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Thermophiles ma microorganisms anobudirira pakupisa kwakanyanya. Kuadzidza kunogona kupa ruzivo rwakakosha nezvekuti hupenyu hunoenderana sei nemamiriro akanyanya. Nekudaro, zvakaoma kuwana tembiricha yepamusoro neyakajairwa optical microscopes. Mhinduro dzinoverengeka dzakagadzirwa nemba dzinobva pane yemuno resistive magetsi kudziyisa dzakakurudzirwa, asi hapana mhinduro iri nyore yekutengesa. Mubepa rino, tinosuma pfungwa ye microscale laser kudziyisa pamusoro penzvimbo ye microscope yekuona kupa tembiricha yepamusoro yezvidzidzo zve thermophile uku ichichengeta nharaunda yemushandisi yakapfava. Kudziya kwe Microscale pamwero nelaser intensity inogona kuwanikwa uchishandisa goridhe nanoparticle yakavharwa substrate sebiocompatible uye inoshanda mwenje yekunyudza. Zvinogona kuitika zve microscale fluid convection, cell retention, uye centrifugal thermophoretic motion inokurukurwa. Nzira yacho yakaratidzwa mumarudzi maviri: (i) Geobacillus stearothermophilus, bhakitiriya rinoshanda rethermophilic rinobereka paanenge 65 ° C, iyo yatakacherechedza kuti inomera, kukura uye kushambira pasi pekupisa kwe microscale; (ii) Thiobacillus sp., yakanyanya hyperthermophilic archaea. pa 80°C. Iri basa rinovhura nzira yekutarisa yakapusa uye yakachengeteka ye thermophilic microorganisms uchishandisa zvazvino uye zvinokwanisika maturusi emicroscope.
Kupfuura mabhiriyoni emakore, hupenyu hwePasi hwakashanduka kuti huenderane neyakasiyana siyana mamiriro ezvakatipoteredza ayo dzimwe nguva anoonekwa seakanyanyisa kubva pamaonero edu evanhu. Kunyanya, mamwe ma microorganisms e thermophilic (mabhakitiriya, archaea, fungi) anonzi thermophiles anobudirira mumhepo yekushisa kubva pa45 ° C kusvika 122 ° C1, 2, 3, 4. Thermophiles inogara munzvimbo dzakasiyana-siyana, dzakadai semvura yakadzika yegungwa remvura inopisa, zvitubu zvinopisa. kana nzvimbo dzine makomo. Tsvagiridzo yavo yakaunza kufarira kwakawanda mukati memakumi mashoma emakore apfuura nekuda kwezvikonzero zviviri. Kutanga, tinogona kudzidza kubva kwavari, semuenzaniso, kuti thermophiles 5, 6, enzymes 7, 8 uye membrane 9 yakagadzikana sei pakudziya kwakanyanya, kana kuti thermophiles inogona sei kumira yakanyanyisa mwero weradiation10. Chechipiri, ndidzo hwaro hwezvakawanda zvakakosha biotechnological application1,11,12 senge kugadzirwa kwemafuta13,14,15,16, chemical synthesis (dihydro, alcohols, methane, amino acids, etc.)17, biomining18 uye thermostable biocatalysts7 ,11, 13. Kunyanya, iyo parizvino inozivikanwa kwazvo polymerase chain reaction (PCR)19 inosanganisira enzyme (Taq polymerase) yakaparadzaniswa neiyo thermophilic bhakitiriya Thermus aquaticus, imwe yekutanga thermophiles kuwanikwa.
Nekudaro, kudzidza kwe thermophiles harisi basa riri nyore uye harigone kuvandudzwa mune chero biological laboratory. Kunyanya, ma<em>thermophiles anorarama haagone kuonekwa mu<em>in vitro chero ipi zvayo yakajairwa maikorosikopu, kunyangwe nemakamuri ekudziya anotengeswa, anowanzo kurongerwa tembiricha yakaderera se40°C. Kubva kuma1990, mapoka mashoma ekutsvagisa akazvipira kuunzwa kwemhando yepamusoro-tembiricha maikorosikopu (HTM) masisitimu. Muna 1994 Glukh et al. Imba yekudziya / inotonhorera yakave yakagadzirwa maererano nekushandiswa kwePeltier cell iyo inodzora kutonhora kwemakona emakona emakona akavharwa kuchengetedza anaerobicity 20. Mudziyo wacho unogona kudziyisa kusvika ku100 °C pamwero we2 °C/s, zvichibvumira vanyori kuti vadzidze motility ye hyperthermophilic bacteria Thermotoga maritima21. Muna 1999 Horn et al. Mudziyo wakada kufanana wakagadzirwa, uchiri kubva pakushandiswa kwemacapillaries anopisa anokodzera kutengeserana maikorosikopu kudzidza kupatsanurwa kwesero / kubatana. Mushure menguva yakareba yekusaita basa, kutsvaga kweHTMs inoshanda kwakatangazve muna 2012, kunyanya maererano nenhevedzano yemapepa neboka reWirth rakashandisa mudziyo wakagadzirwa naHorn et al. Makore gumi nemashanu apfuura, motility yenhamba yakawanda yearchaea, kusanganisira hyperthermophiles, yakadzidzwa pakupisa kusvika ku100 ° C uchishandisa inopisa capillaries23,24. Vakagadziridza zvakare maikorosikopu yepakutanga kuti iwane kupisa nekukurumidza (maminetsi akati wandei pachinzvimbo chemaminitsi makumi matatu nemashanu kusvika patembiricha yakatarwa) uye kuwana mutsara wetembiricha weinodarika 2 cm mukati mepakati. Iyi tembiricha gradient kuumba mudziyo (TGFD) yakashandiswa kudzidza kufamba kweakawanda thermophiles mukati metembiricha gradients pamadaro akakodzera biologically 24, 25.
Kupisa yakavharwa capillaries haisi iyo chete nzira yekuona mhenyu thermophiles. Muna 2012, Kuwabara et al. Dzimba dzePyrex dzinoraswa pamba dzakavharwa neadhesive inodzivirira kupisa (Super X2; Cemedine, Japan) yakashandiswa. Samples akaiswa pane inotengeswa transparent heat plate (Micro Heat Plate, Kitazato Corporation, Japan) inokwanisa kupisa kusvika 110 ° C, asi isati yaitirwa bioimaging. Vanyori vakaona kupatsanurwa kwakanaka kweanaerobic thermophilic bacteria (Thermosipho globiformans, kaviri nguva 24 min) pa65 ° C. Muna 2020, Pulshen et al. Kudziya kwakanaka kwemidziyo yesimbi yekutengeserana (Attofluor TM, Thermofisher) yakaratidzwa uchishandisa zvinhu zviviri zvekupisa zvepamba: chivharo uye nhanho (PCR muchina-yakafuridzirwa configuration). Kubatana uku kunoguma nekudziya kwemvura kwakafanana uye kunodzivirira kubuda uye condensation pasi pechivharo. Kushandiswa kweO-ring kunodzivirira gasi kuchinjana nemhoteredzo. Iyi HTM, inonzi Sulfoscope, yakashandiswa kuratidza Sulfolobus acidocaldarius pa75°C27.
Muganho unocherechedzwa weaya ese masisitimu kwaive kurambidzwa kushandiswa kwezvinangwa zvemhepo, chero kunyudzwa kwemafuta kusingakodzere tembiricha yakakwira kudaro uye yekufungidzira kuburikidza> 1-mm makobvu akajeka masampuli. Muganho unocherechedzwa weaya ese masisitimu kwaive kurambidzwa kushandiswa kwezvinangwa zvemhepo, chero kunyudzwa kwemafuta kusingakodzere tembiricha yakakwira kudaro uye yekufungidzira kuburikidza> 1-mm makobvu akajeka masampuli. Нбщепринанныныным марачениее возд лва Не подходило для дли дозуализззззззззззезезезые Об Kutadza kunozivikanwa kweaya ese masisitimu kwaive kudzikisira kushandiswa kwezvinangwa zvemhepo, sezvo chero kunyudzwa kwemafuta kwanga kusingakodzere tembiricha yakakwira kudaro uye yekuona kuburikidza nemasampuli akajeka> 1 mm gobvu.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. Muganho unocherechedzwa weaya ese masisitimu ndiwo kudzikiswa kwekushandisa girazi rine mhepo, sezvo chero kunyudzwa kwemafuta kusingakodzere kufungidzira masampula akajeka> 1 mm gobvu pakupisa kwakanyanya kudaro. Общепризнанным недостатком всех этих систем является ограниченное использование воздушных объективов, любое иммерсидунно таких высоких температур и визуализации через прозрачные образцы толщиной >1 мм. Chinocherechedzwa dhizaini yemasisitimu ese aya kushandiswa kushoma kwemalenzi emhepo, chero kunyudzwa kwemafuta hakuna kukodzera kudziya kwakanyanya uye kuona kuburikidza nemasampuli akajeka> 1 mm gobvu.Munguva pfupi yapfuura, ichi chinogumira chakasimudzwa naCharles-Orzag et al. 28, akagadzira mudziyo usingachapi kupisa kutenderedza hurongwa hwekufarira, asi zviri mukati megirazi rekuvharira pacharo, rakafukidzwa nehutete hwakajeka hwemushonga wakagadzirwa neITO (indium-tin oxide). Chivharo chinogona kudziyirwa kusvika 75 °C nekupfuura magetsi kuburikidza neyakajeka layer. Zvisinei, munyori anofanirawo kupisa lens kune chinangwa, asi kwete kupfuura 65 ° C, kuti arege kuikuvadza.
Aya mabasa anoratidza kuti kugadzirwa kweiyo yepamusoro-temperature optical microscopy haina kugamuchirwa zvakanyanya, kazhinji inoda midziyo yekumba, uye inowanzoitwa nemutengo wekugadziriswa kwenzvimbo, izvo zvinova zvakakomba kusarudzika nekuti thermophilic microorganisms haina kukura kupfuura mashoma. micrometers. Kudzikisira kudziya kwevhoriyamu ndiyo kiyi yekugadzirisa matambudziko matatu anowanikwa eHTM: kushata kwemamiriro ekunze, yakakwira yekupisa inertia kana sisitimu yapisa, uye kupisa kunokuvadza kwezvinhu zvakatenderedza (oiri yekunyudza, lens yechinangwa… kana maoko emushandisi) pakupisa kwakanyanya. ).
Mubepa rino, tinosuma HTM yekutarisa kwe thermophile iyo isina kubva pakudziisa kudziyisa. Pane kudaro, takawana kudziyisa kwenzvimbo mukati menzvimbo shoma yenzvimbo yekuona maikorosikopu nelaser irradiation ye substrate inotora chiedza. Kugovera tembiricha kwakaonekwa pachishandiswa quantitative phase microscopy (QPM). Kushanda kwenzira iyi kunoratidzwa neGeobacillus stearothermophilus, bhakitiriya remotile thermophilic rinobereka paanenge 65 ° C uye rine nguva pfupi yekupeta kaviri (anenge maminitsi makumi maviri), uye Sulfolobus shibatae, hyperthermophile inokura zvakakwana pa80 ° C (archaea) kuenzanisira. Kazhinji kudzokorora mwero uye kushambira zvakaonekwa sebasa rekushisa. Iyi laser HTM (LA-HTM) haina kuganhurirwa nekukora kwechivharo kana nechimiro chechinangwa (mhepo kana kunyudzwa kwemafuta). Izvi zvinobvumira chero yakakwirira resolution lens pamusika kuti ishandiswe. Iyo zvakare haina kutambura nekudziya kunononoka nekuda kwekupisa inertia (inowana nekukurumidza kupisa pachiyero che millisecond) uye inoshandisa chete zvikamu zvekutengesa. Izvo chete zvitsva zvine chekuita nekuchengetedza zvine chekuita nekuvapo kweakasimba laser matanda (kazhinji anosvika 100 mW) mukati memudziyo uye pamwe nemumaziso, ayo anoda magirazi ekudzivirira.
Nheyo yeLA-HTM ndeyokushandisa laser kupisa sampuli munharaunda mukati memunda wekuona microscope (Fig. 1a). Kuti uite izvi, sampuli inofanira kunge iine chiedza. Kuti tishandise simba relaser rinonzwisisika (risingasviki zana mW), isu hatina kuvimba nekutorwa kwechiedza nesvikiro remvura, asi nekugadzira takawedzera kunyura kwemuenzaniso nekuputira substrate negoridhe nanoparticles (Fig. 1c). Kupisa goridhe nanoparticles nechiedza kwakakosha kumunda wezvinopisa plasmonics, nekutarisirwa kushandiswa mubiomedicine, nanochemistry kana kukohwa kwezuva29,30,31. Kwemakore mashoma apfuura, takashandisa iyi LA-HTM muzvidzidzo zvakati wandei zvine chekuita nekupisa kweplasma application mufizikisi, chemistry uye biology. Chinetso chikuru neiyi nzira iri mukuratidza tembiricha yekupedzisira, sezvo tembiricha yakasimudzwa inongogumira kune microscale dunhu mukati memuenzaniso. Isu takaratidza kuti tembiricha mepu inogona kuwanikwa neina-wavelength transverse shear interferometer, iri nyore, yakakwirira-resolution, uye yakanyanya kunzwisiswa nzira yehuwandu hwechikamu microscopy inoenderana nekushandiswa kwemaviri-dimensional diffraction gratings (inozivikanwawo secross gratings) 33,34,35,36. Kuvimbika kweiyi tekinoroji microscopy tekinoroji, yakavakirwa pakuyambuka grating wavefront microscopy (CGM), kwakaratidzwa mumapepa gumi nemaviri akaburitswa mumakore gumi apfuura37,38,39,40,41,42,43.
Scheme yekuisa parallel laser kupisa, kuumbwa uye tembiricha microscope. b Sample geometry inoumbwa nekamuri yeAttofluor TM ine chivharo chakavharwa negoridhe nanoparticles. c Tarisa zvakanyanya pamuenzaniso (kwete kuyera). d inomiririra iyo yunifomu laser beam mbiri uye (e) inoteedzerwa inotevera tembiricha kugovera pane sampuli ndege yegoridhe nanoparticles. f is annular laser beam profile yakakodzera kugadzira tembiricha yakafanana sezvakaratidzwa mukuenzanisa kwemhedzisiro yekugovera tembiricha inoratidzwa mu (g). Scale bar: 30 µm.
Kunyanya, isu munguva pfupi yapfuura takawana kupisa kwemasero emamalia ane LA-HTM neCGM uye takateedzera cellular kupisa kuvhunduka kwemhinduro mumhando ye37-42 ° C, zvichiratidza kushanda kwehunyanzvi uhu kukufungidzira kwesero mhenyu. Zvisinei, kushandiswa kweLA-HTM pakudzidza kwezvipembenene pakupisa kwakanyanya hakusi pachena, sezvo zvinoda kuchenjerera zvakanyanya kana zvichienzaniswa nemasero emammalian: kutanga, kupisa pasi pepakati nemakumi emadhigirii (pane madhigirii mashomanana) kunotungamirira. kusvika kune yakasimba vertical tembiricha gradient. inogona kugadzira fluid convection 44 iyo, kana isina kubatanidzwa zvakasimba kune substrate, inogona kukonzera kufamba kusingafadzi uye kusanganiswa kwebhakitiriya. Iyi convection inogona kubviswa nekudzikisa ukobvu hweiyo mvura layer. Nokuda kwechikonzero ichi, mukuedza kwose kwakaratidzwa pasi apa, kusungirirwa kwebhakitiriya kwakaiswa pakati pezvivharo zviviri zvinenge 15 µm gobvu yakaiswa mukati mekapu yesimbi (Attofluor TM, Thermofisher, Fig. 1b, c). Mumusimboti, convection inogona kudzivirirwa kana ukobvu hwemvura iri idiki pane danda saizi yekudziya laser. Chechipiri, kushanda mugeometry shoma yakadaro kunogona kukanganisa aerobic organisms (ona Fig. S2). Dambudziko iri rinogona kudziviswa nekushandisa substrate iyo inopindira kune okisijeni (kana chero imwe gasi inokosha), nekusiya mabhubhu emhepo akavharirwa mukati mevhavha, kana nekuchera maburi muvharo yepamusoro (ona Fig. S1) 45. Muchidzidzo ichi, takasarudza mhinduro yekupedzisira (Figures 1b uye S1). Pakupedzisira, laser kupisa haipe yunifomu tembiricha kugovera. Kunyangwe pakusimba kwakafanana kweiyo laser danda (Fig. 1d), kugoverwa kwekushisa hakusi yunifomu, asi kunofanana nekugoverwa kweGaussian nekuda kwekupararira kwekupisa (Fig. 1e). Kana chinangwa chiri chekumisikidza chaiyo tembiricha mumunda wekuona yekudzidza biological masisitimu, asina kuenzana profiles haana kunaka uye anogona zvakare kutungamirira kune thermophoretic kufamba kwebhakitiriya kana vasinganamati kune substrate (ona Fig. S3, S4)39. Kuti izvi zviitike, takashandisa spatial light modulator (SLM) kuumba infrared laser beam zvinoenderana nechimiro chemhete (Fig. 1f) mundege yesample kuti tiwane kugovera tembiricha yakakwana mukati menzvimbo yakapihwa geometric, pasinei nekupararira kwemafuta (Fig. 1d) 39, 42, 46. Isa chivharo chepamusoro pamusoro pedhishi resimbi (Mufananidzo 1b) kuti udzivise evaporation yepakati uye chengeta kwemazuva mashoma. Nekuti iyi yepamusoro inovhara haina kuvharwa, yekuwedzera yepakati inogona kuwedzerwa nyore chero nguva kana zvichidikanwa.
Kuratidza mashandiro anoita LA-HTM uye kuratidza kushanda kwayo mukutsvagisa thermophilic, takadzidza bhakitiriya aerobic Geobacillus stearothermophilus, ine tembiricha yekukura yakakwana inosvika 60-65°C. Bhakitiriya zvakare ine flagella uye kugona kushambira, ichipa imwe chiratidzo cheyakajairika cellular chiitiko.
Samples (Fig. 1b) yakamboiswa pa60 ° C kweawa imwe chete ndokuzoiswa muLA-HTM sampuli inobata. Iyi pre-incubation ndeyekusarudza, asi ichiri kubatsira, nekuda kwezvikonzero zviviri: Chekutanga, kana laser yabatidzwa, inoita kuti maseru akure nekukamurana (ona bhaisikopo M1 muSupplementary Materials). Pasina pre-incubation, kukura kwebhakitiriya kunowanzo kunonoka neanenge maminetsi makumi mana nguva yega yega nzvimbo yekutarisa painopiswa pamuenzaniso. Chechipiri, 1 awa pre-incubation yakasimudzira kunamatira kwebhakitiriya kune chivharo, kudzivirira masero kubva kunze kwemunda wekuona nekuda kwe thermophoresis apo laser yakashandurwa (ona firimu M2 muSupplementary Materials). Thermophoresis kufamba kwezvimedu kana mamorekuru patembiricha gradient, kazhinji kubva kunopisa kusvika kuchando, uye mabhakitiriya haana musiyano43,47. Izvi zvisingadiwe mhedzisiro inobviswa pamusoro penzvimbo yakapihwa nekushandisa SLM kuumba laser beam uye kuwana yakadzika tembiricha kugovera.
Pamusoro pemuonde. Mufananidzo 2 unoratidza kugoverwa kwetembiricha kuyerwa neCGM yakawanikwa nekudiridza girazi substrate yakavharwa negoridhe nanoparticles ine annular laser beam (Fig. 1f). Kugovera tembiricha yakatsetseka kwakaonekwa pamusoro penzvimbo yese yakafukidzwa nelaser beam. Iyi zoni yakaiswa ku65°C, iyo yakanyanya tembiricha yekukura. Kunze kwenzvimbo iyi, tembiricha curve inongowira ku \(1/r\) (uko \(r\) ndiyo radial coordinate).
Mepu yeTembiricha yezviyero zveCGM zvakawanikwa nekushandisa annular laser danda kuvhenekera dhiri regoridhe nanoparticles kuti uwane tembiricha yakatsetseka pamusoro penzvimbo yakatenderedzwa. b Isotherm yemepu yetembiricha (a). Iyo contour yelaser beam inomiririrwa negrey dotted denderedzwa. Kuedza kwakadzokororwa kaviri (ona Supplementary Materials, Figure S4).
Kubudirira kwemasero ebhakitiriya kwakatariswa kwemaawa akati wandei uchishandisa LA-HTM. Pamusoro pemuonde. 3 inoratidza nguva yenguva yemifananidzo mina yakatorwa kubva ku3 awa 20 miniti bhaisikopo (Movie M3, Supplementary Information). Utachiona hwakaonekwa huchiwedzera kuwedzera mukati menzvimbo yedenderedzwa inotsanangurwa nelaser apo tembiricha yaive yakakwana, ichisvika 65°C. Kusiyana neizvi, kukura kwesero kwakaderedzwa zvakanyanya apo tembiricha yakawira pasi pe50 ° C kwe10 s.
Mifananidzo yakadzama yeG. stearothermophilus bacteria inokura mushure mekupisa nelaser panguva dzakasiyana, (a) t = 0 min, (b) 1 h 10 min, (c) 2 h 20 min, (d) 3 h 20 min, kunze kwe 200 Yakatorwa kubva mufirimu reminiti imwe chete (M3 firimu rakapihwa muSupplementary Information) yakasimudzwa pamepu yekushisa inoenderana. Laser inobatidzwa nenguva \(t=0\). Isotherms yakawedzerwa kumufananidzo wekusimba.
Kuti tiwedzere kuyera kukura kwesero uye kuvimba kwayo nekushisa, takayera kuwedzera kwebiomass yezvikamu zvakasiyana-siyana zvekutanga mabhakitiriya ari oga muFirimu M3 yekuona (Fig. 4). Mabhakitiriya emubereki akasarudzwa pakutanga kweiyo mini colony forming unit (mCFU) inoratidzwa mumufananidzo S6. Dry mass zviyero zvakatorwa neCGM 48 kamera iyo yakashandiswa kumepu yekugovera tembiricha. Iko kugona kweCGM kuyera huremu hwakaoma uye tembiricha isimba reLA-HTM. Sezvaitarisirwa, kupisa kwakanyanya kwakakonzera kukurumidza kukura kwebhakitiriya (Fig. 4a). Sezvinoratidzwa muchikamu chechikamu muFig. 4b, kukura patembiricha yese kunotevera kukura kweexponential, uko data rinoshandisa exponential function \(m={m}_{0}{10}^{t/\ tau }+ {{\mbox{cst}}}\), apo \(\tau {{{{\rm{log }}}}}}}2\) - nguva yechizvarwa (kana nguva yakapetwa kaviri), \( g =1/ \tau\) - chiyero chekukura (nhamba yezvikamu payuniti nguva). Pamusoro pemuonde. 4c inoratidza huwandu hwekukura uye nguva yechizvarwa sebasa rekushisa. Inokurumidza kukura mCFUs inoratidzirwa nekuzara kwekukura mushure memaawa maviri, hunhu hunotarisirwa nekuda kwehuwandu hwehutachiona hwehutachiona (yakafanana neyakamira chikamu mune yekirasi yemvura tsika). Chimiro chese \(g\left(T\right)\) (Fig. 4c) inoenderana neinotarisirwa yezvikamu zviviri curve yeG. stearothermophilus ine optimal growth rate around 60-65°C. Matanidza data uchishandisa cardinal model (Figure S5)49 apo \(\left({{G}_{0}{;\;T}}_{{\min }};{T}_{{opt} } ;{T}_{{\max}}}\kurudyi)\) = (0.70 ± 0.2; 40 ± 4; 65 ± 1.6; 67 ± 3) °C, inobvumirana zvakanaka nedzimwe tsika dzakadudzwa muzvinyorwa49. Kunyangwe tembiricha inotsamira paramita ichigonekazve, mwero wekukura we \({G}_{0}\) unogona kusiyana kubva pakuedzwa kuenda kune kumwe (ona manhamba S7-S9 nebhaisikopo M4). Kusiyana nemamiriro ekushisa akakodzera, ayo anofanira kunge ari pasi rose, hukuru hwekukura hunoenderana nehupfumi hwepakati (kuwanikwa kwezvokudya, kusanganiswa kweokisijeni) mukati mekucherechedza microscale geometry.
a Microbial kukura patembiricha dzakasiyana. mCFU: Miniature Colony Kugadzira Units. Dhata yakawanikwa kubva muvhidhiyo yebhakitiriya rimwe chete rinokura mukupisa tembiricha (bhaisikopo M3). b Zvakafanana ne(a), semi-logarithmic scale. c Chiyero chekukura\(\ tau\) uye nguva yechizvarwa\(g\) yakaverengerwa kubva pakudzokororwa kwemutsara (b). Horizontal kukanganisa mabara: tembiricha yemhando pamusoro peiyo mCFUs yakawedzera mundima yekuona panguva yekukura. Vertical error bars: linear regression standard error.
Pamusoro pekukura kwakajairika, mamwe mabhakitiriya dzimwe nguva aifamba achionekwa panguva yekudziya kwelaser, inova maitiro anotarisirwa kune mabhakitiriya ane flagella. Iyo firimu M5 mune rumwe ruzivo inoratidza zviitiko zvekushambira zvakadaro. Muchiyedzo ichi, yunifomu laser radiation yakashandiswa kugadzira tembiricha gradient, sezvakaratidzwa muFigure 1d, e uye S3. Mufananidzo 5 unoratidza maviri emifananidzo akateedzana akasarudzwa kubva muM5 bhaisikopo rinoratidza kuti bhakitiriya rimwe rinoratidza mafambiro anotungamira nepo mamwe mabhakitiriya anoramba asingafambi.
Mafuremu maviri enguva (a) na (b) anoratidza kushambira kwemabhakitiriya maviri akasiyana akaiswa madenderedzwa ane madoti. Mifananidzo yacho yakatorwa kubva kuM5 bhaisikopo (yakapihwa seyekuwedzera zvinhu).
Muchiitiko cheG. stearothermophilus, kufamba kunoshanda kwebhakitiriya (Fig. 5) yakatanga masekondi mashomanana mushure mokunge danda re laser rakashandurwa. Kucherechedza uku kunosimbisa mhinduro yenguva yeiyi thermophilic microorganism pakuwedzera kwekushisa, sezvakatoonekwa naMora et al. 24 . Musoro webhakitiriya motility uye kunyange thermotaxis inogona kuongororwa zvakare uchishandisa LA-HTM.
Kushambira kweMicrobial hakufanirwe kuvhiringika nemamwe marudzi ekufamba kwemuviri, anoti (i) Brownian motion, inoita senge chaotic motion isina gwara chairo, (ii) convection 50 uye thermophoresis 43, inosanganisirwa nekufamba kwese kwese kwese kwe tembiricha. gradient.
. Kana mamiriro ezvemhoteredzo akave akanaka zvakare, spores inomera, ichiita masero mapenyu uye kutangazve kukura. Kunyangwe iyi sporulation / kumera maitiro ichizivikanwa, haina kumbobvira yaonekwa munguva chaiyo. Tichishandisa LA-HTM, tinoshuma pano kutarisa kwekutanga kwezviitiko zvekumera muG. stearothermophilus.
Pamusoro pemuonde. 6a inoratidza nguva-inopera mifananidzo ye optical deep (OT) yakawanikwa uchishandisa CGM seti ye13 spores. Kwenguva yese yekuunganidza (15 h 6 min, \(t=0\) - kutanga kwekudziya kwelaser), 4 kubva pagumi nenhatu spores dzakamera, panguva dzakatevedzana nguva \(t=2\) h, \( 3\ ) h \(10 \)', \(9\) h \(40\)' uye \(11\) h \(30\)'. Kunyangwe chimwe chete chezviitiko izvi chinoratidzwa muMufananidzo 6, 4 zviitiko zvekumera zvinogona kucherechedzwa mufirimu reM6 mune yekuwedzera zvinhu. Sezvineiwo, kumera kunoratidzika kunge kwakangoerekana kwaitika: hazvisi zvese zvinomera uye hazvimereri panguva imwe chete, kunyangwe kuchinja kwakafanana mumamiriro ezvakatipoteredza.
a Time-lapse ine 8 OT mifananidzo (kunyudzwa kwemafuta, 60x, 1.25 NA chinangwa) uye (b) biomass evolution yeG. stearothermophilus aggregates. c (b) Yakadhirowa pachikamu chehafu-logi kuratidza mutsara wekukura kwekukura (dashed line).
Pamusoro pemuonde. 6b,c inoratidza biomass yehuwandu hwemasero mumunda wekuona sekushanda kwenguva panguva yese yekuunganidza data. Kukurumidza kuora kwekuoma kwakaonekwa pa \(t=5\)h mufig. 6b, c, nekuda kwekubuda kwemamwe masero kubva kumunda wekuona. Kukura kwezviitiko zvina izvi \(0.77\pm 0.1\) h-1. Kukosha uku kwakakwirira kudarika chiyero chekukura chinosanganiswa neMufananidzo 3. 3 uye 4, apo masero anokura kazhinji. Chikonzero chekuwedzera kwekukura kweG. stearothermophilus kubva kuspores hachina kujeka, asi zviyero izvi zvinoratidzira kufarira kweLA-HTM uye kushanda pachikamu chimwe chete chesero (kana pachikamu chimwe chemCFU) kuti udzidze zvakawanda pamusoro pemasimba ehupenyu hwesero. .
Kuti tiwedzere kuratidza kushanduka-siyana kweLA-HTM uye kushanda kwayo pakupisa kwakanyanya, takaongorora kukura kweSulfolobus shibatae, hyperthermophilic acidophilic archaea ine tembiricha yekukura ye80 ° C51. Zvichienzaniswa neG. stearothermophilus, idzi archaea dzinewo morphology yakasiyana zvikuru, yakafanana ne 1 micron spheres (cocci) pane matanda akareba (bacilli).
Mufananidzo 7a une sequential optical deep mifananidzo yeS. shibatae mCFU yakawanikwa uchishandisa CGM (ona M7 feature film in Supplementary Materials). Iyi mCFU inokura inosvika 73°C, pazasi petembiricha yakanaka ye80°C, asi mukati metembiricha yekukura kwekushanda. Takaona zviitiko zvakawanda zvefission izvo zvakaita kuti mCFUs iite senge micrograpes yearchaea mushure memaawa mashoma. Kubva pamifananidzo iyi yeOT, mCFU biomass yakayerwa nekufamba kwenguva uye yakaunzwa muMufananidzo 7b. Sezvineiwo, S. shibatae mCFUs yakaratidza kukura kwemutsara kwete kukura kweexponential kunoonekwa neG. stearothermophilus mCFUs. Pakave nenhaurirano yenguva refu 52 pamusoro pemamiriro ekukura kwemasero: nepo zvimwe zvidzidzo zvinorondedzera kukura kwehutachiona hunoenderana nehukuru hwavo (exponential growth), vamwe vanoratidza chiyero chinoramba chiripo (mutsara kana bilinear kukura). Sezvakatsanangurwa naTzur et al.53, kusiyanisa pakati peexponential uye (bi) kukura kwemutsara kunoda kunyatsojeka kwe <6% muzviyero zvebiomass, izvo zvisingasviki kune dzakawanda zveQPM maitiro, kunyange kusanganisira interferometry. Sezvakatsanangurwa naTzur et al.53, kusiyanisa pakati peexponential uye (bi) kukura kwemutsara kunoda kunyatsojeka kwe <6% muzviyero zvebiomass, izvo zvisingasviki kune dzakawanda zveQPM maitiro, kunyange kusanganisira interferometry. Как объяснили Цур и др.53, различение экспоненциального и (би)линейного роста требует точности <6% в измерениях биомасниального, биомассия, биомассия дов QPM, даже с использованием интерферометрии. Sezvakatsanangurwa naZur et al.53, kusiyanisa pakati peexponential uye (bi) kukura kwemutsara kunoda <6% kunyatsoita zviyero zvebiomass, izvo zvisingawaniki kune nzira dzakawanda dzeQPM, kunyange kushandisa interferometry.Sezvakatsanangurwa naZur et al. 53, kusiyanisa pakati peexponential uye (bi) mutsara kukura kunoda zvishoma kudarika 6% kunyatsoita zviyero zvebiomass, izvo zvisingawaniki kune nzira dzakawanda dzeQPM, kunyange kana interferometry inoshandiswa. CGM inozadzikisa kurongeka uku ne-sub-pg kunyatsoita muzviyero zvebiomass36,48.
a Time-lapse ine 6 OT mifananidzo (kunyudzwa kwemafuta, 60x, NA chinangwa 1.25) uye (b) micro-CFU biomass evolution yakayerwa neCGM. Ona bhaisikopo M7 kuti uwane rumwe ruzivo.
Kukura kwakanyatso mutsara kweS. shibatae kwakanga kusingatarisirwi uye hakusati kwataurwa. Nekudaro, kukura kwakanyanya kunotarisirwa, nekuti nekufamba kwenguva, kupatsanurwa kwakawanda kwe2, 4, 8, 16 ... masero anofanira kuitika. Isu takafungidzira kuti kukura kwemutsara kunogona kunge kuri nekuda kwekudzivirirwa kwesero nekuda kwekuomerwa kwesero kurongedza, sekukura kwesero kunodzikira uye kunopedzisira kwasvika payakarara kana density yesero yanyanya.
Isu tinopedzisa nekukurukura zvinotevera zvishanu zvibodzwa zvechido: kuderedzwa kwekudziya vhoriyamu, kuderedzwa kwekupisa inertia, kufarira kwegoridhe nanoparticles, kufarira kwehuwandu hwechikamu microscopy, uye inogoneka tembiricha renji umo LA-HTM inogona kushandiswa.
Kuenzaniswa nekudziisa kudziyisa, kupisa kwelaser kunoshandiswa kusimudzira HTM kunopa zvakati wandei zvakanakira, izvo zvatinoratidzira muchidzidzo chino. Kunyanya, mumagetsi emvura mumunda wekuona microscope, vhoriyamu yekudziya inochengetwa mukati mezvishoma (10 μm) 3 mavhoriyamu. Nenzira iyi, hutachiona hunoonekwa chete hunoshanda, nepo mamwe mabhakitiriya akarara uye anogona kushandiswa kuenderera mberi nekudzidza sampu - hapana chikonzero chekuchinja sampu nguva dzese tembiricha itsva inoda kuongororwa. Mukuwedzera, kupisa kwema microscale kunobvumira kuongorora kwakananga kwehuwandu hwakawanda hwekushisa: Mufananidzo 4c wakawanikwa kubva mufirimu yemaawa matatu (Movie M3), iyo inowanzoda kugadzirira uye kuongororwa kwemasampuli akawanda - imwe neimwe yemuenzaniso uri kudzidza. y tembiricha inomiririra huwandu hwemazuva mukuyedza. Kuderedza vhoriyamu inopisa zvakare inochengeta zvese zvakatenderedza optical zvikamu zve microscope, kunyanya iyo chinangwa lens, pakupisa kwekamuri, iro rave dambudziko guru rakatarisana nenharaunda kusvika zvino. LA-HTM inogona kushandiswa nechero lens, kusanganisira mafuta ekunyudza lenses, uye ichagara pakamuri tembiricha kunyangwe nekupisa kwakanyanya mumunda wekuona. Mhedzisiro huru yelaser yekudziya nzira yatinoshuma muchidzidzo ichi ndeyekuti masero asinganamate kana kuyangarara anogona kunge ari kure nemunda wekuona uye akaoma kudzidza. A workaround inogona kunge iri kushandisa yakaderera magnification lenzi kuwana yakakura tembiricha inokwira inodarika mazana mashoma mamicrons. Kuchenjerera uku kunoperekedzwa nekuderera kwekugadziriswa kwenzvimbo, asi kana chinangwa chiri chekudzidza kufamba kwema microorganisms, yakakwirira nzvimbo yekugadzirisa haidiwi.
Chiyero chenguva yekudziya (uye kutonhora) system \({{{{\rm{\tau }}}}}}}}_{{{\mbox{D}}}}\) zvinoenderana nehukuru hwayo , maererano nemutemo \({{({\rm{\tau }}}}}}}_{{{\mbox{D}}}}}={L}^{2}/D\), kupi \ (L\ ) ndiyo saizi yehunhu hwenzvimbo yekupisa (iyo dhayamita yelaser beam muchidzidzo chedu \(L\ about 100\) μm), \(D\) ndiko kusiyanisa kwekupisa kwenzvimbo (avhareji mune yedu. kesi, girazi nemvura Diffusion rate\(D\ about 2\fold {10}^{-7}\) m2/s Naizvozvo, muchidzidzo ichi, nguva mhinduro dzehurongwa hwe50 ms, kureva, quasi-instantaneous). Kuchinja kwetembiricha, kunogona kutarisirwa nekukasira uku kusimudzwa kwekukwira kwetembiricha hakungopfupiki nguva yekuyedza, asi zvakare inobvumira nguva chaiyo \(t=0\) yechero chidzidzo chine simba chemhedzisiro.
Nzira yedu yakarongwa inoshanda kune chero mwenje-inonwisa substrate (semuenzaniso, masampula ekutengesa ane ITO coating). Nekudaro, goridhe nanoparticles inokwanisa kupa yakanyanya kunyura mune infrared uye yakaderera kunyura mune inooneka renji, iwo ekupedzisira maitiro ayo ari ekufarirwa kweanobudirira optical kucherechedzwa muchikamu chinooneka, kunyanya kana uchishandisa fluorescence. Pamusoro pezvo, goridhe inoenderana ne biocompatible, makemikari inert, density yemaziso inogona kugadziridzwa kubva pa530 nm kusvika padhuze infrared, uye gadziriro yemuenzaniso iri nyore uye inodhura29.
Transverse grating wavefront microscopy (CGM) inobvumira kwete tembiricha mepu paiyo microscale, asiwo biomass yekutarisa, ichiita kuti inyanyo batsira (kana zvisingaite) musanganiswa neLA-HTM. Mumakore gumi apfuura, mamwe tembiricha tembiricha mamicroscope akagadzirwa, kunyanya mumunda we bioimaging, uye mazhinji acho anoda kushandiswa kwekushisa-sensitive fluorescent probes54,55. Nekudaro, nzira idzi dzakashoropodzwa uye mimwe mishumo yakayera kusingaite tembiricha shanduko mukati memasero, pamwe nekuda kwekuti fluorescence inoenderana nezvimwe zvinhu zvakawanda kunze kwekupisa. Mukuwedzera, maprobes akawanda efluorescent haana kugadzikana pakupisa kwakanyanya. Naizvozvo, QPM uye kunyanya CGM inomiririra tembiricha tembiricha microscope yekudzidza hupenyu patembiricha yakakwira uchishandisa optical microscopy.
Zvidzidzo zveS. shibatae, zvinogara zvakakwana pa80 ° C, zvinoratidza kuti LA-HTM inogona kushandiswa pakudzidza hyperthermophiles, kwete chete thermophiles iri nyore. Muchidimbu, hapana muganho kune huwandu hwetembiricha hunogona kusvikwa uchishandisa LA-HTM, uye kunyangwe tembiricha iri pamusoro pe100 ° C inogona kusvikwa pamhepo yemhepo isina kuvira, sezvakaratidzwa neboka redu re38 mune hydrothermal chemistry application pamhepo. pressure A. Laser inoshandiswa pakudziisa goridhe nanoparticles 40 nenzira imwechete. Saka, LA-HTM ine mukana wekushandiswa kutarisa zvisati zvamboitika hyperthermophiles ine yakajairwa high resolution optical microscopy pasi pemamiriro akajairwa (kureva pasi pekunetseka kwezvakatipoteredza).
Zvese zviedzo zvakaitwa pachishandiswa maikorosikopu yekumba, kusanganisira Köhler illumination (ine LED, M625L3, Thorlabs, 700 mW), mubati wemuenzaniso ane bhuku rekufambisa xy, zvinangwa (Olympus, 60x, 0.7 NA, mhepo, LUCPlanFLN60X kana 60x, NA25, Oil. , UPLFLN60XOI), CGM kamera (QLSI cross grating, 39 µm pitch, 0.87 mm kubva Andor Zyla camera sensor) kupa kusimba uye wavefront imaging, uye sCMOS kamera (ORCA Flash 4.0 V3, 16-bit mode, kubva Hamamatsu) kurekodha iyo data inoratidzwa muMufananidzo 5 (kushambira kwebhakitiriya). Iyo dichroic beam splitter ndeye 749 nm BrightLine edge (Semrock, FF749-SDi01). Sefa iri kumberi kwekamera ndeye 694 pfupi pass sefa (FF02-694/SP-25, Semrock). Titanium sapphire laser (Laser Verdi G10, 532 nm, 10 W, pumped tsunami laser cavity, Spectra-Physics muFig. 2-5, yakatsiviwa neMillenia laser, Spectraphysics 10 W, pumped Mira laser cavity, Coherent, yeFig. 2 -5). 6 ne7) dzakaiswa kune wavelength \({{({\rm{\lambda }}}}}}}=800\) nm, iyo inoenderana neplasmon resonance spectrum yegoridhe nanoparticles. Spatial light modulators (1920 × 1152 pixels) akatengwa kubva kuMeadowlark Optics akaverengerwa achishandisa Gerchberg-Saxton algorithm sekutsanangurwa kwazvinoitwa.
Cross grating wavefront microscopy (CGM) inyanzvi yekuona microscopy yakavakirwa pakusanganisa maviri-dimensional diffraction grating (inozivikanwawo sekuyambuka grating) chinhambwe chemirimita imwe kubva kune yakajairwa kamera sensor. Muenzaniso wakajairika weCGM watakashandisa muchidzidzo ichi unonzi four-wavelength transverse shift interferometer (QLSI), apo kuyambuka-grating kunosanganisira intensity/phase checkerboard pattern yakaunzwa uye ine patented naPrimot et al. mu 200034. Mitsara yakasimudzika uye yakatwasuka inogadzira grid-yakafanana mimvuri pane sensor, kukanganisa kwayo kunogona kuverengerwa nhamba munguva chaiyo kuti uwane optical wavefront distortion (kana yakaenzana chikamu profile) chechiitiko chiedza. Kana ikashandiswa pamaikorosikopu, kamera yeCGM inogona kuratidza musiyano wenzira yekuona yechinhu chine mufananidzo, inozivikanwawo seOptical deep (OT), ine sensitivity pahurongwa hwenanometers36. Mune chero kuyerwa kweCGM, kuti ubvise chero chakakanganisika muzvikamu zvemaziso kana matanda, mufananidzo wekutanga weOT unofanirwa kutorwa uye kubviswa kubva kune chero inotevera mifananidzo.
Tembiricha microscope yakaitwa uchishandisa CGM kamera sezvinotsanangurwa mureferensi. 32. Muchidimbu, kupisa mvura inoshandura iyo refractive index, ichigadzira thermal lens effect iyo inokanganisa chiitiko danda. Uku kukanganiswa kwewavefront kunoyerwa neCGM uye kugadziridzwa uchishandisa deconvolution algorithm kuwana matatu-dimensional tembiricha kugovera mune yemvura yepakati. Kana iyo nanoparticles yegoridhe yakagovaniswa zvakaenzana mumuenzaniso wese, tembiricha mepu inogona kuitwa munzvimbo dzisina mabhakitiriya kuburitsa mifananidzo iri nani, ndizvo zvatinoita dzimwe nguva. Iyo referensi CGM mufananidzo wakawanikwa usina kudziyisa (nelaser off) uye wozotorwa panzvimbo imwe chete mumufananidzo nelaser on.
Dry misa kuyerwa kunowanikwa uchishandisa yakafanana CGM kamera inoshandiswa kufungidzira tembiricha. CGM referensi mifananidzo yakawanikwa nekukurumidza kufambisa sampu mu x uye y panguva yekuratidzwa senzira yekuenzanisa chero inhomogeneity muOT nekuda kwekuvapo kwebhakitiriya. Kubva kumifananidzo yeOT yemabhakitiriya, biomass yavo yakawanikwa pachishandiswa ensemble yemifananidzo pamusoro penzvimbo dzakasarudzwa uchishandisa Matlab's homemade segmentation algorithm (ona chikamu "Numerical code"), zvichitevera maitiro anotsanangurwa muref. 48. Muchidimbu, tinoshandisa hukama \(m={\ alpha}^{-1}\iint {{\mbox{OT}}}\left(x,y\right){{\mbox{d}} } x{{\mbox{d}}}y\), apo \({{\mbox{OT}}}\kuruboshwe(x,y\kurudyi)\) pane mufananidzo wakadzama, \(m\) uri huremu hwakaoma uye \({{{{\rm{\ alpha }}}}}}}\) inogara iripo. Takasarudza \({{{{\rm{\alpha)))))))=0.18\) µm3/pg, chinova chinogara chiripo chemasero mapenyu.
Chivharo chinotsvedza 25 mm muhupamhi uye 150 µm gobvu chakavharwa negoridhe nanoparticles chakaiswa muAttofluorTM chamber (Thermofisher) ine nanoparticles dzegoridhe dzakatarisa kumusoro. Geobacillus stearothermophilus yakagadzirwa usiku hwose muLB medium (200 rpm, 60 ° C) zuva rega rega rekuedza risati rasvika. Donhwe re5 µl rekumiswa kweG. stearothermophilus ine optical density (OD) ye 0.3 kusvika 0.5 yakaiswa pabutiro slip negoridhe nanoparticles. Zvadaro, chivharo chevhavha chakatenderera 18 mm muhupamhi negomba 5 mm muhupamhi pakati chakadonhedzwa padonhwe, uye 5 μl yebhakitiriya yakamiswa neiyo yakafanana optical density yakashandiswa kakawanda pakati pegomba. Matsime pazvivharo akagadzirwa maererano nemaitiro anotsanangurwa muref. 45 (ona Supplementary Information kuti uwane rumwe ruzivo). Wobva wawedzera 1 ml yeLB yepakati kune inovhara kudzivirira iyo yemvura layer kubva pakuoma. Iyo yekupedzisira inovhara inoiswa pamusoro pevhavha yakavharwa yekamuri yeAttofluor ™ kudzivirira kubuda kwepakati panguva yekunyudzwa. Pakuedza kumera, takashandisa spores, iyo, mushure mekuedza kwakajairika, dzimwe nguva yakavhara pamusoro pekuvhara. Mutoo wakafanana wakashandiswa kuwana Sulfolobus shibatae. Mazuva matatu (200 rpm, 75 ° C) ekutanga kurimwa kweThiobacillus serrata akaitwa pakati pe182 (DSMZ).
Sampuli dzegoridhe nanoparticles dzakagadzirwa nemicellar block copolymer lithography. Maitiro aya anotsanangurwa zvakadzama muChap. 60. Muchidimbu, micelles yakavharira maion egoridhe akagadzirwa nekusanganisa copolymer neHAuCl4 mutoluene. Mafukidziro akacheneswa akabva anyudzwa mumushonga uye akabatwa ne UV irradiation pamberi peanodzikisira agent kuti awane mhodzi dzegoridhe. Pakupedzisira, mhodzi dzegoridhe dzakakura nekubata chivharo chine mvura ine mvura yeKAuCl4 uye ethanolamine kwemaminetsi gumi nematanhatu, izvo zvakakonzera quasi-periodic uye yakanyatso yunifomu kurongeka kweasina-spherical goridhe nanoparticles mupedyo infrared.
Kushandura mainterferograms kuita mifananidzo yeOT, isu takashandisa algorithm yekumba, sekutsanangurwa kwazvino mune chinongedzo. 33 uye inowanikwa seMatlab package mune inotevera yeruzhinji repository: https://github.com/baffou/CGMprocess. Iyo pasuru inogona kuverenga kusimba uye mifananidzo yeOT zvichibva pane akarekodhwa interferograms (kusanganisira referensi mifananidzo) uye kamera array madaro.
Kuti tiverenge chiyero chechikamu chakaiswa kuSLM kuti tiwane tembiricha yakapihwa, takashandisa yakambogadzirwa kumba algorithm39,42 inowanikwa mune inotevera yeruzhinji repository: https://github.com/baffou/SLM_temperatureShaping. Iyo yekupinza ndiyo inodiwa tembiricha ndima, iyo inogona kusetwa digitally kana kuburikidza ne monochrome bmp mufananidzo.
Kupatsanura maseru uye kuyera huremu hwawo hwakaoma, takashandisa Matlab algorithm yedu yakaburitswa mune inotevera veruzhinji repository: https://github.com/baffou/CGM_magicWandSegmentation. Pamufananidzo wega wega, mushandisi anofanira kudzvanya pabhakitiriya kana mCFU yekufarira, gadzirisa wand senitivity, uye kusimbisa kusarudzwa.
Kuti uwane rumwe ruzivo nezve dhizaini yekudzidza, ona Nature Research Report abstract yakabatana nechinyorwa ichi.
Data inotsigira zvakabuda muchidzidzo ichi inowanika kubva kune vanyori vakasiyana kana chikumbiro chine musoro.
Kodhi yakashandiswa muchidzidzo ichi yakatsanangurwa muchikamu cheNzira, uye shanduro dzekugadzirisa dzinogona kutorwa kubva ku https://github.com/baffou/ mune dzinotevera repositories: SLM_temperatureShaping, CGMprocess, uye CGM_magicWandSegmentation.
Mehta, R., Singhal, P., Singh, H., Damle, D. & Sharma, AK Insight mune thermophiles uye yavo yakakura-spectrum application. Mehta, R., Singhal, P., Singh, H., Damle, D. & Sharma, AK Insight mune thermophiles uye yavo yakakura-spectrum application.Mehta, R., Singhal, P., Singh, H., Damle, D. naSharma, AK Overview ye thermophiles uye kushanda kwavo kwakakura. 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. uye Sharma AK Kunzwisisa kwakadzama kwe thermophiles uye zvakasiyana-siyana zvekushandisa.3 Biotechnology 6, 81 (2016).
Nguva yekutumira: Sep-26-2022