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Thermophiles su ne ƙananan ƙwayoyin cuta waɗanda ke bunƙasa a yanayin zafi. Yin nazarin su zai iya ba da bayanai masu mahimmanci game da yadda rayuwa ta dace da matsananciyar yanayi. Duk da haka, yana da wuya a cimma yanayin zafi mai girma tare da na'urorin gani na al'ada. An ba da shawarar mafita na gida da yawa dangane da dumama wutar lantarki na gida, amma babu wata hanyar kasuwanci mai sauƙi. A cikin wannan takarda, mun gabatar da ra'ayi na microscale Laser dumama a kan na'ura mai ba da hanya tsakanin hanyoyin sadarwa don samar da yanayin zafi mai zafi don nazarin thermophile yayin kiyaye yanayin mai amfani. Microscale dumama a matsakaici Laser tsanani za a iya samu ta amfani da zinariya nanoparticle rufi substrate a matsayin biocompatible da ingantaccen haske absorber. Abubuwan da za a iya haifar da convection na ruwa na microscale, riƙewar tantanin halitta, da motsi na thermophoretic na centrifugal. An nuna hanyar a cikin nau'i biyu: (i) Geobacillus stearothermophilus, kwayar cutar thermophilic mai aiki wanda ke haifuwa a kimanin 65 ° C, wanda muka lura don tsiro, girma da yin iyo a karkashin microscale dumama; (ii) Thiobacillus sp., mafi kyawun yanayin hyperthermophilic archaea. da 80°C. Wannan aikin yana buɗe hanya don sauƙi da amintaccen lura da ƙananan ƙwayoyin cuta na thermophilic ta amfani da kayan aikin microscopy na zamani da araha.
Fiye da biliyoyin shekaru, rayuwa a duniya ta samo asali don dacewa da yanayin yanayin muhalli da yawa waɗanda wasu lokuta ana ɗaukar matsananci daga mahallin ɗan adam. Musamman ma, wasu kwayoyin halitta na thermophilic (kwayoyin cuta, archaea, fungi) da ake kira thermophiles suna bunƙasa a cikin yanayin zafin jiki daga 45 ° C zuwa 122 ° C1, 2, 3, 4. Thermophiles suna rayuwa a cikin yanayi daban-daban, irin su zurfin ruwa na hydrothermal vents, ruwan zafi mai zafi. ko yankunan volcanic. Binciken su ya haifar da sha'awa mai yawa a cikin 'yan shekarun da suka gabata don akalla dalilai biyu. Na farko, za mu iya koyo daga gare su, alal misali, yadda thermophiles 5, 6, enzymes 7, 8 da membranes 9 suke da ƙarfi a irin wannan yanayin zafi, ko kuma yadda thermophiles ke iya jure matsanancin matakan radiation10. Na biyu, su ne ginshiƙi da yawa muhimman aikace-aikace biotechnological1,11,12 kamar man fetur samar13,14,15,16, sinadaran kira (dihydro, alcohols, methane, amino acid, da dai sauransu)17, biomining18 da thermostable biocatalysts7 ,11, 13. Musamman, sanannen sanannen sarkar polymerase (PCR) 19 ya ƙunshi wani enzyme (Taq polymerase) wanda ya keɓe daga ƙwayar cuta mai zafi Thermus aquaticus, ɗayan thermophiles na farko da aka gano.
Koyaya, binciken thermophiles ba aiki bane mai sauƙi kuma ba za'a iya inganta shi ba a cikin kowane dakin gwaje-gwaje na halitta. Musamman, ba za a iya lura da ma'aunin zafi da sanyio a cikin vitro tare da kowane ma'aunin haske na haske ba, har ma da ɗakunan dumama na kasuwanci, yawanci ana ƙididdige yanayin zafi ƙasa da 40 ° C. Tun daga 1990s, ƙungiyoyin bincike kaɗan ne kawai suka sadaukar da kansu don ƙaddamar da tsarin ƙananan ƙananan zafin jiki (HTM). A cikin 1994 Glukh et al. An yi la'akari da ɗakin dumama / sanyaya bisa ga amfani da tantanin halitta na Peltier wanda ke sarrafa zafin jiki na capillaries rectangular rufe don kula da anaerobicity 20. Ana iya yin zafi da na'urar har zuwa 100 ° C a kan ƙimar 2 ° C/s, yana ba wa marubuta damar yin nazarin motsin ƙwayar cutar hyperthermophilic Thermotoga maritima21. A cikin 1999 Horn et al. An ƙirƙiri na'ura mai kama da ita, har yanzu ta dogara ne akan amfani da zazzafan capillaries masu dacewa da microscopy na kasuwanci don nazarin rabo/haɗin sel. Bayan dogon lokaci na rashin aiki na dangi, an ci gaba da neman ingantattun HTMs a cikin 2012, musamman dangane da jerin takaddun da kungiyar Wirth ta yi amfani da na'urar da Horn et al. Shekaru goma sha biyar da suka wuce, an yi nazarin motsi na babban adadin archaea, ciki har da hyperthermophiles, a yanayin zafi har zuwa 100 ° C ta amfani da capillaries masu zafi23,24. Sun kuma gyara na'urar microscope na asali don cimma saurin dumama (mintuna da yawa maimakon mintuna 35 don isa ga yanayin da aka saita) da cimma madaidaicin zafin jiki na sama da 2 cm a fadin matsakaici. An yi amfani da wannan na'urar siffa mai siffar zafin jiki (TGFD) don nazarin motsi na thermophiles da yawa a cikin ma'aunin zafin jiki a nesa mai dacewa da ilimin halitta 24, 25.
Dumama rufaffiyar capillaries ba ita ce kawai hanyar lura da rayayyun thermophiles ba. A cikin 2012, Kuwabara et al. An yi amfani da ɗakunan Pyrex da za a zubar da su a cikin gida da aka rufe da manne mai jurewa zafi (Super X2; Cemedine, Japan). An sanya samfuran akan farantin dumama na kasuwanci (Micro Heat Plate, Kamfanin Kitazato, Japan) mai iya dumama har zuwa 110 ° C, amma ba a yi niyya da asali don nazarin halittu ba. Marubutan sun lura da ingantaccen rabo na kwayoyin cutar anaerobic thermophilic (Thermosipho globiformans, sau biyu lokaci 24 min) a 65 ° C. A cikin 2020, Pulshen et al. An nuna ingantaccen dumama jita-jita na ƙarfe na kasuwanci (Attofluor TM, Thermofisher) ta amfani da abubuwa biyu na dumama na gida: murfi da mataki (na'urar da aka yi wahayi zuwa PCR). Wannan ƙungiyar tana haifar da daidaitaccen zafin jiki na ruwa kuma yana hana ƙazantar da ruwa a ƙasan murfi. Yin amfani da zobe na O-ring yana guje wa musayar gas tare da yanayi. An yi amfani da wannan HTM, wanda ake kira Sulfoscope, don yin hoton Sulfolobus acidocaldarius a 75°C27.
Ƙayyadaddun ƙayyadaddun ƙayyadaddun waɗannan tsarin shine ƙuntatawa ga amfani da manufofin iska, duk wani nutsewar mai bai dace da irin wannan zafin jiki ba kuma don yin hoto ta hanyar> 1-mm samfurori masu kauri. Ƙayyadaddun ƙayyadaddun ƙayyadaddun waɗannan tsarin shine ƙuntatawa ga amfani da manufofin iska, duk wani nutsewar mai bai dace da irin wannan zafin jiki ba kuma don yin hoto ta hanyar> 1-mm samfurori masu kauri. Общепризнаныm nedostatkom vsеh эtyh system bыlo ограничение на использование воздушныh объектов, е погружение в масло не подходило для такой высокой температуры. Rashin gazawar duk waɗannan tsarin shine ƙayyadaddun amfani da manufofin iska, tunda duk wani nutsewar mai bai dace da irin wannan yanayin zafi ba kuma don gani ta hanyar samfuran m> kauri 1 mm.所有这些系统的一个公认限制是限制使用空气物镜，任何油浸都不认限制是限制使用空气物镜。厚的透明样品成像。 Ƙayyadaddun ƙayyadaddun ƙayyadaddun waɗannan tsarin shine iyakancewar yin amfani da madubi mai iska, kamar yadda duk wani nutsewar mai bai dace ba don ɗaukar samfurori masu haske> 1 mm lokacin farin ciki a irin wannan yanayin zafi. Общепризнаныm nedostatkom vseh эtyh system yavlyatsya sãɓãwar launukansa, скачать видео - Alamar da aka sani na duk waɗannan tsarin shine iyakancewar amfani da ruwan tabarau na iska, duk wani nutsewar mai bai dace da irin wannan yanayin zafi da gani ba ta samfuran m> 1 mm lokacin farin ciki.Kwanan nan, Charles-Orzag et al ya ɗaga wannan iyakance. 28, wanda ya ƙera na'urar da ta daina ba da zafi a kusa da tsarin sha'awa, amma a cikin gilashin murfin kanta, an rufe shi da wani bakin ciki mai haske na resistor da aka yi da ITO (indium-tin oxide). Za a iya dumama murfi har zuwa 75 ° C ta hanyar wucewar wutar lantarki ta cikin fili mai haske. Duk da haka, marubucin dole ne kuma ya dumama ruwan tabarau zuwa manufa, amma kada ya wuce 65 ° C, don kada ya lalata shi.
Wadannan ayyukan sun nuna cewa ba a sami karbuwa sosai ba a ko'ina ba a yi amfani da ci gaban ingantaccen yanayin zafi na gani na gani ba, sau da yawa yana buƙatar kayan aikin gida, kuma ana samun sau da yawa a farashi na ƙudurin sararin samaniya, wanda shine babban hasara idan aka ba da cewa ƙananan ƙwayoyin thermophilic ba su da girma fiye da kaɗan. micrometers. Rage ƙarar dumama shine mabuɗin don magance matsaloli guda uku na HTM: ƙarancin ƙudurin sarari, babban zafin zafi lokacin da tsarin yayi zafi, da dumama abubuwa masu cutarwa (man nutsewa, ruwan tabarau na haƙiƙa… ko hannun mai amfani) a matsanancin zafi. ).
A cikin wannan takarda, mun gabatar da HTM don kallon thermophile wanda bai dogara da dumama mai juriya ba. Madadin haka, mun sami dumamar yanayi a cikin ƙayyadaddun yanki na filin kallon microscope ta hanyar saka iska mai ɗaukar haske na Laser. An hango rarraba zafin jiki ta hanyar amfani da ƙayyadaddun ƙayyadaddun lokaci (QPM). Ana nuna tasirin wannan hanyar ta hanyar Geobacillus stearothermophilus, ƙwayoyin cuta mai motsi na thermophilic wanda ke haifuwa a kusan 65 ° C kuma yana da ɗan gajeren lokaci sau biyu (kimanin mintuna 20), da Sulfolobus shibatae, hyperthermophile wanda ke tsiro da kyau a 80 ° C (archaea) don kwatanta. An lura da yawan kwafi na yau da kullun da yin iyo azaman yanayin zafin jiki. Wannan Laser HTM (LA-HTM) baya iyakance da kauri daga cikin murfin ko ta yanayin haƙiƙa (iska ko mai nutsewa). Wannan yana ba da damar yin amfani da kowane babban ruwan tabarau a kasuwa. Hakanan baya fama da jinkirin dumama saboda rashin kuzarin zafi (yana samun dumama nan take akan sikelin millisecond) kuma yana amfani da abubuwan kasuwanci kawai. Sabbin abubuwan da ke damun aminci kawai suna da alaƙa da kasancewar filayen filaye masu ƙarfi na Laser (yawanci har zuwa 100mW) a cikin na'urar kuma wataƙila ta idanu, waɗanda ke buƙatar tabarau masu kariya.
Ka'idar LA-HTM ita ce amfani da Laser don dumama samfurin a cikin gida a cikin filin kallon microscope (Fig. 1a). Don yin wannan, samfurin dole ne ya zama mai ɗaukar haske. Don amfani da m Laser ikon (kasa da 100 mW), ba mu dogara a kan sha na haske ta hanyar ruwa matsakaici, amma artificially ƙara sha na samfurin ta shafa substrate da zinariya nanoparticles (Fig. 1c). Dumama nanoparticles na zinariya tare da haske yana da mahimmancin mahimmanci ga filin plasmonics na thermal, tare da aikace-aikacen da ake sa ran a cikin biomedicine, nanochemistry ko girbin hasken rana29,30,31. A cikin 'yan shekarun da suka gabata, mun yi amfani da wannan LA-HTM a cikin bincike da yawa da suka shafi aikace-aikacen plasma na thermal a cikin ilimin lissafi, sunadarai da ilmin halitta. Babban wahala tare da wannan hanya shine a nuna bayanin martabar zafin jiki na ƙarshe, tun da girman zafin jiki yana iyakance ga yanki na microscale a cikin samfurin. Mun nuna cewa za a iya samun taswirar zafin jiki tare da interferometer mai juyi mai tsayi huɗu, hanya mai sauƙi, mai girma, kuma mai matukar mahimmanci na ƙirar ƙididdiga na lokaci mai ƙididdigewa dangane da amfani da nau'i mai nau'i biyu (wanda kuma aka sani da giciye gratings) 33,34,35,36. An tabbatar da amincin wannan dabarar maƙasudin ma'aunin zafi da sanyio, wanda ya danganta da ƙetaren ra'ayi na raƙuman raƙuman ruwa (CGM), a cikin takardu goma sha biyu da aka buga cikin shekaru goma da suka gabata37,38,39,40,41,42,43.
Tsarin shigar da dumama Laser daidaici, siffata da yanayin zafi. b Samfurin lissafi wanda ya ƙunshi ɗakin AttofluorTM mai ɗauke da murfin murfin da aka lulluɓe da nanoparticles na gwal. c Dubi samfurin (ba don sikelin ba). d yana wakiltar bayanin martabar katako na laser iri ɗaya da (e) da aka kwaikwayi rabon zafin jiki na gaba akan samfurin jirgin saman nanoparticles na gwal. f shine bayanin martabar katako na laser na shekara-shekara wanda ya dace don samar da daidaitaccen zafin jiki kamar yadda aka nuna a cikin kwaikwaiyon sakamakon rarraba zazzabi da aka nuna a cikin (g). Matsakaicin girman: 30µm.
Musamman, kwanan nan mun sami nasarar dumama ƙwayoyin dabbobi masu shayarwa tare da LA-HTM da CGM tare da bin diddigin martanin girgiza zafin salula a cikin kewayon 37-42°C, yana nuna fa'idar wannan dabara ga hoton tantanin halitta guda ɗaya. Duk da haka, aikace-aikacen LA-HTM don nazarin ƙananan ƙwayoyin cuta a yanayin zafi ba abin mamaki ba ne, saboda yana buƙatar ƙarin taka tsantsan idan aka kwatanta da kwayoyin dabbobi masu shayarwa: da farko, dumama ƙasa na matsakaici da dubun digiri (maimakon ƴan digiri) yana kaiwa. zuwa ƙaƙƙarfan gradient zafin jiki mai ƙarfi. zai iya haifar da convection na ruwa 44 wanda, idan ba a haɗe shi da substrate ba, zai iya haifar da motsi maras so da haɗuwa da kwayoyin cuta. Ana iya kawar da wannan convection ta hanyar rage kauri daga cikin ruwa. Don wannan dalili, a cikin duk gwaje-gwajen da aka gabatar a ƙasa, an sanya dakatarwar kwayan cuta tsakanin murfin biyu kamar 15 µm lokacin farin ciki da aka sanya a cikin kofin karfe (Attofluor TM, Thermofisher, Fig. 1b, c). A ka'ida, za a iya kauce wa convection idan kauri daga cikin ruwa ne karami fiye da katako girman Laser dumama. Abu na biyu, yin aiki a cikin irin wannan ƙayyadaddun lissafi na iya shaƙa kwayoyin halitta aerobic (duba siffa S2). Ana iya guje wa wannan matsala ta hanyar yin amfani da abin da zai iya shiga cikin iskar oxygen (ko duk wani iskar gas mai mahimmanci), ta hanyar barin kumfa mai kumfa a cikin murfin, ko ta hanyar hako ramuka a cikin murfin saman (duba hoto S1) 45 . A cikin wannan binciken, mun zaɓi mafita na ƙarshe (Figures 1b da S1). A karshe, Laser dumama ba ya samar da uniform zazzabi rarraba. Ko da a daidai wannan ƙarfin na laser (Fig. 1d), rarraba zafin jiki ba daidai ba ne, amma yayi kama da rarraba Gaussian saboda yaduwar thermal (Fig. 1e). Lokacin da makasudin shine don kafa madaidaicin yanayin zafi a fagen ra'ayi don nazarin tsarin ilimin halitta, bayanan martaba marasa daidaituwa ba su da kyau kuma suna iya haifar da motsi na thermophoretic na ƙwayoyin cuta idan ba su manne da substrate (duba siffa S3, S4)39. Don wannan karshen, mun yi amfani da na'ura mai haske na sararin samaniya (SLM) don tsara ƙirar laser infrared bisa ga siffar zobe (Fig. 1f) a cikin jirgin sama na samfurin don cimma daidaitaccen rarraba yanayin zafi a cikin yanki na geometric da aka ba da shi, duk da yaduwar zafi (Fig. 1d) 39, 42, 46. Sanya murfin saman saman a kan tasa na karfe (Hoto na 1b) don kauce wa ƙafewar matsakaici kuma kiyaye akalla 'yan kwanaki. Saboda ba a rufe wannan murfin saman ba, ana iya ƙara ƙarin matsakaici cikin sauƙi a kowane lokaci idan ya cancanta.
Don kwatanta yadda LA-HTM ke aiki da kuma nuna dacewarsa a cikin bincike na thermophilic, mun yi nazarin ƙwayoyin cuta aerobic Geobacillus stearothermophilus, waɗanda ke da mafi kyawun yanayin girma na kusan 60-65 ° C. Kwayar cutar kuma tana da flagella da ikon yin iyo, tana ba da wata alama ta al'ada ta salula.
Samfurori (Fig. 1b) an riga an haɗa su a 60 ° C na sa'a daya sannan a sanya su a cikin ma'aunin samfurin LA-HTM. Wannan pre-cubation na zaɓi ne, amma har yanzu yana da amfani, saboda dalilai guda biyu: Na farko, lokacin da aka kunna Laser, yana sa sel su girma da rarrabuwa nan da nan (duba fim ɗin M1 a cikin ƙarin kayan aiki). Ba tare da riga-kafi ba, yawanci ana jinkirin haɓakar ƙwayoyin cuta da kusan mintuna 40 a duk lokacin da sabon wurin kallo ya yi zafi akan samfurin. Na biyu, 1 hour pre-cubation inganta manne kwayoyin zuwa murfin, hana sel daga ficewar daga fagen gani saboda thermophoresis lokacin da Laser aka kunna (duba fim M2 a Ƙarin Materials). Thermophoresis shine motsi na barbashi ko kwayoyin halitta tare da yanayin zafin jiki, yawanci daga zafi zuwa sanyi, kuma ƙwayoyin cuta ba su da banbanci43,47. An kawar da wannan tasirin da ba a so a kan wani yanki da aka ba da shi ta hanyar amfani da SLM don tsara katako na Laser da kuma cimma daidaitattun rarraba zafin jiki.
A kan fig. Hoto 2 yana nuna yawan zafin jiki da aka auna ta hanyar CGM da aka samu ta hanyar haskaka gilashin gilashin da aka rufe da nanoparticles na zinari tare da katako na laser annular (Fig. 1f). An lura da rarraba zafin jiki mai lebur akan duk yankin da katakon Laser ya rufe. An saita wannan yanki zuwa 65 ° C, mafi kyawun zafin jiki na girma. A wajen wannan yanki, yanayin zafin jiki a zahiri ya faɗi zuwa \(1/r\) (inda \(r\) shine haɗin gwiwar radial).
taswirar zafin jiki na ma'aunin CGM da aka samu ta hanyar amfani da katako na laser na annular don haskaka wani Layer na nanoparticles na gwal don samun bayanin yanayin zafin jiki a kan wani yanki mai madauwari. b Isotherm na taswirar zafin jiki (a). Kwancen katakon Laser yana wakilta da da'irar launin toka mai digo. An maimaita gwajin sau biyu (duba Ƙarin Kayayyakin, Hoto S4).
An kula da yuwuwar ƙwayoyin ƙwayoyin cuta na sa'o'i da yawa ta amfani da LA-HTM. A kan fig. 3 yana nuna tazarar lokaci don hotuna huɗu da aka ɗauka daga fim ɗin awa 3 na mintuna 20 (Fim M3, Ƙarin Bayani). An lura da ƙwayoyin cuta suna yaduwa sosai a cikin yankin madauwari da aka ayyana ta hanyar laser inda zafin jiki ya kasance mafi kyau, yana gabatowa 65 ° C. Sabanin haka, haɓakar tantanin halitta ya ragu sosai lokacin da zafin jiki ya faɗi ƙasa da 50 ° C na 10 s.
Hotunan zurfin gani na kwayoyin G. stearothermophilus suna girma bayan dumama laser a lokuta daban-daban, (a) t = 0 min, (b) 1 h 10 min, (c) 2 h 20 min, (d) 3 h 20 min, daga 200 Ciro daga fim na minti ɗaya (fim ɗin M3 da aka bayar a Ƙarin Bayani) wanda aka sanya akan taswirar zafin jiki daidai. Laser yana kunna a lokaci \ (t=0 \). An ƙara Isotherms zuwa hoton tsanani.
Don ƙarin ƙididdige ci gaban tantanin halitta da dogaro da zafin jiki, mun auna haɓakar haɓakar halittu na yankuna daban-daban na ƙwayoyin cuta na farko a cikin filin kallo na Movie M3 (Fig. 4). Bakteriyar iyaye da aka zaɓa a farkon ƙirar ƙaramin yanki (mCFU) ana nuna su a hoto S6. An ɗauki ma'auni bushe tare da kyamarar CGM 48 wacce aka yi amfani da ita don taswirar rarraba zafin jiki. Ikon CGM don auna bushe bushe da zafin jiki shine ƙarfin LA-HTM. Kamar yadda ake tsammani, yawan zafin jiki ya haifar da saurin girma na ƙwayoyin cuta (Fig. 4a). Kamar yadda aka nuna a cikin siffa ta 4b, girma a duk yanayin zafi yana biye da girma mai girma, inda bayanai ke amfani da aikin juzu'i \(m={m}_{0}{10}^{t/\ tau}+ {{ \mbox{cst}}}\), inda \(\tau {{{{\rm{log }}}}}2\) - lokacin tsara (ko lokacin sau biyu), \( g =1/ \tau\) - ƙimar girma (yawan rarrabuwa a kowane lokaci naúrar). A kan fig. 4c yana nuna girman girman girma da lokacin tsarawa azaman aikin zafin jiki. MCFUs masu girma da sauri suna da alaƙa da jikewa na girma bayan sa'o'i biyu, halayen da ake tsammani saboda yawan ƙwayar ƙwayoyin cuta (mai kama da lokacin tsayawa a cikin al'adun ruwa na gargajiya). Siffar gabaɗaya \ (g \ hagu (T \ dama) \) (Fig. 4c) yayi daidai da yanayin da ake tsammani sau biyu don G. stearothermophilus tare da ƙimar girma mafi kyau a kusa da 60-65 ° C. Daidaita bayanan ta amfani da samfurin kadinal (Hoto S5)49 inda \(\hagu ({{G}_{0}{;\;T}}_{{\min }};{T}_{{opt}}) ; {T}_{{\max}} \ dama) \) = (0.70 ± 0.2; 40 ± 4; 65 ± 1.6; 67 ± 3) °C, wanda ya yi daidai da sauran dabi'u da aka ambata a cikin adabi49. Kodayake ma'auni masu dogaro da zafin jiki ana iya sakewa, matsakaicin girman girman \({G}_{0}\) na iya bambanta daga gwaji ɗaya zuwa wani (duba adadi S7-S9 da fim ɗin M4). Ya bambanta da sigogi masu dacewa da zafin jiki, wanda ya kamata ya zama na duniya, matsakaicin girman girma ya dogara da kaddarorin matsakaici (samuwar abubuwan gina jiki, ƙaddamar da iskar oxygen) a cikin ma'auni na microscale.
ci gaban ƙananan ƙwayoyin cuta a yanayin zafi daban-daban. mCFU: Ƙananan Ƙungiyoyin Ƙirƙirar Mulki. Bayanan da aka samo daga bidiyon kwayar cuta guda daya da ke girma a cikin yanayin zafi (fim M3). b Daidai da (a), ma'aunin Semi-logarithmic. c Ƙimar girma \ (\ tau \) da lokacin tsarawa \ (g \) ƙididdiga daga komawar layi (b). Matsakaicin kuskure a kwance: kewayon zafin jiki wanda mCFUs ya faɗaɗa cikin fagen kallo yayin girma. Sandunan Kuskuren Tsaye: Kuskuren mizani na mizani.
Baya ga ci gaban al'ada, wasu ƙwayoyin cuta a wasu lokuta suna shawagi zuwa gani yayin dumama Laser, wanda shine halayen da ake tsammani ga ƙwayoyin cuta tare da flagella. Fim ɗin M5 a cikin ƙarin bayani yana nuna irin waɗannan ayyukan ninkaya. A cikin wannan gwaji, an yi amfani da hasken laser iri ɗaya don ƙirƙirar yanayin zafin jiki, kamar yadda aka nuna a Figures 1d, e da S3. Hoto na 5 yana nuna jerin hotuna guda biyu da aka zaɓa daga fim ɗin M5 suna nuna cewa ƙwayoyin cuta guda ɗaya suna nuna motsin kwatance yayin da sauran ƙwayoyin cuta ba su da motsi.
Matsalolin lokaci guda biyu (a) da (b) suna nuna ninkaya na ƙwayoyin cuta daban-daban guda biyu masu alamar da'irar dige-dige. An ciro Hotunan daga fim ɗin M5 (wanda aka bayar azaman ƙarin kayan).
A cikin yanayin G. stearothermophilus, motsi mai aiki na ƙwayoyin cuta (Fig. 5) ya fara ƴan daƙiƙa kaɗan bayan an kunna katako na laser. Wannan abin lura yana jaddada martanin ɗan lokaci na wannan ƙananan ƙwayoyin cuta na thermophilic zuwa haɓakar zafin jiki, kamar yadda Mora et al ya rigaya ya lura. 24 . Za a iya ƙara bincika batun motsin ƙwayoyin cuta har ma da thermotaxis ta amfani da LA-HTM.
Bai kamata a rikita yin iyo na ƙananan ƙwayoyin cuta tare da wasu nau'ikan motsin jiki ba, wato (i) motsin Brownian, wanda ya bayyana motsi ne mai ruɗani ba tare da tabbataccen jagora ba, (ii) convection 50 da thermophoresis 43, wanda ya ƙunshi motsi na yau da kullun tare da zafin jiki. gradient.
G. stearothermophilus sananne ne don ikonsa na samar da ƙwanƙwasa masu juriya sosai (ƙirar spore) lokacin da aka fallasa yanayin yanayi mara kyau a matsayin tsaro. Lokacin da yanayin muhalli ya sake zama mai kyau, spores suna tsiro, suna samar da sel masu rai kuma suna dawo da girma. Ko da yake wannan tsari na sporulation/germination sananne ne, ba a taɓa ganin shi a ainihin lokaci ba. Yin amfani da LA-HTM, muna bayar da rahoto anan farkon abin lura da abubuwan da suka faru a cikin G. stearothermophilus.
A kan fig. 6a yana nuna hotuna na tsawon lokaci na zurfin gani (OT) da aka samu ta amfani da saitin CGM na 13 spores. Domin dukan lokacin tarin (15 h 6 min, \ (t = 0 \) - farkon dumama Laser), 4 daga cikin 13 spores germinated, a jere lokaci maki \ (t = 2 \) h, \ ( 3 \ ). h \(10 \)', \(9\) h \(40\)' da \(11\) h \(30\)'. Ko da yake ɗayan waɗannan abubuwan ne kawai aka nuna a cikin Hoto na 6, ana iya lura da abubuwan germination 4 a cikin fim ɗin M6 a cikin ƙarin kayan. Abin sha'awa shine, germination ya bayyana a matsayin bazuwar: ba duk spores ke tsiro ba kuma ba sa fitowa lokaci guda, duk da canje-canje iri ɗaya a yanayin muhalli.
Tsawon lokaci wanda ya ƙunshi hotunan OT 8 (nutsar da mai, 60x, 1.25 NA haƙiƙa) da (b) juyin halittar halittu na G. stearothermophilus aggregates. c (b) Zana a kan ma'auni na rabin log don haskaka layin girman girman girma (layin dage).
A kan fig. 6b,c yana nuna biomass na yawan adadin tantanin halitta a fagen gani a matsayin aikin lokaci na tsawon lokacin tattara bayanai. Rushewar busasshen busasshen taro da aka gani a \(t=5\) h a fig. 6b, c, saboda ficewar wasu sel daga fagen kallo. Yawan ci gaban waɗannan abubuwan guda huɗu shine \(0.77\pm 0.1 \) h-1. Wannan ƙimar ta fi girma girma da ke hade da Hoto 3. 3 da 4, inda sel ke girma kullum. Dalilin karuwar girma na G. stearothermophilus daga spores ba a sani ba, amma waɗannan ma'auni suna nuna sha'awar LA-HTM kuma suna aiki a matakin tantanin halitta guda ɗaya (ko a matakin mCFU ɗaya) don ƙarin koyo game da yanayin rayuwar tantanin halitta. .
Don ƙara nuna versatility na LA-HTM da aikinta a yanayin zafi mai girma, mun bincika girmar Sulfolobus shibatae, hyperthermophilic acidophilic archaea tare da ingantaccen zafin girma na 80 ° C51. Idan aka kwatanta da G. stearothermophilus, waɗannan archaea kuma suna da nau'in halittar jiki daban-daban, kama da 1 micron spheres (cocci) maimakon elongated sanduna (bacilli).
Hoto 7a ya ƙunshi jerin hotuna zurfin gani na S. shibatae mCFU da aka samu ta amfani da CGM (duba fim ɗin fasalin M7 a cikin Ƙarin Kayayyakin). Wannan mCFU yana girma a kusan 73 ° C, ƙasa da mafi kyawun zafin jiki na 80 ° C, amma a cikin kewayon zafin jiki don haɓaka aiki. Mun lura da al'amuran fission da yawa waɗanda suka sanya mCFUs suyi kama da micrograpes na archaea bayan 'yan sa'o'i. Daga waɗannan hotunan OT, mCFU biomass an auna tsawon lokaci kuma an gabatar da shi a cikin Hoto 7b. Abin sha'awa, S. shibatae mCFUs sun nuna haɓakar layi maimakon haɓakar haɓakar da aka gani tare da G. stearothermophilus mCFUs. An yi tattaunawa mai tsawo 52 game da yanayin haɓakar haɓakar ƙwayoyin sel: yayin da wasu nazarin ke ba da rahoton ƙimar girma na ƙananan ƙwayoyin cuta waɗanda suke daidai da girman su (girman girma), wasu suna nuna ƙimar ci gaba (ci gaban kai tsaye ko bilinear). Kamar yadda Tzur et al.53 ya bayyana, bambanta tsakanin haɓakawa da (bi) girma na layi yana buƙatar daidaitattun <6% a cikin ma'auni na biomass, wanda ba zai iya isa ga yawancin fasahohin QPM ba, har ma da shiga tsakani. Kamar yadda Tzur et al.53 ya bayyana, bambanta tsakanin haɓakawa da (bi) girma na layi yana buƙatar daidaitattun <6% a cikin ma'auni na biomass, wanda ba zai iya isa ga yawancin fasahohin QPM ba, har ma da shiga tsakani. Как объяснили Цур и др.53, Как объяснили Кур и др.53 остижимо для большинства методов QPM, даже с использованием интерферометрии. Kamar yadda Zur et al.53 ya bayyana, bambanta tsakanin girma da (bi) girma na layi yana buƙatar <6% daidaito a cikin ma'auni na biomass, wanda ba zai iya samuwa ga mafi yawan hanyoyin QPM ba, har ma da yin amfani da interferometry.Kamar yadda Zur et al. 53, bambance tsakanin girma da (bi) girma na layi yana buƙatar ƙasa da 6% daidaito a ma'aunin halitta, wanda ba zai iya samuwa ga yawancin hanyoyin QPM, koda lokacin da ake amfani da interferometry. CGM ta cimma wannan daidaito tare da daidaiton sub-pg a cikin ma'aunin biomass36,48.
Tsawon lokaci wanda ya ƙunshi hotuna 6 na OT (nutsar da mai, 60x, NA haƙiƙa 1.25) da (b) juyin halitta micro-CFU wanda aka auna tare da CGM. Duba fim ɗin M7 don ƙarin bayani.
Ci gaban madaidaiciyar layin S. shibatae ya kasance ba zato ba tsammani kuma har yanzu ba a ba da rahoton ba. Koyaya, ana tsammanin girma mai ma'ana, aƙalla saboda bayan lokaci, rarrabuwa da yawa na 2, 4, 8, 16… dole ne su faru. Mun yi hasashe cewa haɓakar layi ɗaya na iya kasancewa saboda hanawar tantanin halitta saboda tarin ƙwayoyin sel masu yawa, kamar yadda haɓakar tantanin halitta ke raguwa kuma a ƙarshe ya kai matsayi na barci lokacin da yawan ƙwayar tantanin halitta ya yi yawa.
Mun kammala ta hanyar tattauna abubuwan da ke biyo baya biyar na sha'awa bi da bi: raguwa a cikin ƙarar dumama, raguwa a cikin inertia na thermal, sha'awar nanoparticles na zinare, sha'awar microscopy na lokaci mai ƙididdigewa, da kuma yiwuwar zafin jiki wanda za'a iya amfani da LA-HTM.
Idan aka kwatanta da dumama mai juriya, dumama Laser da ake amfani da shi don haɓaka HTM yana ba da fa'idodi da yawa, waɗanda muka kwatanta a cikin wannan binciken. Musamman, a cikin kafofin watsa labarai na ruwa a fagen kallon na'urar gani, ana adana ƙarar dumama a cikin ƴan (10 μm) 3 kundin. Ta wannan hanyar, kawai ƙananan ƙwayoyin da aka lura suna aiki, yayin da sauran ƙwayoyin cuta suna barci kuma za a iya amfani da su don kara nazarin samfurin - babu buƙatar canza samfurin duk lokacin da sabon zafin jiki ya buƙaci a duba. Bugu da ƙari, microscale dumama yana ba da damar yin nazarin kai tsaye na babban yanayin yanayin zafi: An samo hoton 4c daga fim din 3-hour (Movie M3), wanda yawanci yana buƙatar shirye-shiryen da jarrabawar samfurori da yawa - daya ga kowane samfurin da aka yi nazari. y shine zafin jiki wanda ke wakiltar adadin kwanakin a cikin gwajin. Rage zafi mai zafi yana kuma adana duk abubuwan da ke kewaye da na'urar hangen nesa, musamman ma na'urar hangen nesa, a yanayin zafin dakin, wanda ya kasance babbar matsalar da al'umma ke fuskanta kawo yanzu. Ana iya amfani da LA-HTM tare da kowane ruwan tabarau, gami da ruwan tabarau na nutsewar mai, kuma za su kasance a cikin zafin jiki har ma da matsanancin zafi a fagen kallo. Babban ƙayyadaddun hanyar dumama Laser da muka ba da rahoto a cikin wannan binciken shine cewa ƙwayoyin da ba su bi ko yin iyo ba na iya yin nisa daga fagen kallo kuma suna da wahalar yin nazari. Matsakaicin abin da zai iya zama don amfani da ƙananan ruwan tabarau na haɓakawa don cimma haɓakar zafin jiki mafi girma fiye da 'yan microns ɗari kaɗan. Wannan taka tsantsan yana tare da raguwar ƙudurin sararin samaniya, amma idan makasudin shine nazarin motsi na ƙwayoyin cuta, ba a buƙatar babban ƙudurin sararin samaniya.
Ma'aunin lokacin dumama (da sanyaya) tsarin \({{{{\rm{\tau}}}}}}}_{{{\mbox{D}}}}\) ya dogara da girmansa, bisa ga doka \({{{({\rm{\tau }}}}}}_{{\mbox{D}}}}={L}^{2}/D\), inda \ (L \) shine yanayin yanayin yanayin zafi (diamita na katako na laser a cikin bincikenmu shine \ (L \ game da 100 \) μm), \ (D \) shine yanayin yanayin zafi (matsakaici a cikin mu). case, gilashin da ruwa Yadawa rate\(D \ game da 2 \ ninka {10}^{-7}\) m2/s). Canje-canjen yanayin zafi, ana iya tsammanin wannan kafawar haɓakar zafin jiki ba kawai yana rage tsawon lokacin gwajin ba, har ma yana ba da damar daidaitaccen lokacin \(t=0\) don kowane bincike mai ƙarfi na tasirin zafin jiki.
Hanyar da muka gabatar tana amfani da kowane abu mai ɗaukar haske (misali, samfuran kasuwanci tare da murfin ITO). Duk da haka, ƙananan nanoparticles na zinariya suna iya samar da haɓaka mai girma a cikin infrared da ƙananan sha a cikin kewayon da ake iya gani, halayen na ƙarshe na abin da ke da sha'awa don tasiri mai mahimmanci na gani a cikin bayyane, musamman lokacin amfani da haske. Bugu da kari, zinariya ne bioocompatible, chemically inert, Tantancewar yawa za a iya gyara daga 530 nm zuwa kusa da infrared, da samfurin shiri ne mai sauki da kuma tattalin arziki29.
Matsakaicin raƙuman raƙuman ruwa (CGM) yana ba da damar yin taswirar zafin jiki ba kawai a microscale ba, har ma da saka idanu na biomass, yana sa ya zama mai amfani musamman (idan ba dole ba) a hade tare da LA-HTM. A cikin shekaru goma da suka gabata, an ƙirƙiri wasu fasahohin ƙididdiga na yanayin zafin jiki, musamman a fannin nazarin halittu, kuma galibinsu suna buƙatar amfani da na'urorin bincike masu zafin jiki54,55. Duk da haka, an soki waɗannan hanyoyin kuma wasu rahotanni sun auna canje-canjen yanayin zafi a cikin sel, mai yiwuwa saboda gaskiyar cewa haske ya dogara da abubuwa da yawa ban da zafin jiki. Bugu da kari, mafi yawan masu kyalli ba su da kwanciyar hankali a yanayin zafi. Sabili da haka, QPM da musamman CGM suna wakiltar fasaha mai mahimmanci na yanayin zafi don nazarin rayuwa a yanayin zafi mai zafi ta amfani da microscopy na gani.
Nazarin S. shibatae, wanda ke rayuwa mafi kyau a 80 ° C, ya nuna cewa ana iya amfani da LA-HTM don nazarin hyperthermophiles, ba kawai ƙananan thermophiles ba. A ka'ida, babu iyaka ga kewayon yanayin zafi da za a iya isa ta amfani da LA-HTM, har ma da yanayin zafi sama da 100 ° C za a iya isa a matsa lamba na yanayi ba tare da tafasa ba, kamar yadda ƙungiyarmu ta 38 ta nuna a aikace-aikacen sunadarai na hydrothermal a yanayi. matsa lamba A. Ana amfani da Laser don dumama gwal nanoparticles 40 a cikin hanya guda. Don haka, LA-HTM yana da yuwuwar a yi amfani da shi don lura da hyperthermophiles waɗanda ba a taɓa ganin irin su ba tare da madaidaicin babban ƙayyadaddun ƙirar gani a ƙarƙashin daidaitattun yanayi (watau ƙarƙashin damuwa na muhalli).
An yi duk gwaje-gwajen ta amfani da na'urar microscope na gida, gami da hasken Köhler (tare da LED, M625L3, Thorlabs, 700 mW), mariƙin samfurin tare da motsin xy na hannu, maƙasudi (Olympus, 60x, 0.7 NA, iska, LUCPlanFLN60X ko 60x, 1.25 NA , UPLFLN60XOI), CGM kamara (QLSI giciye grating, 39 µm pitch, 0.87 mm daga Andor Zyla firikwensin kamara) don samar da tsanani da kuma wavefront hoto, da sCMOS kamara (ORCA Flash 4.0 V3, 16-bit yanayin, daga Hamamatsu) don yin rikodin bayanai da aka nuna a Hoto na 5 (wanka na kwayan cuta). Dichroic beam splitter shine 749 nm BrightLine gefen (Semrock, FF749-SDi01). Tace a gaban kamara shine matattarar gajeriyar wucewa ta 694 (FF02-694/SP-25, Semrock). Titanium sapphire Laser (Laser Verdi G10, 532 nm, 10 W, famfo tsunami Laser cavity, Spectra-Physics a cikin siffa 2-5, kara maye gurbinsu da Millenia Laser, Spectraphysics 10 W, famfo Mira Laser cavity, Coherent, don siffa 2. -5). 6 da 7) an saita zuwa tsayin raƙuman raƙuman ruwa \({{({\rm{\lambda }}}}}}=800\) nm, wanda yayi dai-dai da bakan bakan plasmon resonance na gwal. 1152 pixels) an siyi su daga Meadowlark Optics.
Cross grating wavefront microscopy (CGM) dabara ce ta duban gani da ido dangane da haɗa grating mai girma biyu (wanda kuma aka sani da giciye grating) a nesa na millimita ɗaya daga firikwensin kyamara na al'ada. Misali mafi mahimmanci na CGM wanda muka yi amfani da shi a cikin wannan binciken ana kiransa mai tsaka-tsakin tsaka-tsakin motsi mai tsayi hudu (QLSI), inda giciye-giciye ya ƙunshi nau'i mai ƙarfi / lokaci-lokaci checkerboard wanda Primot et al ya gabatar da haƙƙin mallaka. a cikin 200034. Layukan grating na tsaye da a kwance suna haifar da grid-kamar inuwa akan firikwensin, murdiya wanda za'a iya sarrafa su ta ƙididdigewa a ainihin lokacin don samun murdiya na gani (ko bayanin martaba na lokaci daidai) na hasken abin da ya faru. Lokacin amfani da na'ura mai kwakwalwa, kyamarar CGM na iya nuna bambancin hanyar gani na wani abu da aka zana, wanda kuma aka sani da zurfin gani (OT), tare da hankali akan tsari na nanometers36. A cikin kowane ma'aunin CGM, don kawar da duk wani lahani a cikin kayan aikin gani ko katako, dole ne a ɗauki hoton OT na farko kuma a cire shi daga kowane hotuna na gaba.
An yi nazarin yanayin zafi ta amfani da kyamarar CGM kamar yadda aka bayyana a cikin tunani. 32. A taƙaice, dumama ruwa yana canza maƙasudinsa na refractive, yana haifar da tasirin ruwan tabarau na thermal wanda ke karkatar da hasken abin da ya faru. Ana auna wannan murdiya ta gaban igiyar ruwa ta CGM kuma ana sarrafa ta ta amfani da algorithm na deconvolution don samun rarraba zafin jiki mai girma uku a cikin matsakaicin ruwa. Idan an rarraba nanoparticles na zinariya a ko'ina cikin samfurin, za a iya yin taswirar zafin jiki a wuraren da ba tare da kwayoyin cuta ba don samar da hotuna mafi kyau, wanda shine abin da muke yi a wasu lokuta. Hoton CGM da aka ambata an samo shi ba tare da dumama ba (tare da kashe laser) kuma daga baya an kama shi a wuri ɗaya a cikin hoton tare da laser a kunne.
Ana samun ma'aunin bushewa ta amfani da kyamarar CGM iri ɗaya da aka yi amfani da ita don hoton zafin jiki. Hotunan ma'anar CGM an samo su ta hanyar motsi da sauri samfurin a cikin x da y yayin da ake nunawa a matsayin hanyar da za a iya kwatanta duk wani rashin daidaituwa a cikin OT saboda kasancewar kwayoyin cuta. Daga hotunan OT na ƙwayoyin cuta, an samo asalin halittarsu ta hanyar amfani da tarin hotuna akan wuraren da aka zaɓa ta amfani da algorithm rarrabuwar gida na Matlab (duba sashe na “Lambobin Lambobi”), bin hanyar da aka bayyana a ref. 48. A takaice, muna amfani da dangantakar \(m={\alpha}^{-1}\int {{\mbox{OT}} \ hagu(x,y\right){{\mbox{d}} } x{{\mbox{d}}}y\), inda \({{\mbox{OT}}} \ hagu(x,y \ dama)\) shine hoton zurfin gani, \(m\) shine busasshen nauyi da kuma \({{{{\rm{\alpha }}}}}\) akai akai. Mun zaɓi \({{{\rm{\alpha))))))=0.18\) µm3/pg, wanda shine madaidaicin sel masu rai.
An sanya murfin murfin 25 mm a diamita da kauri 150 µm mai rufi da nanoparticles na gwal a cikin ɗakin AttofluorTM (Thermofisher) tare da nanoparticles na gwal suna fuskantar sama. Geobacillus stearothermophilus an riga an tsara shi cikin dare a matsakaicin LB (200 rpm, 60°C) kafin kowace rana ta gwaji. Digo na 5 µl na dakatarwar G. stearothermophilus tare da ƙarancin gani (OD) na 0.3 zuwa 0.5 an sanya shi akan ɗigon murfin tare da gwanayen gwal. Sa'an nan kuma, an jefar da murfin zagaye na 18 mm a diamita tare da rami 5 mm a diamita a tsakiya a kan digo, kuma 5 μl na dakatarwar kwayan cuta tare da nau'in gani iri ɗaya an maimaita akai-akai zuwa tsakiyar ramin. An shirya rijiyoyin da ke kan murfi daidai da tsarin da aka bayyana a cikin ref. 45 (duba Ƙarin Bayani don ƙarin bayani). Sa'an nan kuma ƙara 1 ml na matsakaicin LB zuwa murfin murfin don hana ruwan ruwa daga bushewa. Rufaffen murfin ƙarshe ana sanya shi akan rufaffiyar murfi na ɗakin Attofluor™ don hana ƙafewar matsakaici yayin shiryawa. Don gwaje-gwajen germination, mun yi amfani da spores, wanda, bayan gwaje-gwaje na al'ada, wani lokaci ya rufe murfin saman. An yi amfani da irin wannan hanyar don samun Sulfolobus shibatae. Kwanaki uku (200 rpm, 75 ° C) na farko na noman Thiobacillus serrata an gudanar da su a matsakaicin 182 (DSMZ).
Samfurori na gwal nanoparticles an shirya su ta hanyar micellar block copolymer lithography. An kwatanta wannan tsari daki-daki a Chap. 60. A taƙaice, micelles encapsulating zinariya ions aka hada ta hanyar hada copolymer da HAuCl4 a cikin toluene. Sa'an nan kuma an nutsar da murfin da aka tsaftace a cikin maganin kuma a bi da su tare da hasken UV a gaban wakili mai rage don samun tsaba na zinariya. A ƙarshe, an shuka tsaba na zinariya ta hanyar tuntuɓar murfin murfin tare da maganin ruwa mai ruwa na KAuCl4 da ethanolamine na tsawon mintuna 16, wanda ya haifar da tsari na lokaci-lokaci kuma daidaitaccen tsari na nanoparticles na gwal a cikin infrared na kusa.
Don canza interferograms zuwa hotunan OT, mun yi amfani da algorithm na gida, kamar yadda aka yi dalla-dalla a cikin mahaɗin. 33 kuma yana samuwa azaman kunshin Matlab a cikin ma'ajiyar jama'a mai zuwa: https://github.com/baffou/CGMprocess. Kunshin zai iya ƙididdige ƙarfi da hotunan OT bisa ga rikodin interferograms (gami da hotunan tunani) da nisan tsararrun kyamara.
Don ƙididdige tsarin tsarin da aka yi amfani da shi ga SLM don samun bayanin martabar zafin jiki, mun yi amfani da algorithm39,42 da aka ƙera a baya wanda ke samuwa a cikin ma'ajiyar jama'a mai zuwa: https://github.com/baffou/SLM_temperatureShaping. Shigarwar ita ce filin zafin da ake so, wanda za'a iya saita shi ta lambobi ko ta hoton bmp monochrome.
Don rarraba sel da auna busassun nauyin su, mun yi amfani da algorithm na Matlab da aka buga a cikin ma'ajiyar jama'a mai zuwa: https://github.com/baffou/CGM_magicWandSegmentation. A kan kowane hoto, dole ne mai amfani ya danna kan kwayoyin cuta ko mCFU na sha'awa, daidaita ma'aunin wand, kuma tabbatar da zaɓin.
Don ƙarin bayani game da ƙira na nazari, duba Ƙididdigar Rahoton Bincike mai alaƙa da wannan labarin.
Ana samun bayanan da ke goyan bayan sakamakon wannan binciken daga marubutan bisa ga buƙatu mai ma'ana.
Ƙididdigar lambar tushe da aka yi amfani da ita a cikin wannan binciken an yi cikakken bayani a cikin sashin Hanyoyi, kuma za a iya sauke nau'in gyara kuskure daga https://github.com/baffou/ a cikin ma'ajiyar masu zuwa: SLM_temperatureShaping, CGMprocess, da CGM_magicWandSegmentation.
Mehta, R., Singhal, P., Singh, H., Damle, D. & Sharma, AK Insight cikin thermophiles da faffadan aikace-aikacensu. Mehta, R., Singhal, P., Singh, H., Damle, D. & Sharma, AK Insight cikin thermophiles da faffadan aikace-aikacensu.Mehta, R., Singhal, P., Singh, H., Damle, D. da Sharma, AK Bayani na thermophiles da faffadan aikace-aikacen su. 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. da Sharma AK Zurfafa fahimtar thermophiles da aikace-aikace iri-iri.3 Biotechnology 6, 81 (2016).
Lokacin aikawa: Satumba-26-2022