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Thermophile ni mikorobe ikura ku bushyuhe bwinshi. Kubiga birashobora gutanga amakuru yingirakamaro yukuntu ubuzima buhuza nibihe bikabije. Ariko, biragoye kugera kubushyuhe bwo hejuru hamwe na microscopes isanzwe. Hatanzwe ibyifuzo byinshi byakorewe murugo hashingiwe ku gushyushya amashanyarazi byaho, ariko nta gisubizo cyoroshye cyubucuruzi. Muri iyi nyandiko, turamenyekanisha igitekerezo cya microscale laser yo gushyushya hejuru ya microscope kugirango turebe ubushyuhe bwinshi kubushakashatsi bwa thermophile mugihe ibidukikije byumukoresha byoroheje. Gushyushya Microscale ku gipimo giciriritse cya laser birashobora kugerwaho ukoresheje nanoparticle ya zahabu yubatswe munsi ya biocompatible kandi ikora neza. Ingaruka zishobora guterwa na microscale fluid convection, kugumana selile, hamwe na centrifugal thermophoretic igenda iraganirwaho. Uburyo bwerekanwe mu moko abiri: (i) Geobacillus stearothermophilus, bacteri ikora ya termofilike ikora hafi ya 65 ° C, twabonye ko imera, gukura no koga munsi yubushyuhe bwa microscale; (ii) Thiobacillus sp., hyperthermophilic archaea nziza. kuri 80 ° C. Aka kazi gatanga inzira yo kwitegereza byoroheje kandi byizewe bya mikorobe mikorobe ikoresheje ibikoresho bya kijyambere kandi bihendutse.
Mu myaka miriyari, ubuzima bwo ku isi bwagiye buhinduka kugira ngo buhuze n’ibidukikije bitandukanye usanga rimwe na rimwe bifatwa nkibikabije mubitekerezo byabantu. By'umwihariko, mikorobe zimwe na zimwe ziterwa na mikorobe (bagiteri, archaea, fungi) bita thermophile zitera imbere mu bushyuhe buri hagati ya 45 ° C kugeza 122 ° C1, 2, 3, 4. Thermophile iba mu bidukikije bitandukanye, nk'imyanda miremire yo mu nyanja, amasoko ashyushye. cyangwa ahantu h'ibirunga. Ubushakashatsi bwabo bwatanze inyungu nyinshi mumyaka mike ishize byibuze kubwimpamvu ebyiri. Ubwa mbere, turashobora kubigiraho, kurugero, uburyo thermofile 5, 6, enzymes 7, 8 na membrane 9 ihagaze neza mubushyuhe bwinshi, cyangwa uburyo thermofile ishobora kwihanganira imishwarara ikabije10. Icya kabiri, nizo shingiro ryibikorwa byinshi byikoranabuhanga bikoreshwa1,11,12 nkumusaruro wa lisansi13,14,15,16, synthèse chimique (dihydro, alcool, metani, acide amino, nibindi) 17, biomining18 hamwe na biocatalysis ya thermostable7, 11, 13. By'umwihariko, ibyamamare bizwi cyane bya polymerase (PCR) 19 birimo enzyme (Taq polymerase) itandukanijwe na bagiteri ya thermophilique Thermus aquaticus, imwe muri thermofile ya mbere yavumbuwe.
Nyamara, ubushakashatsi bwa thermophile ntabwo ari umurimo woroshye kandi ntibushobora gutezwa imbere muri laboratoire y'ibinyabuzima. By'umwihariko, thermophile nzima ntishobora kugaragara muri vitro hamwe na microscope isanzwe yumucyo, kabone niyo haba hari ibyumba bishyushya biboneka mubucuruzi, ubusanzwe bipima ubushyuhe buri munsi ya 40 ° C. Kuva mu myaka ya za 90, amatsinda make yubushakashatsi yitangiye kwinjiza sisitemu yo hejuru yubushyuhe bwo hejuru (HTM). Muri 1994 Glukh n'abandi. Icyumba cyo gushyushya / gukonjesha cyatekerejwe hashingiwe ku gukoresha selile ya Peltier igenzura ubushyuhe bwa capillaries y'urukiramende ifunze kugirango ikomeze anaerobicity 20. Igikoresho kirashobora gushyuha kugeza kuri 100 ° C ku gipimo cya 2 ° C / s, bigatuma abanditsi biga umuvuduko wa bacteri ya hyperthermophilique Thermotoga maritima21. Mu 1999 Ihembe n'abandi. Igikoresho gisa cyane cyarakozwe, kiracyashingira kumikoreshereze ya capillaries zishyushye zibereye microscopi yubucuruzi kugirango bigabanye kugabana / guhuza. Nyuma yigihe kirekire cyo kudakora ugereranije, gushakisha HTMs nziza byasubukuwe muri 2012, cyane cyane bijyanye nuruhererekane rwimpapuro nitsinda rya Wirth ryakoresheje igikoresho cyahimbwe na Horn nabandi. Imyaka 15 irashize, umuvuduko wa archaea nyinshi, harimo na hyperthermophile, wize ku bushyuhe bugera kuri 100 ° C ukoresheje capillaries zishyushye 23,24. Bahinduye kandi microscope yumwimerere kugirango bagere ku bushyuhe bwihuse (iminota mike aho kuba iminota 35 kugirango bagere ku bushyuhe bwashyizweho) kandi bagera ku bushyuhe buringaniye buringaniye bwa cm zirenga 2 hagati. Iki gipimo cyerekana ubushyuhe (TGFD) cyakoreshejwe mukwiga kugendagenda kwa thermofile nyinshi mubipimo byubushyuhe ku ntera ijyanye n’ibinyabuzima intera 24, 25.
Gushyushya capillaries zifunze ntabwo aribwo buryo bwonyine bwo kureba thermophile nzima. Muri 2012, Kuwabara n'abandi. Byumba byakoreshwaga mu cyumba cya Pyrex bifunze hamwe n’ibiti birwanya ubushyuhe (Super X2; Cemedine, Ubuyapani) byakoreshejwe. Izi ngero zashyizwe ku bicuruzwa bishyushya ibicuruzwa biboneka mu bucuruzi (Micro Heat Plate, Kitazato Corporation, Ubuyapani) bushobora gushyuha kugeza kuri 110 ° C, ariko ntabwo byari bigenewe bioimaging. Abanditsi babonye igabanywa ryiza rya bagiteri ya anaerobic thermophilic (Thermosipho globiformans, inshuro ebyiri 24 min) kuri 65 ° C. Muri 2020, Pulshen n'abandi. Gushyushya neza ibyokurya byubucuruzi byubucuruzi (AttofluorTM, Thermofisher) byerekanwe hakoreshejwe ibintu bibiri byo gushyushya urugo: umupfundikizo hamwe na stade (iboneza imashini ya PCR). Iri shyirahamwe ritanga ubushyuhe bumwe bwamazi kandi birinda guhumeka no guhunika munsi yumupfundikizo. Gukoresha O-impeta birinda guhanahana gaze nibidukikije. Iyi HTM, yitwa Sulfoscope, yakoreshejwe mu gushushanya Sulfolobus acideocaldarius kuri 75 ° C27.
Ikizwi cyemewe muri sisitemu zose kwari ukubuza gukoresha intego z’ikirere, kwibiza amavuta ayo ari yo yose bidakwiriye kubera ubushyuhe bwo hejuru no gushushanya binyuze muri> mm-1 z'ubugari bubonerana. Ikizwi cyemewe muri sisitemu zose kwari ukubuza gukoresha intego z’ikirere, kwibiza amavuta ayo ari yo yose bidakwiriye kubera ubushyuhe bwo hejuru no gushushanya binyuze muri> mm-1 z'ubugari bubonerana. Общепризныным недостатком всех этих было ограничение на использование воздушных ока овов, поскольку любое иммер черное погружение в масло не подходило для тако вчокы? Ikimenyane cyagaragaye muri ubwo buryo bwose ni imbogamizi yo gukoresha intego z’ikirere, kubera ko kwibiza amavuta ayo ari yo yose bitari bikwiranye n’ubushyuhe bwo hejuru ndetse no kubonerana binyuze mu ngero ziboneye> mm 1 z'ubugari.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。 Imipaka izwi muri sisitemu zose nimbogamizi yo gukoresha indorerwamo ihumeka ikirere, kuko kwibiza amavuta byose ntibikwiriye kwerekanwa ingero zibonerana> mm 1 z'ubugari kuri ubwo bushyuhe bwinshi. Общепризнанным недостатком всех этих с является ограниченное использование воздушных огов, любое иммер черное погружение в масло непригодно для таких вискоких зощик? Ikimenyane kizwi muri sisitemu zose ni ugukoresha imipaka yo mu kirere, kwibiza amavuta ayo ari yo yose ntibikwiriye kubera ubushyuhe bwo hejuru no kubonerana binyuze mu ngero ziboneye> mm 1 z'ubugari.Vuba aha, iyi mbogamizi yakuweho na Charles-Orzag n'abandi. 28, wateguye igikoresho kitagitanga ubushyuhe hafi ya sisitemu yinyungu, ahubwo imbere yikirahure gitwikiriye ubwacyo, gitwikiriwe nigice cyoroshye kibonerana cya résistoriste ikozwe na ITO (indium-tin oxyde). Umupfundikizo urashobora gushyuha kugeza kuri 75 ° C unyuze mumashanyarazi unyuze murwego rubonerana. Ariko, umwanditsi agomba kandi gushyushya lens ku ntego, ariko ntibirenze 65 ° C, kugirango bitayangiza.
Iyi mirimo yerekana ko iterambere rya microscopi yubushyuhe bwo hejuru yubushyuhe butigeze ryemerwa cyane, akenshi risaba ibikoresho byakorewe murugo, kandi akenshi bigerwaho mugiciro cyo gukemura ahantu, bikaba ari bibi cyane dore ko mikorobe ya termofilique itarenze bike. micrometero. Kugabanya ingano yubushyuhe nurufunguzo rwo gukemura ibibazo bitatu byihariye bya HTM: imiterere idahwitse yumwanya, inertie yumuriro mwinshi iyo sisitemu ishyushye, hamwe nubushyuhe bwangiza bwibintu bikikije (amavuta yo kwibiza, lens lens… cyangwa amaboko yabakoresha) mubushyuhe bukabije. ).
Muri iyi nyandiko, turamenyekanisha HTM yo kwitegereza thermophile idashingiye ku gushyushya birwanya. Ahubwo, twageze ku bushyuhe bwaho mukarere gato ka microscope yo kureba dukoresheje lazeri ya lazeri ya substrate ikurura urumuri. Ikwirakwizwa ry'ubushyuhe ryagaragaye hifashishijwe microscopi yo mu cyiciro (QPM). Imikorere yubu buryo igaragazwa na Geobacillus stearothermophilus, bacterium ya moteri ya termofilique yororoka igera kuri 65 ° C kandi ikagira igihe gito cyo gukuba kabiri (iminota 20), na Sulfolobus shibatae, hyperthermophile ikura neza kuri 80 ° C (archaea) Kugereranya. Igipimo gisanzwe cyo kwigana no koga byagaragaye nkigikorwa cyubushyuhe. Iyi lazeri HTM (LA-HTM) ntabwo igarukira kubunini bwikibiriti cyangwa kumiterere yintego (kwibiza umwuka cyangwa amavuta). Ibi bituma lens iyo ari yo yose ihanitse ku isoko ikoreshwa. Ntabwo kandi ibabazwa no gushyuha gahoro bitewe nubushuhe bwumuriro (igera kubushuhe bwihuse kurwego rwa milisegonda) kandi ikoresha gusa ibice biboneka mubucuruzi. Gusa impungenge nshya z'umutekano zijyanye no kuba hariho urumuri rukomeye rwa lazeri (ubusanzwe rugera kuri mW 100) imbere yigikoresho kandi birashoboka binyuze mumaso, bisaba indorerwamo zo gukingira.
Ihame rya LA-HTM nugukoresha laser kugirango ushushe icyitegererezo murwego rwo kureba microscope (Ishusho 1a). Kugirango ukore ibi, icyitegererezo kigomba kuba gikurura urumuri. Kugira ngo dukoreshe ingufu za lazeri (munsi ya mW 100), ntitwashingiye ku kwinjiza urumuri hakoreshejwe uburyo bwamazi, ariko twongereye muburyo bwogukora uburyo bwo kwinjiza icyitegererezo dusiga substrate hamwe na nanoparticles ya zahabu (Ishusho 1c). Gushyushya zahabu nanoparticles hamwe nurumuri bifite akamaro kanini mubijyanye na plasmonique yumuriro, hamwe nibiteganijwe gukoreshwa muri biomedicine, nanochemie cyangwa gusarura izuba 29,30,31. Mu myaka mike ishize, twakoresheje iyi LA-HTM mubushakashatsi bwinshi bujyanye no gukoresha plasma yumuriro muri fiziki, chimie na biologiya. Ingorane nyamukuru hamwe nubu buryo ni mukugaragaza imiterere yubushyuhe bwa nyuma, kubera ko ubushyuhe bwo hejuru bugarukira mu karere ka microscale muri sample. Twerekanye ko ikarita yubushyuhe ishobora kugerwaho hamwe na bine-yumurambararo wa transar shear interferometero, uburyo bworoshye, bukemurwa cyane, hamwe nuburyo bworoshye cyane bwa microscopi ya fonctionnement ishingiye ku gukoresha ibice bibiri-bitandukanya ibice (bizwi kandi no kwambukiranya umusaraba) 33,34,35,36. Ubwizerwe bwubu buhanga bwa microscopi yumuriro, bushingiye kuri microscopi yambukiranya imipaka (CGM), bwerekanwe mu mpapuro icumi zasohotse mu myaka icumi ishize 37,38,39,40,41,42,43.
Gahunda yo gushiraho ubushyuhe bwa laser, gushushanya nubushyuhe bwa microscope. b Icyitegererezo cya geometrie igizwe nicyumba cya AttofluorTM kirimo igifuniko cyometseho zahabu nanoparticles. c Reba neza kuri sample (ntabwo ari igipimo). d yerekana umwirondoro umwe wa laser beam na (e) ikwirakwizwa ryubushyuhe bwakurikiyeho ku ndege ntangarugero ya zahabu nanoparticles. f ni umwirondoro wa lazeri ya buri mwaka ikwiranye no kubyara ubushyuhe bumwe nkuko bigaragara mu kwigana ubushyuhe bwavuyemo bwerekanwe muri (g). Umwanya munini: 30 µm.
By'umwihariko, duherutse kugera ku gushyushya ingirangingo z’inyamabere hamwe na LA-HTM na CGM kandi dukurikirana ibisubizo by’ubushyuhe bwa selile biri hagati ya 37-42 ° C, byerekana ko ubwo buryo bukoreshwa mu kwerekana amashusho amwe. Nyamara, ikoreshwa rya LA-HTM mu kwiga mikorobe ku bushyuhe bwo hejuru ntirisobanutse, kuko risaba ubwitonzi bwinshi ugereranije n’uturemangingo tw’inyamabere: icya mbere, gushyushya munsi yikigereranyo na dogere icumi (aho kuba dogere nkeya) ziyobora Kuri Ubushyuhe bukomeye bwa vertical gradient. Irashobora gukora flux convection 44 iyo, iyo idafatanye neza na substrate, irashobora gutera ingendo itifuzwa no kuvanga bagiteri. Iyi convection irashobora kuvaho mugabanya ubunini bwurwego rwamazi. Kubwiyi ntego, mubushakashatsi bwose bwerekanwe hepfo, guhagarika bagiteri byashyizwe hagati yububiko bubiri hafi 15 µm z'uburebure bwashyizwe imbere mugikombe cyicyuma (AttofluorTM, Thermofisher, Ishusho 1b, c). Ihame, convection irashobora kwirindwa mugihe umubyimba wamazi ari muto ugereranije nubunini bwa lazeri yo gushyushya. Icya kabiri, gukora muri geometrie ntoya birashobora guhumeka ibinyabuzima byo mu kirere (reba Ishusho S2). Iki kibazo kirashobora kwirindwa ukoresheje substrate yinjira muri ogisijeni (cyangwa gaze iyindi yose yingirakamaro), mugusiga ibyuka bihumeka byugarijwe imbere yigitwikirizo, cyangwa mugucukura umwobo mumifuniko yo hejuru (reba Ishusho S1) 45. Muri ubu bushakashatsi, twahisemo igisubizo cya nyuma (Ishusho 1b na S1). Hanyuma, gushyushya lazeri ntabwo bitanga ubushyuhe bumwe. Ndetse no muburemere bumwe bwurumuri rwa lazeri (Igishusho 1d), ikwirakwizwa ryubushyuhe ntirihuje, ahubwo risa nogukwirakwizwa kwa Gaussiya kubera gukwirakwizwa nubushyuhe (Ishusho 1e). Iyo intego ari ugushiraho ubushyuhe nyabwo murwego rwo kureba ubushakashatsi bwibinyabuzima, imyirondoro idahwanye ntabwo ari nziza kandi irashobora no gutuma habaho kwimuka kwa bagiteri iyo idakurikiza insimburangingo (reba Ishusho S3, S4) 39. Kugira ngo ibyo bishoboke, twakoresheje moderi yumucyo (SLM) kugirango dushyireho urumuri rwa lazeri dukurikije imiterere yimpeta (Igishusho 1f) mu ndege yicyitegererezo kugirango tugere ku bushyuhe bwubushyuhe bumwe mubice runaka bya geometrike, nubwo ikwirakwizwa ryumuriro (Igishusho 1d) 39, 42, 46. Shyira igipfundikizo cyo hejuru hejuru yisahani yicyuma (Igicapo 1b) kugirango wirinde guhumeka kandi urebe byibuze iminsi mike. Kuberako iyi mpfundikizo yo hejuru idafunze, uburyo bwinyongera burashobora kongerwaho byoroshye mugihe icyo aricyo cyose bibaye ngombwa.
Kugirango tugaragaze uburyo LA-HTM ikora kandi yerekana ko ikoreshwa mubushakashatsi bwa termofilique, twize bacteri zo mu kirere Geobacillus stearothermophilus, zifite ubushyuhe bwiza bwo gukura bwa dogere 60-65 ° C. Bagiteri ifite kandi flagella nubushobozi bwo koga, itanga ikindi kimenyetso cyibikorwa bisanzwe bya selile.
Ingero (Igishusho 1b) zabanje kubikwa kuri 60 ° C kumasaha imwe hanyuma zishyirwa mubitabo bya LA-HTM. Iyi pre-incubation irahitamo, ariko iracyafite akamaro, kubwimpamvu ebyiri: Icya mbere, iyo laser ifunguye, itera selile guhita zikura no kugabana (reba firime M1 mubikoresho byiyongera). Hatabayeho pre-incubation, gukura kwa bagiteri mubisanzwe gutinda niminota 40 buri gihe ahantu hashya hashyushye kurugero. Icya kabiri, isaha 1 mbere yubushakashatsi bwateje imbere gufatira kwa bagiteri ku gipfukisho, bikabuza ingirabuzimafatizo gutembera mu murima bitewe na thermoforesis igihe lazeri yafungurwaga (reba firime M2 mu bikoresho by'inyongera). Thermophoresis ni urujya n'uruza rw'ibice cyangwa molekile ku gipimo cy'ubushyuhe, ubusanzwe kuva ubushyuhe n'ubukonje, kandi na bagiteri na zo ntizisanzwe 43,47. Izi ngaruka zitifuzwa zivanwaho ahantu runaka ukoresheje SLM kugirango ushire urumuri rwa laser kandi ugere kubushyuhe bukabije.
Ku mutini. Igishushanyo cya 2 cyerekana igabanywa ry'ubushyuhe ryapimwe na CGM ryabonetse mu kurasa ikirahuri cy'ikirahuri cyometseho zahabu ya nanoparticles hamwe na lazeri ya buri mwaka (Ishusho 1f). Ikwirakwizwa ry'ubushyuhe buringaniye ryagaragaye ahantu hose hatwikiriwe na laser. Aka karere kashyizwe kuri 65 ° C, ubushyuhe bwiza bwo gukura. Hanze y'aka karere, ubushyuhe bwo kugabanuka busanzwe bugwa kuri \ (1 / r \) (aho \ (r \) ni umuhuzabikorwa wa radiyo).
ikarita yubushyuhe bwibipimo bya CGM byabonetse ukoresheje urumuri rwa lazeri yumwaka kugirango urabagirane urwego rwa zahabu nanoparticles kugirango ubone ubushyuhe buringaniye hejuru yumuzingi. b Isotherm yikarita yubushyuhe (a). Ibice bya lazeri bigereranwa nuruziga rudasanzwe. Ubushakashatsi bwasubiwemo kabiri (reba Ibikoresho by'inyongera, Ishusho S4).
Imikorere ya selile ya bagiteri yakurikiranwe amasaha menshi ikoresheje LA-HTM. Ku mutini. 3 yerekana intera yigihe cyamashusho ane yakuwe mumasaha 3 yiminota 20 (Filime M3, Amakuru yinyongera). Indwara ya bagiteri yagaragaye cyane mu gice cyizengurutswe na lazeri aho ubushyuhe bwari bwiza, bugera kuri 65 ° C. Ibinyuranye, imikurire ya selile yagabanutse cyane mugihe ubushyuhe bwagabanutse munsi ya 50 ° C kuri s 10.
Amashusho yimbitse ya bacteri ya G. stearothermophilus akura nyuma yo gushyushya lazeri mubihe bitandukanye, (a) t = 0 min, (b) 1 h 10 min, (c) 2 h 20 min, (d) 3 h 20 min, hanze 200 Yakuwe muri firime yumunota umwe (M3 film yatanzwe mumakuru yinyongera) hejuru yikarita yubushyuhe. Lazeri ifungura mugihe \ (t = 0 \). Isotherms yongewe kumashusho yimbaraga.
Kugirango turusheho kugereranya imikurire y'uturemangingo no guterwa n'ubushyuhe, twapimye ubwiyongere bwa biomass ya koloni zitandukanye za bagiteri zabanje kwigunga mu gice cya Filime M3 (Ishusho 4). Indwara ya bagiteri yatoranijwe mugitangira cyo gushiraho mini coloni igizwe (mCFU) irerekanwa mumashusho S6. Ibipimo byumye byafashwe hamwe na kamera ya CGM 48 yakoreshejwe mugushushanya ikwirakwizwa ryubushyuhe. Ubushobozi bwa CGM bwo gupima uburemere bwumye nubushyuhe nimbaraga za LA-HTM. Nkuko byari byitezwe, ubushyuhe bwinshi bwatumye imikurire ya bagiteri yihuta (Ishusho 4a). Nkuko bigaragara mu gice cya kabiri cyibiti ku gishushanyo cya 4b, gukura ku bushyuhe bwose bikurikira gukura gukabije, aho amakuru akoresha imikorere yerekana \ (m = {m} _ {0} {10} ^ {t / \ tau} + {{\ mbox {cst}}} \), aho \ (\ tau {{{{{\ rm {log}}}}}} 2 \) - igihe cyibisekuruza (cyangwa inshuro ebyiri), \ (g = 1 / \ tau \) - umuvuduko wo gukura (umubare wamacakubiri mugihe cyigihe). Ku mutini. 4c yerekana umuvuduko ukura nigihe cyigihe nkibikorwa byubushyuhe. MCFU ikura vuba irangwa no kuzura gukura nyuma yamasaha abiri, imyitwarire iteganijwe kubera ubwinshi bwa bagiteri (bisa nicyiciro gihagaze mumico ya kera ya fluid). Imiterere rusange \ (g \ ibumoso (T \ iburyo)) Huza amakuru ukoresheje icyitegererezo gikaridinali (Igicapo S5) 49 aho \ (\ ibumoso ({{G} _ {0} {; \; T}} _ {{\ min}}; {T} _ {{opt}} ; {T} _ {{\ max}} \ iburyo) \) = (0.70 ± 0.2; 40 ± 4; 65 ± 1.6; 67 ± 3) ° C, ibyo bikaba bihuza neza n’izindi ndangagaciro zavuzwe mu bitabo 49. Nubwo ibipimo biterwa nubushyuhe byororoka, umuvuduko mwinshi wo gukura wa \ ({G} _ {0} \) urashobora gutandukana mubushakashatsi bumwe nubundi (reba imibare S7-S9 na firime M4). Bitandukanye nubushyuhe bukwiranye nubushyuhe, bugomba kuba kuri bose, umuvuduko ntarengwa wubwiyongere bushingiye kumiterere yikigereranyo (kuboneka intungamubiri, intungamubiri za ogisijeni) muri geometrike yagaragaye.
imikurire ya Microbial ku bushyuhe butandukanye. mCFU: Ibice bigizwe na Miniature. Amakuru yakuwe muri videwo ya bagiteri imwe ikura mubushyuhe (firime M3). b Kimwe na (a), igipimo cya-logarithmic. c Igipimo cyubwiyongere \ (\ tau \) nigihe cyigihe \ (g \) kibarwa uhereye kumurongo ugaruka (b). Utubari twibeshya: ubushyuhe burenze mCFUs yagutse murwego rwo kureba mugihe cyo gukura. Ikosa rihagaritse umurongo: ikosa risubira inyuma.
Usibye gukura bisanzwe, bagiteri zimwe na zimwe zireremba mugihe cyo gushyushya laser, iyi ikaba ari imyitwarire iteganijwe kuri bagiteri hamwe na flagella. Filime M5 mumakuru yinyongera yerekana ibikorwa nkibi byo koga. Muri ubu bushakashatsi, imirasire imwe ya laser yakoreshejwe kugirango habeho ubushyuhe, nkuko bigaragara ku gishushanyo 1d, e na S3. Igicapo 5 cerekana amashusho abiri akurikirana yatoranijwe muri firime M5 yerekana ko bagiteri imwe yerekana icyerekezo mugihe izindi bagiteri zose zikomeza kugenda.
Ibihe bibiri (a) na (b) byerekana koga kwa bagiteri ebyiri zitandukanye zirangwa nuruziga. Amashusho yakuwe muri firime M5 (yatanzwe nkibikoresho byiyongera).
Ku bijyanye na G. stearothermophilus, kugenda kwa bagiteri (Ishusho 5) byatangiye amasegonda make nyuma yo gufungura urumuri rwa lazeri. Iri genzura ryibanda ku gisubizo cy’agateganyo cy’iyi mikorobe mvaruganda yiyongera ku bushyuhe, nkuko bimaze kugaragara na Mora n'abandi. 24. Ingingo ya moteri ya bagiteri ndetse na thermotaxis irashobora gushakishwa cyane ukoresheje LA-HTM.
Koga kwa mikorobe ntigomba kwitiranwa nubundi bwoko bwimikorere yumubiri, aribyo (i) icyerekezo cya Brownian, bigaragara ko ari akajagari katagira icyerekezo gifatika, (ii) convection 50 na thermophoresis 43, bigizwe no gutembera kwimuka kubushyuhe. icyiciro.
G. stearothermophilus izwiho ubushobozi bwo gukora spore idashobora kwihanganira cyane (spore formation) iyo ihuye nibidukikije bidukikije nkubwirinzi. Iyo ibidukikije byongeye kuba byiza, intanga zimera, zigakora selile nzima kandi zigakomeza gukura. Nubwo ubu buryo bwo gutera / kumera buzwi, ntabwo bwigeze buboneka mugihe nyacyo. Dukoresheje LA-HTM, turatanga raporo hano kwambere kwitegereza ibyabaye kumera muri G. stearothermophilus.
Ku mutini. 6a yerekana amashusho yatakaye yuburebure bwa optique (OT) yabonetse ukoresheje CGM igizwe na spore 13. Igihe cyose cyo gukusanya (15 h 6 min, \ (t = 0 \) - intangiriro yo gushyushya lazeri), 4 kuri 13 za spore zimaze kumera, mugihe gikurikiranye \ (t = 2 \) h, \ (3 \ ) h \ (10 \) ', \ (9 \) h \ (40 \)' na \ (11 \) h \ (30 \) '. Nubwo kimwe gusa muri ibyo bintu cyerekanwe ku gishushanyo cya 6, ibintu 4 byo kumera bishobora kugaragara muri firime ya M6 mubikoresho byiyongera. Igishimishije, kumera bisa nkaho bidashoboka: intanga zose ntizimera kandi ntizimera icyarimwe, nubwo impinduka zimwe zidukikije.
igihe-kigizwe n'amashusho 8 ya OT (kwibiza amavuta, 60x, 1.25 NA intego) na (b) ubwihindurize bwa biomass ya G. stearothermophilus agregate. c (b) Gushushanya ku gice cya kabiri cyo kugereranya kugirango ugaragaze umurongo wikigereranyo cyikura (umurongo ucagaguye).
Ku mutini. 6b, c yerekana biomass yabaturage ba selile murwego rwo kureba nkigikorwa cyigihe mugihe cyose cyo gukusanya amakuru. Kwangirika kwinshi kwa misa yumye byagaragaye kuri \ (t = 5 \) h mumitini. 6b, c, kubera gusohoka kwa selile zimwe murwego rwo kureba. Iterambere ryibi bintu bine ni \ (0.77 \ pm 0.1 \) h-1. Agaciro karenze umuvuduko wo gukura ujyanye nishusho 3.3 na 4, aho selile zikura bisanzwe. Impamvu yo kwiyongera kwiterambere rya G. stearothermophilus kuva spores ntisobanutse, ariko ibi bipimo byerekana inyungu za LA-HTM kandi bigakorera kurwego rumwe (cyangwa kurwego rumwe rwa mCFU) kugirango umenye byinshi kubyerekeranye nubuzima bwimikorere ya selile .
Kugirango turusheho kwerekana byinshi bya LA-HTM n'imikorere yayo ku bushyuhe bwinshi, twasuzumye imikurire ya Sulfolobus shibatae, hyperthermophilic acideophilique archaea ifite ubushyuhe bwiza bwo gukura bwa 80 ° C51. Ugereranije na G. stearothermophilus, izi archaea nazo zifite morphologie zitandukanye cyane, zisa na micron 1 (cocci) aho kuba inkoni ndende (bacilli).
Igicapo 7a kigizwe nubushakashatsi bwimbitse bwa optique ya S. shibatae mCFU yabonetse ukoresheje CGM (reba firime ya M7 yerekana ibikoresho byiyongera). Iyi mCFU ikura kuri 73 ° C, munsi yubushyuhe bwiza bwa 80 ° C, ariko mubushyuhe bwo gukura neza. Twabonye ibintu byinshi byacitse bituma mCFUs isa na micrograpes ya archaea nyuma yamasaha make. Uhereye kuri aya mashusho ya OT, biomass ya mCFU yapimwe mugihe kandi yerekanwe mubishusho 7b. Igishimishije, S. shibatae mCFUs yerekanye imikurire yumurongo aho gukura gukura kugaragara hamwe na G. stearothermophilus mCFUs. Habayeho ibiganiro bimaze igihe kinini 52 byerekeye imiterere yikura ryingirabuzimafatizo: mugihe ubushakashatsi bumwe buvuga ko umuvuduko wubwiyongere bwa mikorobe ugereranije nubunini bwazo (gukura kwerekanwa), ibindi byerekana umuvuduko uhoraho (gukura kumurongo cyangwa bilinear). Nkuko byasobanuwe na Tzur et al.53, gutandukanya imikurire ya exponential na (bi) bisaba umurongo wa <6% mubipimo bya biomass, bidashoboka kubuhanga bwinshi bwa QPM, ndetse harimo na interferometrie. Nkuko byasobanuwe na Tzur et al.53, gutandukanya imikurire ya exponential na (bi) bisaba umurongo wa <6% mubipimo bya biomass, bidashoboka kubuhanga bwinshi bwa QPM, ndetse harimo na interferometrie. Как ояяннили Цур и др.53, различение башонен чиного и (би) кигого роста требует точности <6% в измерениях биомассы, что недостижимо для большинствааов QPM, daже с еппрочов. Nkuko byasobanuwe na Zur et al.53, gutandukanya gukura kwerekanwa na (bi) kumurongo bisaba <6% byukuri mubipimo bya biomass, bikaba bitagerwaho muburyo bwinshi bwa QPM, ndetse hakoreshejwe interferometrie.Nkuko byasobanuwe na Zur n'abandi. 53, gutandukanya imikurire yerekana na (bi) kumurongo bisaba kutarenza 6% mubipimo bya biomass, ibyo bikaba bitagerwaho muburyo bwinshi bwa QPM, kabone niyo interferometrie ikoreshwa. CGM igera kuri ubu busobanuro hamwe na sub-pg mubipimo bya biomass36,48.
igihe-kigizwe n'amashusho 6 ya OT (kwibiza amavuta, 60x, intego ya NA 1.25) na (b) ubwihindurize bwa biomass ya CFU yapimwe na CGM. Reba firime M7 kubindi bisobanuro.
Iterambere ryiza cyane rya S. shibatae ntiryari ryitezwe kandi ntiriratangazwa. Ariko, gukura kwinshi guteganijwe, byibuze kuko mugihe kirenze, ibice byinshi bya 2, 4, 8, 16… selile bigomba kubaho. Twakekereje ko gukura kumurongo bishobora guterwa no kubuza ingirabuzimafatizo bitewe no gupakira ingirabuzimafatizo, nkuko imikurire y'utugari itinda kandi amaherezo ikagera aho isinziriye iyo ubwinshi bw'utugari buri hejuru cyane.
Twasoje tuganira ku ngingo eshanu zikurikira zishimishije: kugabanya ingano yubushyuhe, kugabanya inertie yumuriro, inyungu muri nanoparticles ya zahabu, inyungu za microscopi yicyiciro, hamwe nubushyuhe bushoboka LA-HTM ishobora gukoreshwa.
Ugereranije no gushyushya birwanya, gushyushya laser bikoreshwa mugutezimbere HTM bitanga ibyiza byinshi, ibyo turabigaragaza muri ubu bushakashatsi. By'umwihariko, mubitangazamakuru byamazi murwego rwo kureba microscope, ingano yo gushyushya ibikwa mubice bike (10 μ m) 3. Muri ubu buryo, gusa mikorobe zarebwa zirakora, mugihe izindi bagiteri zisinziriye kandi zirashobora gukoreshwa mugukomeza kwiga icyitegererezo - nta mpamvu yo guhindura icyitegererezo igihe cyose hagomba gusuzumwa ubushyuhe bushya. Byongeye kandi, gushyushya microscale bituma hasuzumwa mu buryo butaziguye ubushyuhe bwinshi: Igicapo 4c cyakuwe muri firime yamasaha 3 (Movie M3), ubusanzwe bisaba gutegura no gusuzuma ingero nyinshi - imwe kuri buri cyitegererezo kirimo kwigwa. y nubushyuhe bugaragaza umubare wiminsi mubigeragezo. Kugabanya amajwi ashyushye kandi bituma ibintu byose bikikije optique ya microscope, cyane cyane lens lens, mubushyuhe bwicyumba, kikaba aricyo kibazo gikomeye abaturage bahuye nacyo kugeza ubu. LA-HTM irashobora gukoreshwa ninzira iyo ari yo yose, harimo linzira yo kwibiza amavuta, kandi izaguma ku bushyuhe bwicyumba ndetse nubushyuhe bukabije murwego rwo kureba. Inzitizi nyamukuru yuburyo bwo gushyushya laser twatanze muri ubu bushakashatsi nuko selile zidakurikiza cyangwa zireremba zishobora kuba kure yumwanya wo kureba kandi bigoye kwiga. Igikorwa gishobora kuba ugukoresha lensike ntoya kugirango ugere ku bushyuhe bunini burenze microne magana. Uku kwitonda guherekezwa no kugabanuka kwikemurwa ryahantu, ariko niba intego ari ukwiga urujya n'uruza rw'ibinyabuzima, imiterere ihanitse ntigisabwa.
Igipimo cyigihe cyo gushyushya (no gukonjesha) sisitemu \ ({{{{{\ rm {\ tau}}}}}}}} _ {{{\ mbox {D}}}} \) biterwa nubunini bwayo, ukurikije amategeko \ ({{{({\ rm {\ tau}}}}}}} _ {{{\ mbox {D}}}} = {L} ^ {2} / D \), aho \ . urubanza, ikirahuri n'amazi Igipimo cya Diffusion \ (D \ hafi 2 \ inshuro {10} ^ {- 7} \) m2 / s) impinduka zubushyuhe, zirashobora gutegurwa Uku gushiraho ako kanya kuzamuka kwubushyuhe ntigabanya gusa igihe cyibigeragezo, ahubwo binemerera igihe nyacyo \ (t = 0 \) kubushakashatsi ubwo aribwo bwose bwingaruka zubushyuhe.
Uburyo twasabye burakoreshwa muburyo ubwo aribwo bwose bukurura urumuri (urugero, ingero z'ubucuruzi hamwe na ITO). Nyamara, nanoparticles ya zahabu irashobora gutanga kwinjiza cyane muri infragre no kwinjirira hasi murwego rugaragara, ibimenyetso byanyuma biranga inyungu zo kwitegereza neza muburyo bugaragara, cyane cyane iyo ukoresheje fluorescence. Byongeye kandi, zahabu ni biocompatable, chimique inert, ubwinshi bwa optique burashobora guhinduka kuva kuri 530 nm kugera hafi ya infragre, kandi gutegura icyitegererezo biroroshye kandi byubukungu29.
Transcopi ya grating wavefront microscopie (CGM) ntishobora gusa gushushanya ikarita yubushyuhe kuri microscale, ariko kandi ikanakurikirana biomass, bigatuma iba ingirakamaro cyane (niba atari ngombwa) ifatanije na LA-HTM. Mu myaka icumi ishize, ubundi buryo bwa microscopi yubushyuhe bwatejwe imbere, cyane cyane mubijyanye na bioimaging, kandi inyinshi murizo zisaba gukoresha ubushakashatsi bwangiza ubushyuhe bwa fluorescent 544.55. Nyamara, ubu buryo bwaranenzwe kandi raporo zimwe zapimye ihinduka ry’ubushyuhe budasanzwe muri selile, bishoboka ko biterwa nuko fluorescence iterwa nibintu byinshi bitari ubushyuhe. Byongeye kandi, probe nyinshi za fluorescent ntizihinduka mubushyuhe bwinshi. Kubwibyo, QPM na CGM byumwihariko byerekana ubushyuhe bwiza bwa microscopi tekinike yo kwiga ubuzima mubushyuhe bwinshi ukoresheje microscopi optique.
Ubushakashatsi bwakozwe na S. shibatae, butuye neza kuri 80 ° C, bwerekana ko LA-HTM ishobora gukoreshwa mukwiga hyperthermophile, ntabwo ari thermophile gusa. Ihame, nta karimbi k'ubushyuhe bushobora kugerwaho ukoresheje LA-HTM, ndetse n'ubushyuhe buri hejuru ya 100 ° C burashobora kugerwaho n'umuvuduko w'ikirere utabanje kubira, nkuko byagaragajwe n'itsinda ryacu rya 38 mu gukoresha imiti ya hydrothermal chimique ikoreshwa mu kirere. igitutu A. Lazeri ikoreshwa mugushyushya zahabu nanoparticles 40 muburyo bumwe. Niyo mpamvu, LA-HTM ifite ubushobozi bwo gukoreshwa mu kureba hyperthermophile itigeze ibaho hamwe na microscopi isanzwe ihanitse cyane (urugero nko guhangayikishwa n’ibidukikije).
Ubushakashatsi bwose bwakozwe hakoreshejwe microscope yakozwe murugo, harimo kumurika Köhler (hamwe na LED, M625L3, Thorlabs, 700 mW), ufite icyitegererezo hamwe nintoki za xy, intego (Olympus, 60x, 0.7 NA, ikirere, LUCPlanFLN60X cyangwa 60x, 1.25 NA, Amavuta . amakuru yerekanwe ku gishushanyo cya 5 (koga ya bagiteri). Gutandukanya ibiti bibiri ni 749 nm BrightLine (Semrock, FF749-SDi01). Akayunguruzo imbere ya kamera ni 694 ngufi ya filteri (FF02-694 / SP-25, Semrock). Lazeri ya titanium (Laser Verdi G10, 532 nm, 10 W, yavomye tsunami laser cavity, Spectra-Physics ku gishushanyo cya 2-5, ikomeza gusimburwa na Millenia laser, Spectraphysics 10 W, yavomye Mira laser cavity, Coherent, ku gishushanyo cya 2. -5). 6 na 7) zashyizwe kumurongo wumurongo wa \ 1152 pigiseli) yaguzwe muri Meadowlark Optics. Hologramamu yabazwe ukoresheje algorithm ya Gerchberg-Saxton nkuko byasobanuwe kuri 39.
Microscopi ya cross griting wavefront (CGM) nubuhanga bwa microscopi optique bushingiye ku guhuza ibice bibiri byo gutandukanya ibice (bizwi kandi no kwambuka umusaraba) ku ntera ya milimetero imwe na sensor isanzwe ya kamera. Urugero rusanzwe rwa CGM twakoresheje muri ubu bushakashatsi rwitwa enye-yumurambararo wa transvers shift interferometero (QLSI), aho kwambukiranya ibice bigizwe nimbaraga / icyiciro cyo kugenzura cyatangijwe kandi cyatanzwe na Primot et al. mumwaka wa 200034.Imirongo ihanamye kandi itambitse irema igicucu kimeze nkigicucu kuri sensor, kugoreka kwayo birashobora gutunganyirizwa mubare mugihe nyacyo kugirango ubone kugoreka kwa optique (cyangwa icyiciro gihwanye) cyerekana urumuri rwabaye. Iyo ikoreshejwe kuri microscope, kamera ya CGM irashobora kwerekana itandukaniro ryinzira nziza yikintu cyashushanijwe, kizwi kandi nkuburebure bwa optique (OT), hamwe na sensitivite kuri gahunda ya nanometero36. Mu bipimo byose bya CGM, kugirango ukureho inenge iyo ari yo yose igizwe n'ibikoresho bya optique cyangwa ibiti, ishusho y'ibanze ya OT igomba gufatwa kandi igakurwa ku mashusho ayo ari yo yose yakurikiyeho.
Ubushyuhe bwa microscopi bwakozwe hakoreshejwe kamera ya CGM nkuko byasobanuwe. 32. Muri make, gushyushya amazi bihindura igipimo cyayo cyangiritse, bigatera ingaruka zumuriro ugoreka ibyabaye. Uku kugoreka kumurongo byapimwe na CGM kandi bigatunganywa hakoreshejwe algorithm ya deconvolution kugirango haboneke ubushyuhe bwibipimo bitatu muburyo bwamazi. Niba nanoparticles ya zahabu igabanijwe neza murugero rwose, ikarita yubushyuhe irashobora gukorerwa ahantu hatarimo bagiteri kugirango itange amashusho meza, nibyo dukora rimwe na rimwe. Ishusho ya CGM yabonetse idashyushye (hamwe na laser yazimye) hanyuma ifatwa ahantu hamwe mumashusho hamwe na laser kuri.
Gupima misa yumye bigerwaho hifashishijwe kamera imwe ya CGM ikoreshwa mugushushanya ubushyuhe. Amashusho ya CGM yabonetse mukwimura byihuse icyitegererezo muri x na y mugihe cyo kwerekana nkuburyo bwo kugereranya inomomogeneité muri OT bitewe na bagiteri. Uhereye ku mashusho ya OT ya bagiteri, biomass yabonetse hakoreshejwe itsinda ry’amashusho hejuru y’ahantu hatoranijwe hakoreshejwe algorithm yo mu rugo ya Matlab (reba agace “Kode Numero”), ukurikije inzira yasobanuwe muri ref. 48. Muri make, dukoresha isano \ (m = {\ alpha} ^ {- 1} \ iint {{\ mbox {OT}}} \ ibumoso (x, y \ iburyo) {{\ mbox {d}} } x {{\ mbox {d}}} y \), aho \ ({{\ mbox {OT}}} \ ibumoso (x, y \ iburyo) \) ni ishusho yimbitse ya optique, \ (m \) ni uburemere bwumye kandi \ ({{{{{\ rm {\ alpha}}}}}}} \) ni ihoraho. Twahisemo \ ({{{{\ rm {\ alpha))))))) = 0.18 \) µm3 / pg, ni ibintu bisanzwe bihoraho kuri selile nzima.
Igipfukisho c'igifuniko cya mm 25 z'umurambararo na 150 µm z'ubugari busize hamwe na zahabu ya nanoparticles yashyizwe mu cyumba cya AttofluorTM (Thermofisher) hamwe na nanoparticles ya zahabu ireba hejuru. Geobacillus stearothermophilus yabanjirijwe ijoro ryose hagati ya LB (200 rpm, 60 ° C) mbere ya buri munsi wubushakashatsi. Igitonyanga cya 5 µl cyo guhagarikwa kwa G. stearothermophilus hamwe nubucucike bwa optique (OD) bwa 0.3 kugeza 0.5 yashyizwe kumpapuro zifunitse hamwe na nanoparticles ya zahabu. Hanyuma, igipfundikizo kizengurutswe cya mm 18 z'umurambararo hamwe n'umwobo wa mm 5 z'umurambararo hagati rwagabanutse ku gitonyanga, hanyuma 5 μl yo guhagarika bagiteri hamwe n'ubucucike bumwe bwakoreshejwe inshuro nyinshi hagati mu mwobo. Amariba ku gipfukisho yateguwe akurikije inzira yasobanuwe muri ref. 45 (reba Amakuru yinyongera kubindi bisobanuro). Noneho shyiramo ml 1 ya LB iciriritse mugipfundikizo kugirango wirinde amazi yumye. Igifuniko cya nyuma gishyirwa hejuru yumupfundikizo wurugero rwa Attofluor ™ kugirango wirinde guhumeka hagati mugihe cya incububasi. Kubushakashatsi bwo kumera, twakoresheje spores, nyuma yubushakashatsi busanzwe, rimwe na rimwe yatwikiriye igifuniko cyo hejuru. Uburyo busa bwakoreshejwe kugirango ubone Sulfolobus shibatae. Iminsi itatu (200 rpm, 75 ° C) yo guhinga mbere ya seri ya Thiobacillus yakozwe hagati ya 182 (DSMZ).
Ingero za zahabu nanoparticles zateguwe na micellar block copolymer lithographie. Iyi nzira yasobanuwe muburyo burambuye muri Chap. 60. Muri make, micelles ikubiyemo ion zahabu zashizwe hamwe no kuvanga copolymer na HAuCl4 muri toluene. Ibifuniko bisukuye byahise byinjizwa mubisubizo hanyuma bivurwa na irrasiyo ya UV imbere yumukozi ugabanya imbuto za zahabu. Hanyuma, imbuto za zahabu zahinzwe no guhuza igifuniko hamwe nigisubizo cyamazi cya KAuCl4 na etanolamine muminota 16, ibyo bikaba byaviriyemo gahunda ya quasi-periodic kandi ihuriweho cyane na nanoparticles ya zahabu itari serefegitura hafi ya infragre.
Guhindura interferogrammes kumashusho ya OT, twakoresheje algorithm yo murugo, nkuko bisobanuwe murirusange. 33 kandi iraboneka nkigikoresho cya Matlab mububiko rusange bukurikira: https://github.com/baffou/CGMprocess. Ipaki irashobora kubara ubukana namashusho ya OT ashingiye kuri interferogrammes zafashwe amajwi (harimo amashusho yerekana) hamwe nintera ya kamera.
Kugirango tubare icyiciro cyakoreshejwe kuri SLM kugirango tubone umwirondoro wubushyuhe watanzwe, twakoresheje algorithm yakozwe murugo mbere 39,42 iboneka mububiko rusange bukurikira: https://github.com/baffou/SLM_temperatureShaping. Iyinjiza nubushyuhe bwubushyuhe bwifuzwa, bushobora gushyirwaho muburyo bwa digitale cyangwa binyuze mumashusho ya monochrome bmp.
Kugabanya selile no gupima uburemere bwumye, twakoresheje algorithm ya Matlab yasohotse mububiko rusange bukurikira: https://github.com/baffou/CGM_magicWandSegmentation. Kuri buri shusho, uyikoresha agomba gukanda kuri bagiteri cyangwa mCFU yinyungu, guhindura sensibilité ya wand, no kwemeza guhitamo.
Kubindi bisobanuro kubijyanye nigishushanyo mbonera, reba Raporo yubushakashatsi bwibidukikije ifitanye isano niyi ngingo.
Amakuru ashyigikira ibisubizo byubu bushakashatsi araboneka kubanditsi babisabwe babisabye.
Inkomoko yinkomoko yakoreshejwe muri ubu bushakashatsi irambuye mu gice cya Methods, kandi verisiyo yo gukuramo irashobora gukurwa kuri https://github.com/baffou/ mububiko bukurikira: SLM_temperatureShaping, CGMprocess, na CGM_magicWandSegmentation.
Mehta, R., Singhal, P., Singh, H., Damle, D. & Sharma, AK Ubushishozi muri thermophile hamwe nibikorwa byabo byinshi. Mehta, R., Singhal, P., Singh, H., Damle, D. & Sharma, AK Ubushishozi muri thermophile hamwe nibikorwa byabo byinshi.Mehta, R., Singhal, P., Singh, H., Damle, D. na Sharma, AK Incamake ya thermophile hamwe nuburyo bukoreshwa. 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. na Sharma AK Gusobanukirwa byimbitse ya termofile hamwe nuburyo bwinshi bwo gukoresha.3 Ibinyabuzima 6, 81 (2016).
Igihe cyo kohereza: Nzeri-26-2022