lokushisa elisha lidinga ukuhlolwa. Ukwengeza, ukushisa kwe-microscale kuvumela ukuhlolwa okuqondile kwebanga elikhulu lamazinga okushisa: Umfanekiso we-4c utholwe ku-movie yamahora angu-3 (i-Movie M3), ngokuvamile edinga ukulungiswa nokuhlolwa kwamasampula amaningana - eyodwa yesampula ngayinye efundwayo. y izinga lokushisa elimelela inani lezinsuku ekuhlolweni. Ukunciphisa umthamo oshisayo futhi kugcina zonke izingxenye ze-optical ezizungezile ze-microscope, ikakhulukazi i-lens lens, ekamelweni lokushisa, okuye kwaba yinkinga enkulu umphakathi obhekene nayo kuze kube manje. I-LA-HTM ingasetshenziswa nanoma iyiphi i-lens, okuhlanganisa amalensi okucwiliswa kawoyela, futhi izohlala ekamelweni lokushisa ngisho namazinga okushisa aphakeme kakhulu emkhakheni wokubuka. Umkhawulo oyinhloko wendlela yokushisisa nge-laser esiyibikayo kulolu cwaningo ukuthi amaseli anganamatheli noma angantanti angase abe kude nomkhakha wokubuka futhi kube nzima ukuwafunda. Indlela yokusebenza ingaba ukusebenzisa amalensi okukhulisa okuphansi ukuze ufinyelele izinga lokushisa elikhudlwana elingaphezu kwama-microns angamakhulu ambalwa. Lesi sixwayiso sihambisana nokuncipha kwesinqumo sendawo, kodwa uma umgomo uwukufunda ukunyakaza kwama-microorganisms, ukulungiswa kwendawo ephezulu akudingeki.
Isikali sesikhathi sokushisisa (nokupholisa) isistimu \({{{{\rm{\tau }}}}}}}_{{{\mbox{D}}}}\) sincike kusayizi wayo , ngokomthetho \({{({\rm{\tau }}}}}}}_{{{\mbox{D}}}}}={L}^{2}/D\), lapho \ (L\ ) ubukhulu besici bomthombo wokushisa (ububanzi be-laser beam esifundweni sethu ngu-\(L\ about 100\) μm), \(D\) ukuhluka okushisayo kwemvelo (isilinganiso esilinganisweni sethu Ikesi, ingilazi namanzi Izinga lokusabalalisa\(D\ about 2\fold {10}^{-7}\) m2/s Ngakho-ke, kulolu cwaningo, izimpendulo zesikhathi ze-oda lika-50 ms, okungukuthi, i-quasi-instantaneous). ukushintsha kwezinga lokushisa, kungalindelwa lokhu kumiswa ngokushesha kokunyuka kwezinga lokushisa akufinyezi nje kuphela ubude besikhathi sokuhlola, kodwa futhi kuvumela isikhathi esinembayo \(t=0\) sanoma yisiphi isifundo esishukumisayo semiphumela yezinga lokushisa.
Indlela yethu ehlongozwayo iyasebenza kunoma iyiphi i-substrate emunca ukukhanya (isibonelo, amasampula okuthengisa ane-ITO coating). Kodwa-ke, ama-nanoparticles egolide ayakwazi ukunikeza ukumuncwa okuphezulu ku-infrared ne-low absorption ebangeni elibonakalayo, izici zokugcina ezithakazelisa ukubhekwa okuphumelelayo kwe-optical ebangeni elibonakalayo, ikakhulukazi uma usebenzisa i-fluorescence. Ukwengeza, igolide liyavumelana ne-biocompatible, inert amakhemikhali, ukuminyana kwe-optical kungalungiswa ukusuka ku-530 nm ukuya eduze kwe-infrared, futhi ukulungiswa kwesampula kulula futhi ukonga29.
I-Transverse grating wavefront microscopy (CGM) ayivumeli kuphela imephu yokushisa ku-microscale, kodwa futhi nokugadwa kwe-biomass, okuyenza ibe wusizo ngokukhethekile (uma kungenasidingo) kuhlanganiswa ne-LA-HTM. Kule minyaka eyishumi edlule, kuye kwathuthukiswa ezinye izindlela zokusebenzisa i-microscopy yokushisa, ikakhulukazi emkhakheni we-bioimaging, futhi eziningi zazo zidinga ukusetshenziswa kwe-fluorescent probes ezwela ukushisa okungama-54,55. Kodwa-ke, lezi zindlela ziye zagxekwa futhi eminye imibiko iye yakala ukushintsha kwezinga lokushisa okungelona iqiniso ngaphakathi kwamaseli, mhlawumbe ngenxa yokuthi i-fluorescence incike ezintweni eziningi ngaphandle kwezinga lokushisa. Ngaphezu kwalokho, ama-probe amaningi e-fluorescent awazinzile emazingeni okushisa aphezulu. Ngakho-ke, i-QPM futhi ikakhulukazi i-CGM imelela indlela efanelekile ye-microscopy yokushisa yokufunda ukuphila emazingeni okushisa aphezulu kusetshenziswa i-optical microscopy.
Ucwaningo lwe-S. shibatae, oluphila ngokugcwele ku-80°C, lubonisa ukuthi i-LA-HTM ingasetshenziswa ekutadisheni ama-hyperthermophiles, hhayi nje ama-thermophiles alula. Empeleni, awukho umkhawulo ebangeni lamazinga okushisa angafinyelelwa kusetshenziswa i-LA-HTM, futhi ngisho namazinga okushisa angaphezu kuka-100 ° C angafinyelelwa kungcindezi yasemkhathini ngaphandle kokubilisa, njengoba kuboniswa iqembu lethu lama-38 ekusetshenzisweni kwamakhemikhali e-hydrothermal atmospheric. ingcindezi A. Ilaser isetshenziselwa ukushisisa ama-nanoparticles egolide angu-40 ngendlela efanayo. Ngakho-ke, i-LA-HTM inamandla okusetshenziselwa ukubuka ama-hyperthermophiles angakaze abonwe ane-microscopy yokubona ephezulu yokucaca ngaphansi kwezimo ezijwayelekile (okungukuthi ngaphansi kokucindezeleka kwemvelo).
Konke ukuhlola kwenziwa kusetshenziswa isibonakhulu sasekhaya, okuhlanganisa ukukhanya kwe-Köhler (ene-LED, M625L3, Thorlabs, 700 mW), isibambi sesifanekiso esinokunyakaza kwe-xy okwenziwa ngesandla, izinjongo (i-Olympus, 60x, 0.7 NA, umoya, LUCPlanFLN60X noma 60x, NA25, O1. , UPLFLN60XOI), ikhamera ye-CGM (QLSI cross grating, 39 µm pitch, 0.87 mm kusukela kunzwa yekhamera ye-Andor Zyla) ukuze unikeze ukuqina nokucabanga okungaphambili kwegagasi, kanye nekhamera ye-sCMOS (ORCA Flash 4.0 V3, imodi ye-16-bit , kusukela ku-Hamamatsu) ukurekhoda idatha eboniswe kuMfanekiso 5 (ukubhukuda kwamagciwane). I-dichroic beam splitter iwumkhawulo we-749 nm BrightLine (Semrock, FF749-SDi01). Isihlungi esingaphambili kwekhamera siyisihlungi sokudlula esifushane esingu-694 (FF02-694/SP-25, Semrock). I-Titanium sapphire laser (Laser Verdi G10, 532 nm, 10 W, i-pump tsunami laser cavity, i-Spectra-Physics ku-Fig. 2-5, iphinde yathathelwa indawo yi-Millenia laser, i-Spectraphysics 10 W, i-Mira laser cavity empontshiwe, i-Coherent, ye-Fig. 2 -5). 6 kanye no-7) zisethelwe kubude begagasi \({{({\rm{\lambda }}}}}}}=800\) nm, obuhambisana ne-plasmon resonance spectrum yama-nanoparticles egolide. Amamojula okukhanya kwendawo (1920 × 1152 pixels) athengwe ku-Meadowlark Optics Amahologramu abalwe kusetshenziswa i-algorithm ye-Gerchberg-Saxton njengoba kuchazwe kusixhumanisi.
I-Cross grating wavefront microscopy (CGM) iwubuchule be-optical microscopy obusekelwe ekuhlanganiseni igridi yokuhlukanisa enezinhlangothi ezimbili (eyaziwa nangokuthi i-cross grating) ebangeni eliyimilimitha elilodwa ukusuka kunzwa yekhamera evamile. Isibonelo esivame kakhulu se-CGM esisisebenzisile kulolu cwaningo sibizwa ngokuthi i-interferometer ye-wavelength eguquguqukayo ye-four-wavelength (QLSI), lapho i-cross-grating iqukethe iphethini yokuhlola ye-intensity/isigaba eyethulwe futhi inelungelo lobunikazi ngu-Primot et al. ngo-200034. Imigqa yokugezela eqondile nevundlile idala ithunzi elifana negridi kunzwa, ukuhlanekezela okungacutshungulwa ngenombolo ngesikhathi sangempela ukuze kutholwe ukuhlanekezelwa kwe-wavefront ye-optical (noma iphrofayela yesigaba esilinganayo) yokukhanya kwesigameko. Uma isetshenziswa kusibonakhulu, ikhamera ye-CGM ingabonisa umehluko wendlela yokubona yento enesithombe, eyaziwa nangokuthi ukujula kokubona (OT), nokuzwela ngokohlelo lwama-nanometers36. Kunoma isiphi isilinganiso se-CGM, ukuze kuqedwe noma yikuphi ukukhubazeka ezingxenyeni ezibonayo noma imishayo, isithombe esiyinhloko se-OT esiyisethenjwa kufanele sithathwe futhi sikhishwe kunoma yiziphi izithombe ezilandelayo.
I-microscopy yezinga lokushisa yenziwe kusetshenziswa ikhamera ye-CGM njengoba kuchazwe kusithenjwa. 32. Ngamafuphi, ukushisa uketshezi kushintsha inkomba yayo ye-refractive, kudala umphumela we-lens oshisayo ohlanekezela umsebe wesigameko. Lokhu kuhlanekezela kwe-wavefront kukalwa yi-CGM futhi kusetshenzwe kusetshenziswa i-algorithm ye-deconvolution ukuze kutholwe ukusatshalaliswa kwezinga lokushisa okunezinhlangothi ezintathu ku-liquid medium. Uma ama-nanoparticles egolide asatshalaliswa ngokulinganayo kulo lonke isampula, ukuqoshwa kwemephu kwezinga lokushisa kungenziwa ezindaweni ezingenazo amagciwane ukuze kukhiqizwe izithombe ezingcono, okuyinto esiyenza ngezinye izikhathi. Isithombe esiyisethenjwa se-CGM sitholwe ngaphandle kokushisisa (nge-laser icishiwe) futhi ngemva kwalokho sathwetshulwa endaweni efanayo esithombeni kusetshenziswa i-laser.
Isilinganiso sesisindo esomile sitholwa kusetshenziswa ikhamera ye-CGM efanayo esetshenziselwa ukuthwebula izinga lokushisa. Izithombe zereferensi ye-CGM zitholwe ngokuhambisa ngokushesha isampula ku-x kanye no-y ngesikhathi sokuchayeka njengendlela yokulinganisa noma yikuphi ukungahambi kahle ku-OT ngenxa yokuba khona kwamagciwane. Kusuka ezithombeni ze-OT zamabhaktheriya, i-biomass yawo itholwe kusetshenziswa iqoqo lezithombe ezindaweni ezikhethiwe kusetshenziswa i-algorithm yesegimenti eyenziwe ekhaya kaMatlab (bona isigatshana esithi “Ikhodi Yezinombolo”), kulandelwa inqubo echazwe kunkomba. 48. Ngamafuphi, sisebenzisa ukuhlobana \(m={\alpha}^{-1}\iint {{\mbox{OT}}}\left(x,y\right){{\mbox{d}} } x{{\mbox{d}}}y\), lapho \({{\mbox{OT}}}\left(x,y\right)\) okuyisithombe sokujula kokubona, \(m\) isisindo esomile futhi \({{{{\rm{\ alpha }}}}}}\) ayiguquki. Sikhethe \({{{{\rm{\alpha)))))))=0.18\) µm3/pg, okuyisimo esijwayelekile samaseli aphilayo.
Isiliphu sekhava esingu-25 mm ububanzi no-150 µm obukhulu obumbozwe ngama-nanoparticles esagolide safakwa egunjini le-AttofluorTM (Thermofisher) ama-nanoparticles egolide abheke phezulu. I-Geobacillus stearothermophilus yatshalwa ngaphambili ngobusuku ku-LB medium (200 rpm, 60°C) ngaphambi kosuku ngalunye lokuhlolwa. Ukwehla okungu-5 µl kokumiswa kwe-G. stearothermophilus ene-optical density (OD) engu-0.3 kuya ku-0.5 kubekwe kusiliphu sekhava esinama-nanoparticles egolide. Khona-ke, isiliphu sekhava esiyindilinga esingu-18 mm ububanzi esinembobo engu-5 mm ububanzi phakathi nendawo sawiswa ethonsini, futhi u-5 μl wokumiswa kwebhaktheriya okunomthamo ofanayo wokubona wasetshenziswa ngokuphindaphindiwe phakathi nendawo yembobo. Imithombo kumakhava yalungiswa ngokuhambisana nenqubo echazwe ku-ref. 45 (bheka Ulwazi Olwengeziwe ukuze uthole ulwazi olwengeziwe). Bese wengeza u-1 ml we-LB medium ku-coverlip ukuze uvimbele ungqimba oluwuketshezi ukuthi lungomi. I-coverlip yokugcina ibekwe phezu kwesivalo esivaliwe se-Attofluor™ chamber ukuvimbela ukuhwamuka kwendawo ephakathi ngesikhathi sokufukamela. Ukuhlola ukuhluma, sasebenzisa ama-spores, okuthi, ngemva kokuhlolwa okuvamile, ngezinye izikhathi amboze isiliphu esiphezulu. Kwasetshenziswa indlela efanayo ukuze kutholwe i-Sulfolobus shibatae. Izinsuku ezintathu (200 rpm, 75°C) zokutshalwa kwe-Thiobacillus serrata kwenziwa endaweni eyi-182 ephakathi (DSMZ).
Amasampula ama-nanoparticles egolide alungiswa nge-micellar block copolymer lithography. Le nqubo ichazwe ngokuningiliziwe ku-Chap. 60. Kafushane, ama-micelles afaka ama-ion egolide ahlanganiswa ngokuxuba i-copolymer ne-HAuCl4 ku-toluene. Izembozo ezihlanziwe zabe sezicwiliswa kusixazululo futhi ziphathwa ngemisebe ye-UV phambi kwe-ejenti yokunciphisa ukuze kutholwe imbewu yegolide. Ekugcineni, imbewu yegolide yatshalwa ngokuthinta i-coverslip enesisombululo esinamanzi se-KAuCl4 ne-ethanolamine imizuzu engu-16, okuholele ekuhlelweni kwe-quasi-periodic nokufana kakhulu kwama-nanoparticles egolide angewona ayindilinga endaweni eseduze ye-infrared.
Ukuguqula ama-interferogram abe yizithombe ze-OT, sisebenzise i-algorithm eyenziwe ekhaya, njengoba kuchaziwe kusixhumanisi. 33 futhi iyatholakala njengephakeji leMatlab endaweni yokugcina yomphakathi elandelayo: https://github.com/baffou/CGMprocess. Iphakheji ingabala ukushuba kanye nezithombe ze-OT ngokusekelwe kuma-interferogram arekhodiwe (okuhlanganisa nezithombe eziyisithenjwa) kanye namabanga afanayo ekhamera.
Ukuze ubale iphethini yesigaba esetshenziswe ku-SLM ukuze sithole iphrofayela yezinga lokushisa enikeziwe, sisebenzise i-algorithm39,42 eyenziwe ekhaya eyakhiwe ngaphambilini etholakala endaweni yokugcina yomphakathi elandelayo: https://github.com/baffou/SLM_temperatureShaping. Okokufaka kuyinkambu yezinga lokushisa elifiswayo, elingasethwa ngokwedijithali noma ngesithombe se-bmp esinombala ogqamile.
Ukuze sihlukanise amaseli futhi silinganise isisindo sawo esomile, sisebenzise i-algorithm yethu ye-Matlab eshicilelwe endaweni yokugcina yomphakathi elandelayo: https://github.com/baffou/CGM_magicWandSegmentation. Esithombeni ngasinye, umsebenzisi kufanele achofoze kubhaktheriya noma i-mCFU ayithandayo, alungise ukuzwela kwe-wand, futhi aqinisekise ukukhetha.
Ukuze uthole ulwazi olwengeziwe mayelana nesakhiwo socwaningo, bheka i-Nature Research Report abstract exhunywe kulesi sihloko.
Idatha esekela imiphumela yalolu cwaningo iyatholakala kubabhali abafanele uma kunesicelo esifanele.
Ikhodi yomthombo esetshenziswe kulolu cwaningo inemininingwane esigabeni Sezindlela, futhi izinguqulo zokususa iphutha zingalandwa kusukela ku-https://github.com/baffou/ kumakhosombe alandelayo: SLM_temperatureShaping, CGMprocess, kanye ne-CGM_magicWandSegmentation.
I-Mehta, R., Singhal, P., Singh, H., Damle, D. & Sharma, AK Insight into thermophiles kanye nokusetshenziswa kwawo okubanzi. I-Mehta, R., Singhal, P., Singh, H., Damle, D. & Sharma, AK Insight into thermophiles kanye nokusetshenziswa kwawo okubanzi.Mehta, R., Singhal, P., Singh, H., Damle, D. and Sharma, AK Uhlolojikelele lwama-thermophiles kanye nokusebenza kwawo 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.U-Mehta R., Singhal P., Singh H., Damle D. kanye no-Sharma AK Ukuqonda okujulile kwama-thermophiles kanye nenhlobonhlobo yezinhlelo zokusebenza.3 I-Biotechnology 6, 81 (2016).
Isikhathi sokuthumela: Sep-26-2022