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Thermophiles ndi tizilombo tating'onoting'ono tomwe timakula bwino pakatentha kwambiri. Kuwawerenga kungapereke chidziwitso chofunikira cha momwe moyo umasinthira ku mikhalidwe yovuta kwambiri. Komabe, ndizovuta kukwaniritsa kutentha kwambiri ndi ma microscopes owoneka bwino. Njira zingapo zopangira kunyumba zotengera kutenthetsa kwamagetsi kwapanyumba zaperekedwa, koma palibe njira yosavuta yochitira malonda. Mu pepala ili, tikuyambitsa lingaliro la kutentha kwa laser ya microscale pamwamba pa malo owonera maikulosikopu kuti apereke kutentha kwakukulu kwa maphunziro a thermophile ndikusunga chilengedwe cha wosuta. Kutentha kwa Microscale pamlingo wocheperako wa laser kumatha kutheka pogwiritsa ntchito gawo lapansi lagolide lokutidwa ndi nanoparticle monga choyezera chowunikira komanso chothandiza. Zotsatira zomwe zingatheke za microscale fluid convection, retention cell, ndi centrifugal thermophoretic motion zimakambidwa. Njirayi yawonetsedwa mu mitundu iwiri: (i) Geobacillus stearothermophilus, bakiteriya yogwira ntchito ya thermophilic yomwe imabereka pafupifupi 65 ° C, yomwe tawona kuti imamera, kukula ndi kusambira pansi pa kutentha kwazing'ono; (ii) Thiobacillus sp., optimally hyperthermophilic archaea. pa 80°C. Ntchitoyi imatsegula njira yowunikira kosavuta komanso kotetezeka kwa tizilombo tating'onoting'ono ta thermophilic pogwiritsa ntchito zida zamakono komanso zotsika mtengo zowonera ma microscopy.
Kwa zaka mabiliyoni ambiri, zamoyo Padziko Lapansi zasintha kuti zigwirizane ndi zochitika zosiyanasiyana zachilengedwe zomwe nthawi zina zimaonedwa kuti ndizovuta kwambiri malinga ndi momwe timaonera anthu. Makamaka, tizilombo tating'onoting'ono ta thermophilic (mabakiteriya, archaea, bowa) otchedwa thermophiles timakula bwino mu kutentha kwapakati pa 45 ° C mpaka 122 ° C1, 2, 3, 4. Thermophiles amakhala m'malo osiyanasiyana, monga nyanja yakuya ya hydrothermal vents, akasupe otentha. kapena madera ophulika. Kafukufuku wawo wabweretsa chidwi chachikulu pazaka makumi angapo zapitazi pazifukwa zosachepera ziwiri. Choyamba, tingaphunzire kuchokera kwa iwo, mwachitsanzo, momwe ma thermophiles 5, 6, michere 7, 8 ndi nembanemba 9 ali okhazikika pa kutentha kotereku, kapena momwe ma thermophiles amatha kupirira milingo yayikulu kwambiri ya radiation10. Chachiwiri, ndiwo maziko a ntchito zambiri zofunika pazachilengedwe1,11,12 monga kupanga mafuta13,14,15,16, kaphatikizidwe ka mankhwala (dihydro, alcohols, methane, amino acid, etc.)17, biomining18 and thermostable biocatalysts7 ,11, 13. Makamaka, polymerase chain reaction (PCR)19 yodziwika bwino yomwe panopo imakhudza enzyme (Taq polymerase) yomwe ili kutali ndi thermophilic bacterium Thermus aquaticus, imodzi mwa ma thermophiles oyamba kupezeka.
Komabe, kuphunzira za thermophiles si ntchito yophweka ndipo sikungapangidwe bwino mu labotale iliyonse yazachilengedwe. Makamaka, ma thermophiles okhala ndi moyo sangathe kuwonedwa mu vitro ndi maikulosikopu yopepuka yanthawi zonse, ngakhale ndi zipinda zotenthetsera zomwe zimapezeka pamalonda, zomwe nthawi zambiri zimayikidwa kuti zizitentha mpaka 40°C. Kuyambira m'zaka za m'ma 1990, magulu ofufuza ochepa okha ndi omwe adadzipereka kuti akhazikitse machitidwe a microscope (HTM) apamwamba kwambiri. Mu 1994 Glukh et al. Chipinda chotenthetsera / chozizira chinakhazikitsidwa pogwiritsa ntchito selo la Peltier lomwe limayang'anira kutentha kwa ma capillaries a rectangular otsekedwa kuti asunge anaerobicity 20. Chipangizocho chikhoza kutenthedwa mpaka 100 ° C pa mlingo wa 2 ° C / s, kulola olemba kuti aphunzire motility ya hyperthermophilic bacteria Thermotoga maritima21. Mu 1999 Horn et al. Chida chofanana kwambiri chapangidwa, chokhazikika pakugwiritsa ntchito ma capillaries otentha oyenera ma microscopy amalonda kuti aphunzire kugawanika kwa ma cell / kulumikizana. Pambuyo pa nthawi yayitali yosagwira ntchito, kufufuza kwa ma HTM ogwira mtima kunayambiranso mu 2012, makamaka pokhudzana ndi mapepala angapo a gulu la Wirth lomwe linagwiritsa ntchito chipangizo chopangidwa ndi Horn et al. Zaka khumi ndi zisanu zapitazo, kusuntha kwa chiwerengero chachikulu cha archaea, kuphatikizapo hyperthermophiles, chinaphunziridwa pa kutentha mpaka 100 ° C pogwiritsa ntchito capillaries23,24. Anasinthanso maikulosikopu yoyambirira kuti akwaniritse kutentha mwachangu (mphindi zingapo m'malo mwa mphindi 35 kuti afikire kutentha komwe adayikidwa) ndikukwaniritsa kutentha kwamtundu wopitilira 2 cm kudutsa sing'anga. Chida ichi cha kutentha kwa gradient shape (TGFD) chagwiritsidwa ntchito pophunzira kuyenda kwa ma thermophiles ambiri mkati mwa kutentha kwapakati pa mtunda wogwirizana ndi biologically 24, 25.
Kutenthetsa ma capillaries otsekedwa si njira yokhayo yowonera ma thermophiles amoyo. Mu 2012, Kuwabara et al. Zipinda zopangira kunyumba za Pyrex zomata zomatira zosagwira kutentha (Super X2; Cemedine, Japan) zidagwiritsidwa ntchito. Zitsanzozi zinayikidwa pa mbale yotentha yowonekera yomwe ikupezeka pamalonda (Micro Heat Plate, Kitazato Corporation, Japan) yomwe imatha kutentha mpaka 110 ° C, koma osati poyamba kuti apange bioimaging. Olembawo adawona kugawanika koyenera kwa mabakiteriya a anaerobic thermophilic (Thermosipho globiformans, kuwirikiza nthawi 24 min) pa 65 ° C. Mu 2020, Pulshen et al. Kutentha koyenera kwa mbale zazitsulo zamalonda (Attofluor TM, Thermofisher) kunasonyezedwa pogwiritsa ntchito zinthu ziwiri zopangira zopangira: chivindikiro ndi siteji (makonzedwe opangidwa ndi makina a PCR). Kuyanjana kumeneku kumabweretsa kutentha kwamadzimadzi komwe kumalepheretsa kutuluka kwamadzi ndi condensation pansi pa chivindikirocho. Kugwiritsa ntchito mphete ya O kumapewa kusinthanitsa gasi ndi chilengedwe. HTM iyi, yotchedwa Sulfoscope, idagwiritsidwa ntchito kujambula Sulfolobus acidocaldarius pa 75°C27.
Kuletsa kozindikirika kwa machitidwe onsewa kunali kuletsa kugwiritsa ntchito zolinga za mpweya, kumizidwa kulikonse kwamafuta kukhala kosayenera kutentha kotereku komanso kujambula kudzera pazitsanzo zowoneka bwino za> 1-mm. Kuletsa kozindikirika kwa machitidwe onsewa kunali kuletsa kugwiritsa ntchito zolinga za mpweya, kumizidwa kulikonse kwamafuta kukhala kosayenera kutentha kotereku komanso kujambula kudzera pazitsanzo zowoneka bwino za> 1-mm. Momwe mungapangire zolemba zanu pakompyuta yanu, zomwe zimakupatsani mwayi woti mugwiritse ntchito bwino. для такой высокой температуры и для визуализации через прозрачные образцы толщиной > 1 мм. Kulephera kodziwika kwa machitidwe onsewa kunali kuchepetsa kugwiritsa ntchito zolinga za mpweya, chifukwa kumizidwa kulikonse kwa mafuta sikunali koyenera kutentha kwakukulu koteroko komanso kuwonetsera kupyolera mu zitsanzo zowonekera> 1 mm wandiweyani.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 的 的 的 的 的 的 的 的 的 的 的 的 的 的 的 的 Cholepheretsa chodziwika cha machitidwe onsewa ndikuchepetsa kugwiritsa ntchito galasi lokhala ndi mpweya, chifukwa kumizidwa kulikonse kwamafuta sikuli koyenera kuyerekeza zitsanzo zowonekera> 1 mm wandiweyani pa kutentha kotere. Werengani zambiri za momwe mungapangire zolemba zanu pakompyuta yanu, zomwe zikugwirizana ndi zosowa zanu. dziwani momwe mungapangire zolemba zanu > 1 мм. Chodziwika bwino cha machitidwe onsewa ndikugwiritsa ntchito pang'ono kwa ma lens a mpweya, kumizidwa kulikonse kwamafuta sikuli koyenera kutentha kotereku komanso kuwonekera kudzera mu zitsanzo zowonekera> 1 mm wandiweyani.Posachedwapa, izi zidachotsedwa ndi Charles-Orzag et al. 28, yemwe adapanga chipangizo chomwe sichimaperekanso kutentha kuzungulira dongosolo lachidwi, koma mkati mwa galasi lophimba lokha, lophimbidwa ndi wosanjikiza wochepa thupi wa resistor wopangidwa ndi ITO (indium-tin oxide). Chivundikirocho chikhoza kutenthedwa mpaka 75 ° C podutsa mphamvu yamagetsi kupyola munsanjika woonekera. Komabe, wolembayo ayeneranso kutenthetsa mandala ku cholinga, koma osapitirira 65 ° C, kuti asawononge.
Ntchito zimenezi zikusonyeza kuti chitukuko imayenera mkulu-kutentha kuwala kuwala kwa microscopy sichinayambe anatengera ambiri, nthawi zambiri amafuna zipangizo kunyumba, ndipo nthawi zambiri zimatheka pa mtengo wa kusamvana malo, amene ali kuipa kwambiri chifukwa thermophilic tizilombo si zazikulu kuposa ochepa. ma micrometer. Kuchepetsa kutentha kwamphamvu ndiye chinsinsi chothetsera mavuto atatu a HTM: kusasunthika bwino kwa malo, kutentha kwambiri pakatenthetsa makina, ndi kutentha koyipa kwa zinthu zozungulira (mafuta omiza, magalasi owoneka bwino ... kapena manja a ogwiritsa ntchito) pakutentha kwambiri. ).
Mu pepala ili, tikuyambitsa HTM yowonera thermophile yomwe siinakhazikike pakuwotcha kwamphamvu. M'malo mwake, tidapeza zotenthetsera zamtundu wina mkati mwa gawo laling'ono la malo owonera maikulosikopu pogwiritsa ntchito kuwala kwa laser kwa gawo lapansi loyamwa kuwala. Kugawa kwa kutentha kunawonetsedwa pogwiritsa ntchito quantitative phase microscopy (QPM). Kugwira ntchito kwa njirayi kumasonyezedwa ndi Geobacillus stearothermophilus, bakiteriya yotentha ya thermophilic yomwe imabereka pafupifupi 65 ° C ndipo imakhala ndi nthawi yochepa yowirikiza (pafupifupi mphindi 20), ndi Sulfolobus shibatae, hyperthermophile yomwe imakula bwino pa 80 ° C (archaea). kufotokoza. Kuthamanga kwachibadwa ndi kusambira kunawonedwa ngati ntchito ya kutentha. Laser HTM (LA-HTM) iyi siili malire ndi makulidwe a chivundikirocho kapena ndi chikhalidwe cha cholinga (kumiza mpweya kapena mafuta). Izi zimalola magalasi aliwonse apamwamba pamsika kuti agwiritsidwe ntchito. Simavutikanso ndi kutentha pang'onopang'ono chifukwa cha kutenthedwa kwa kutentha (imapeza kutentha pompopompo pa sikelo ya millisecond) ndipo imagwiritsa ntchito zigawo zomwe zimapezeka pamalonda. Zomwe zili zatsopano zokhudzana ndi chitetezo ndizokhudzana ndi kukhalapo kwa matabwa amphamvu a laser (nthawi zambiri mpaka 100 mW) mkati mwa chipangizocho ndipo mwina kudzera m'maso, omwe amafunikira magalasi oteteza.
Mfundo ya LA-HTM ndiyo kugwiritsa ntchito laser kutentha chitsanzo m'deralo mkati mwa malo owonera microscope (mkuyu 1a). Kuti tichite izi, chitsanzocho chiyenera kukhala chopepuka. Kugwiritsa ntchito wololera laser mphamvu (zosakwana 100 mW), sitinadalira mayamwidwe kuwala ndi sing'anga madzi, koma chongopeka anawonjezera mayamwidwe chitsanzo ndi ❖ kuyanika gawo lapansi ndi nanoparticles golide (mkuyu. 1c). Kutenthetsa ma nanoparticles agolide ndi kuwala ndikofunikira kwambiri kumunda wamafuta a plasmonics, omwe akuyembekezeka kugwiritsidwa ntchito mu biomedicine, nanochemistry kapena kukolola kwa dzuwa29,30,31. Pazaka zingapo zapitazi, tagwiritsa ntchito LA-HTM iyi m'maphunziro angapo okhudzana ndi kugwiritsa ntchito mafuta a plasma mufizikiki, chemistry ndi biology. Chovuta chachikulu ndi njirayi ndikuwonetsa mbiri yomaliza ya kutentha, popeza kutentha kwapamwamba kumangokhala kudera laling'ono mkati mwa chitsanzo. Tawonetsa kuti mapu a kutentha amatha kupindula ndi ma interferometer a kutalika kwa mafunde anayi, njira yosavuta, yosasunthika, komanso yovuta kwambiri ya microscopy ya gawo la magawo awiri pogwiritsa ntchito ma gratings awiri a diffraction (omwe amadziwikanso kuti cross gratings) 33,34,35,36. Kudalirika kwa njira yopangira ma microscopy yotenthetsera iyi, yozikidwa pa mawotchi owonera mafunde amphamvu (CGM), zawonetsedwa m'mapepala khumi ndi awiri omwe adasindikizidwa zaka khumi zapitazi37,38,39,40,41,42,43.
Dongosolo la kukhazikitsa kwa parallel laser Kutentha, kuumba ndi microscope kutentha. b Zitsanzo za geometry zokhala ndi chipinda cha Attofluor TM chomwe chili ndi chophimba chokhala ndi nanoparticles zagolide. c Yang'anani mosamalitsa chitsanzo (osati kukulitsa). d imayimira mbiri yofananira ya mtengo wa laser ndi (e) kugawa kofananira kotsatira kwa kutentha pa ndege ya nanoparticles yagolide. f ndi mbiri yamtengo wa laser wa annular yoyenera kupanga kutentha kofanana monga momwe zikuwonetsedwera poyerekezera ndi kugawa kwa kutentha komwe kukuwonetsedwa mu (g). Scale bar: 30 µm.
Makamaka, posachedwapa tapeza kutentha kwa ma cell a mammalian ndi LA-HTM ndi CGM ndikutsata mayankho a kutentha kwa ma cell amtundu wa 37-42 ° C, kuwonetsa kugwiritsidwa ntchito kwa njirayi pamaganizidwe a cell amodzi. Komabe, kugwiritsa ntchito LA-HTM pophunzira tizilombo pa kutentha kwakukulu sikumveka bwino, chifukwa kumafuna kusamala kwambiri poyerekeza ndi maselo a mammalian: choyamba, kutentha pansi pa sing'anga ndi makumi a madigiri (osati madigiri ochepa) amatsogolera. kutsika kwamphamvu kowongoka kwa kutentha. imatha kupanga convection 44 yamadzimadzi, yomwe, ngati sichinaphatikizidwe mwamphamvu ku gawo lapansi, ingayambitse kusuntha kosafunikira ndikusakanikirana kwa mabakiteriya. Convection iyi imatha kuthetsedwa pochepetsa makulidwe amadzimadzi. Pachifukwa ichi, muzoyesera zonse zomwe zaperekedwa pansipa, kuyimitsidwa kwa mabakiteriya kunayikidwa pakati pa zophimba ziwiri pafupifupi 15 µm wandiweyani woyikidwa mkati mwa kapu yachitsulo (Attofluor TM, Thermofisher, Fig. 1b, c). M'malo mwake, convection imatha kupewedwa ngati makulidwe amadzimadzi ndi ang'onoang'ono kuposa kukula kwa mtengo wa laser. Kachiwiri, kugwira ntchito mu geometry yochepa yotere kumatha kusokoneza zamoyo za aerobic (onani mkuyu. S2). Vutoli likhoza kupewedwa pogwiritsa ntchito gawo lapansi lomwe limatha kutulutsa mpweya (kapena mpweya wina uliwonse wofunikira), mwa kusiya mavuvu a mpweya wotsekedwa mkati mwa chivundikirocho, kapena pobowola mabowo pamwamba pa chivundikiro chapamwamba (onani mkuyu S1) 45 . Mu phunziro ili, tasankha njira yomaliza (Zithunzi 1b ndi S1). Pomaliza, kutentha kwa laser sikumapereka kutentha kofananako. Ngakhalenso mphamvu yomweyo ya laser mtengo (mkuyu 1d), kugawa kutentha si yunifolomu, koma amafanana ndi Gaussian kugawa chifukwa matenthedwe kufalikira (mkuyu 1e). Pamene cholinga ndikukhazikitsa kutentha koyenera m'mawonedwe ophunzirira machitidwe achilengedwe, ma profiles osagwirizana sali abwino ndipo angayambitsenso kayendedwe ka thermophoretic kwa mabakiteriya ngati satsatira gawo lapansi (onani mkuyu S3, S4)39. Kuti tichite zimenezi, tinagwiritsa ntchito malo kuwala modulator (SLM) kuumba infuraredi laser mtengo malinga ndi mawonekedwe a mphete (Fig. 1f) mu ndege ya chitsanzo tikwaniritse mwangwiro yunifolomu kutentha kugawa mkati mwa malo opatsidwa geometric, ngakhale kufalikira kwa kutentha (mkuyu 1d) 39, 42, 46. Ikani chophimba pamwamba pa mbale yachitsulo (Chithunzi 1b) kuti mupewe kutuluka kwa sing'anga ndikusunga kwa masiku osachepera. Chifukwa chophimba chakumtundachi sichimasindikizidwa, sing'anga yowonjezera imatha kuwonjezeredwa nthawi iliyonse ngati kuli kofunikira.
Kuti tiwonetse momwe LA-HTM imagwirira ntchito ndikuwonetsa momwe imagwirira ntchito mu kafukufuku wa thermophilic, tidaphunzira za mabakiteriya a aerobic Geobacillus stearothermophilus, omwe ali ndi kutentha kwabwino kwambiri kozungulira 60-65 ° C. Bakiteriyayi imakhalanso ndi flagella komanso imatha kusambira, zomwe zimapereka chizindikiro china cha zochitika zama cell.
Zitsanzo (mkuyu 1b) zidakonzedweratu pa 60 ° C kwa ola limodzi ndikuyikidwa mu LA-HTM chotengera chitsanzo. Izi zisanachitike makulitsidwe ndizosankha, komabe zothandiza, pazifukwa ziwiri: Choyamba, laser ikatsegulidwa, imapangitsa kuti ma cell akule ndikugawa (onani kanema M1 mu Supplementary Materials). Popanda kukulitsidwa, kukula kwa bakiteriya kumachedwa pafupifupi mphindi 40 nthawi iliyonse malo owonera atsopano akatenthedwa pachitsanzocho. Chachiwiri, ola la 1 lisanayambe kukhazikitsidwa limalimbikitsa kumamatira kwa mabakiteriya ku chivundikiro, kuteteza maselo kuti asatuluke m'munda chifukwa cha thermophoresis pamene laser inatsegulidwa (onani filimu M2 mu Supplementary Materials). Thermophoresis ndi kayendedwe ka tinthu tating'ono kapena mamolekyu mozungulira kutentha, nthawi zambiri kuchokera kotentha mpaka kuzizira, ndipo mabakiteriya ndi chimodzimodzi43,47. Zotsatira zosayenerazi zimachotsedwa pamalo operekedwa pogwiritsa ntchito SLM kupanga mtengo wa laser ndikukwaniritsa kugawa kwa kutentha.
Pa mkuyu. Chithunzi 2 chikuwonetsa kugawa kwa kutentha komwe kumayesedwa ndi CGM yopezedwa mwa kuyatsa gawo lapansi lagalasi lokutidwa ndi ma nanoparticles agolide okhala ndi mtengo wa laser annular (mkuyu 1f). Kugawika kwa kutentha kwapansi kunkawoneka pamalo onse ophimbidwa ndi mtengo wa laser. Chigawochi chinakhazikitsidwa ku 65 ° C, kutentha koyenera kukula. Kunja kwa derali, kutentha kwapakati kumagwera mwachibadwa ku \(1/r\) (komwe \(r\) ndi njira yozungulira).
Mapu a Kutentha a miyeso ya CGM yomwe idapezedwa pogwiritsa ntchito mtengo wa laser annular kuti iwunikire golide wosanjikiza wa nanoparticles kuti mupeze mawonekedwe osalala a kutentha pamalo ozungulira. b Isotherm ya mapu a kutentha (a). Kuzungulira kwa mtengo wa laser kumayimiridwa ndi bwalo la madontho imvi. Kuyesera kunabwerezedwa kawiri (onani Zowonjezera Zowonjezera, Chithunzi S4).
Kutheka kwa maselo a bakiteriya kunayang'aniridwa kwa maola angapo pogwiritsa ntchito LA-HTM. Pa mkuyu. 3 ikuwonetsa nthawi yofikira zithunzi zinayi zotengedwa mu kanema wa mphindi 3 ndi mphindi 20 (Movie M3, Zowonjezera Zowonjezera). Mabakiteriya adawonedwa kuti akuchulukirachulukira mkati mwa malo ozungulira omwe amafotokozedwa ndi laser pomwe kutentha kunali koyenera, kuyandikira 65 ° C. Mosiyana ndi zimenezi, kukula kwa maselo kunachepetsedwa kwambiri pamene kutentha kunatsika pansi pa 50 ° C kwa 10 s.
Zithunzi zozama za mabakiteriya a G. stearothermophilus omwe amakula pambuyo pa kutentha kwa laser nthawi zosiyanasiyana, (a) t = 0 min, (b) 1 h 10 min, (c) 2 h 20 min, (d) 3 h 20 min, kuchokera 200 Yotengedwa mufilimu ya mphindi imodzi (filimu ya M3 yoperekedwa mu Supplementary Information) yomwe ili pamwamba pa mapu a kutentha. Laser imayatsa nthawi \(t=0\). Ma Isotherm awonjezedwa ku chithunzi champhamvu.
Kuwonjezera quanticate selo kukula ndi kudalira kutentha, ife anayeza kuwonjezeka zotsalira zazomera zosiyanasiyana madera a mabakiteriya poyamba akutali mu Movie M3 munda view (mkuyu. 4). Mabakiteriya amakolo omwe amasankhidwa poyambira kupanga mini colony forming unit (mCFU) akuwonetsedwa pazithunzi S6. Miyezo youma yowuma idatengedwa ndi kamera ya CGM 48 yomwe idagwiritsidwa ntchito kupanga mapu kugawa kwa kutentha. Kukhoza kwa CGM kuyeza kulemera kowuma ndi kutentha ndi mphamvu ya LA-HTM. Monga kuyembekezera, kutentha kwakukulu kunayambitsa kukula kwa bakiteriya (mkuyu. 4a). Monga momwe zikusonyezedwera mu semi-log plot mu Mkuyu 4b, kukula pa kutentha kulikonse kumatsatira kukula kwachitsanzo, kumene deta imagwiritsa ntchito exponential function \(m={m}_{0}{10}^{t/\ tau }+ {{\mbox{cst}}}}\), kumene \(\tau {{{{\rm{log }}}}}}}2\) - nthawi ya m'badwo (kapena kuwirikiza nthawi), \( g =1/ \tau\) - kukula (chiwerengero cha magawo pa nthawi ya unit). Pa mkuyu. 4c ikuwonetsa kuchuluka kwa kukula ndi nthawi yobadwa ngati ntchito ya kutentha. Ma mCFU omwe akukula mwachangu amadziwikiranso ndikukula pakatha maola awiri, zomwe zimayembekezereka chifukwa chakuchulukirachulukira kwa mabakiteriya (ofanana ndi gawo loyima la zikhalidwe zamadzimadzi). Mawonekedwe ambiri \ (g \ kumanzere (T \ kumanja) \) (Mkuyu 4c) amafanana ndi mayendedwe a magawo awiri omwe amayembekezeredwa kwa G. stearothermophilus ndi kukula kwabwino kozungulira 60-65 ° C. Fananizani deta pogwiritsa ntchito cardinal model (Chithunzi S5)49 pamene \(\ kumanzere({{G}_{0}{;\;T}}_{{\ min }};{T}_{{opt} } } ;{T}_{{\max}}\ kumanja)\) = (0.70 ± 0.2; 40 ± 4; 65 ± 1.6; 67 ± 3) °C, zomwe zimagwirizana bwino ndi mfundo zina zotchulidwa m'mabuku49. Ngakhale magawo odalira kutentha amatha kubwerezedwa, kukula kwakukulu kwa \({G}_{0}\) kungasiyane kuchokera kuyesa kumodzi kupita kwina (onani zithunzi S7-S9 ndi kanema M4). Mosiyana ndi magawo oyenerera kutentha, omwe amayenera kukhala padziko lonse lapansi, kukula kwakukulu kumadalira zomwe zili mkati (kukhalapo kwa zakudya, kusakanikirana kwa okosijeni) mkati mwa geometry ya microscale.
kukula kwa tizilombo tosiyanasiyana pa kutentha kosiyanasiyana. mCFU: Miniature Colony Forming Units. Deta yopezedwa kuchokera mu kanema wa bakiteriya imodzi yomwe ikukula mu kutentha pang'ono (filimu M3). b Zofanana ndi (a), semi-logarithmic sikelo. c Mlingo wa kukula \(\ tau\) ndi nthawi ya mbadwo \ (g\) yowerengedwa kuchokera ku mzere wa mzere (b). Mipiringidzo yopingasa yopingasa: kusiyanasiyana kwa kutentha komwe ma mCFU adakulirakulira m'malo owonera pakukula. Mipiringidzo yolakwika yoyima: cholakwika chokhazikika pamizere.
Kuphatikiza pa kukula kwabwinobwino, mabakiteriya ena nthawi zina amayandama pakuwotcha kwa laser, zomwe zimayembekezereka kwa mabakiteriya okhala ndi flagella. Kanema wa M5 muzowonjezera zikuwonetsa zochitika zosambira zoterezi. Pakuyesaku, ma radiation a laser ayunifolomu adagwiritsidwa ntchito popanga kutentha kwa kutentha, monga momwe tawonetsera pa Zithunzi 1d, e ndi S3. Chithunzi 5 chikuwonetsa magawo awiri azithunzi omwe adasankhidwa mu kanema wa M5 akuwonetsa kuti bakiteriya imodzi imawonetsa kusuntha komwe mabakiteriya ena onse amakhala osasuntha.
Mafelemu awiri a nthawi (a) ndi (b) amasonyeza kusambira kwa mabakiteriya awiri osiyana omwe ali ndi madontho. Zithunzizo zidachotsedwa mu kanema wa M5 (zoperekedwa ngati zowonjezera).
Pankhani ya G. stearothermophilus, kayendetsedwe kake ka mabakiteriya (mkuyu 5) kunayamba masekondi angapo pambuyo poti mtengo wa laser unatsegulidwa. Kuwonetsetsa uku kukugogomezera kuyankha kwakanthawi kwa kachilombo ka thermophilic pakuwonjezeka kwa kutentha, monga momwe Mora et al. 24 . Mutu wa bakiteriya motility ngakhale thermotaxis ukhoza kufufuzidwanso pogwiritsa ntchito LA-HTM.
Kusambira kwa tizilombo toyambitsa matenda sikuyenera kusokonezedwa ndi mitundu ina ya kayendedwe ka thupi, monga (i) Kuyenda kwa Brownian, komwe kumawoneka ngati kusuntha kwachisokonezo popanda njira yeniyeni, (ii) convection 50 ndi thermophoresis 43, yomwe imakhala yoyendayenda nthawi zonse ndi kutentha. gradient.
G. stearothermophilus imadziwika chifukwa cha kuthekera kwake kutulutsa spore zosamva bwino (mapangidwe a spore) akakumana ndi zovuta zachilengedwe ngati chitetezo. Zinthu zachilengedwe zikakhalanso bwino, njerezo zimamera n’kupanga maselo amoyo n’kuyambanso kukula. Ngakhale kuti sporulation / kumera kumeneku kumadziwika bwino, sikunayambe kuwonedwa mu nthawi yeniyeni. Pogwiritsa ntchito LA-HTM, timapereka lipoti pano kuyang'ana koyamba kwa zochitika za kumera mu G. stearothermophilus.
Pa mkuyu. 6a ikuwonetsa zithunzi zanthawi yayitali zakuzama kwa kuwala (OT) zopezedwa pogwiritsa ntchito CGM seti ya 13 spores. Pa nthawi yonse yosonkhanitsa (15 h 6 min, \(t=0\) - chiyambi cha kutentha kwa laser), 4 mwa 13 spores amamera, pa nthawi zotsatizana \(t=2\) h, \( 3\ ) h \(10 \)', \(9\) h \(40\)' ndi \(11\) h \(30\)'. Ngakhale chimodzi chokha mwazochitikazi chikuwonetsedwa mu Chithunzi 6, zochitika 4 za kumera zitha kuwonedwa mu kanema wa M6 muzowonjezera. Chochititsa chidwi n'chakuti, kumera kumawoneka ngati kwachisawawa: si mbewu zonse zomwe zimamera ndipo sizimamera nthawi imodzi, ngakhale kusintha komweko kwa chilengedwe.
a Kutha kwa nthawi yokhala ndi zithunzi za 8 OT (kumiza mafuta, 60x, 1.25 NA cholinga) ndi (b) biomass evolution ya G. stearothermophilus aggregates. c (b) Kujambula pa sikelo ya chipika kuti muwonetse mzere wa kukula (mzere wodutsa).
Pa mkuyu. 6b,c ikuwonetsa kuchuluka kwa ma cell m'malo owonera ngati ntchito yanthawi munthawi yonse yosonkhanitsira deta. Kuwola kofulumira kwa misa yowuma kumawonedwa pa \(t=5\)h mkuyu. 6b, c, chifukwa cha kutuluka kwa maselo ena kuchokera kumalo owonera. Kukula kwa zochitika zinayizi ndi \(0.77\pm 0.1\) h-1. Mtengo uwu ndi wapamwamba kusiyana ndi kukula komwe kumagwirizanitsidwa ndi Chithunzi 3. 3 ndi 4, kumene maselo amakula bwino. Chifukwa cha kuchuluka kwa kukula kwa G. stearothermophilus kuchokera ku spores sichidziwika bwino, koma miyeso iyi imasonyeza chidwi cha LA-HTM ndikugwira ntchito pa selo limodzi (kapena pa mlingo umodzi wa mCFU) kuti mudziwe zambiri za kayendetsedwe ka moyo wa selo. .
Kuti tiwonetsenso kusinthasintha kwa LA-HTM ndi momwe amachitira pa kutentha kwakukulu, tinayang'ana kukula kwa Sulfolobus shibatae, hyperthermophilic acidophilic archaea ndi kutentha kwakukulu kwa 80 ° C51. Poyerekeza ndi G. stearothermophilus, archaea awa alinso ndi morphology yosiyana kwambiri, yofanana ndi 1 micron spheres (cocci) osati ndodo zazitali (bacilli).
Chithunzi 7a chimakhala ndi zithunzi zotsatizana zakuya za S. shibatae mCFU zopezedwa pogwiritsa ntchito CGM (onani filimu ya M7 mu Supplementary Materials). MCFU iyi imakula mozungulira 73 ° C, pansi pa kutentha koyenera kwa 80 ° C, koma mkati mwa kutentha kwa kukula kogwira ntchito. Tidawona zochitika zingapo za fission zomwe zidapangitsa ma mCFU kuwoneka ngati ma micrograpes a archaea patatha maola angapo. Kuchokera pazithunzi za OT izi, biomass ya mCFU idayezedwa pakapita nthawi ndikuwonetsedwa mu Chithunzi 7b. Chochititsa chidwi n'chakuti, S. shibatae mCFUs anawonetsa kukula kwa mzere kusiyana ndi kukula kwakukulu komwe kumawonedwa ndi G. stearothermophilus mCFUs. Pakhala pali kukambirana kwa nthawi yaitali 52 ponena za chikhalidwe cha kukula kwa maselo: pamene maphunziro ena amafotokoza kukula kwa tizilombo toyambitsa matenda tomwe timafanana ndi kukula kwake (kukula kwakukulu), ena amasonyeza mlingo wokhazikika (kukula kwa mzere kapena bilinear). Monga tafotokozera Tzur et al.53, kusiyanitsa pakati pa kukula kwachidziwitso ndi (bi) kukula kwa mzere kumafuna kulondola kwa <6% mu miyeso ya biomass, yomwe ili kutali ndi njira zambiri za QPM, ngakhale kuphatikizapo interferometry. Monga tafotokozera Tzur et al.53, kusiyanitsa pakati pa kukula kwachidziwitso ndi (bi) kukula kwa mzere kumafuna kulondola kwa <6% mu miyeso ya biomass, yomwe ili kutali ndi njira zambiri za QPM, ngakhale kuphatikizapo interferometry. Как объяснили Цур ndi др.53, различение экспоненциального и (би)линейного роста требует точности <6% в измерениях биомассы, песни, песни kuchokera ku QPM, pitilizani kukhala ndi inteрферометрии. Monga momwe Zur et al.53 adafotokozera, kusiyanitsa pakati pa kukula kwachidziwitso ndi (bi) kukula kwa mzere kumafuna <6% yolondola muzitsulo za biomass, zomwe sizingatheke kwa njira zambiri za QPM, ngakhale kugwiritsa ntchito interferometry.Monga adafotokozera Zur et al. 53, kusiyanitsa pakati pa kukula kwakukulu ndi (bi) kukula kwa mzere kumafuna zosakwana 6% zolondola pamiyezo ya biomass, zomwe sizingatheke pa njira zambiri za QPM, ngakhale pamene interferometry imagwiritsidwa ntchito. CGM imakwaniritsa kulondola uku ndi kulondola kwa sub-pg mu miyeso ya biomass36,48.
Kutha kwa nthawi kokhala ndi zithunzi za 6 OT (kumiza mafuta, 60x, NA cholinga 1.25) ndi (b) kusinthika kwa biomass kwa micro-CFU kuyeza ndi CGM. Onani kanema wa M7 kuti mumve zambiri.
Kukula bwino kwa mzere wa S. shibatae kunali kosayembekezereka ndipo sikunafotokozedwebe. Komabe, kukula kwakukulu kumayembekezeredwa, chifukwa pakapita nthawi, magawano angapo a 2, 4, 8, 16 ... ma cell ayenera kuchitika. Tinkaganiza kuti kukula kwa mzere kumatha kukhala chifukwa cha kutsekeka kwa ma cell chifukwa cha kuchulukana kwa maselo, monga momwe ma cell amakulira pang'onopang'ono ndipo pamapeto pake amafika pamalo osagona pomwe kuchuluka kwa ma cell kumakhala kokwera kwambiri.
Timamaliza pokambirana mfundo zisanu zotsatirazi zomwe zimachititsa chidwi: kuchepetsa kutentha kwa voliyumu, kuchepetsa kutentha kwa kutentha, chidwi cha nanoparticles golide, chidwi cha microscopy gawo la kuchuluka, komanso kutentha komwe kungatheke LA-HTM.
Poyerekeza ndi kutentha kwamphamvu, kutentha kwa laser komwe kumagwiritsidwa ntchito popanga HTM kumapereka zabwino zingapo, zomwe tikuwonetsa mu phunziroli. Makamaka, muzinthu zamadzimadzi zomwe zimawonekera pa microscope, voliyumu yotentha imasungidwa mkati mwa ochepa (10 μm) 3 voliyumu. Mwa njira iyi, ma microbes omwe amawonedwa okha ndi omwe akugwira ntchito, pamene mabakiteriya ena amakhala ogona ndipo angagwiritsidwe ntchito kuti apitirize kuphunzira chitsanzo - palibe chifukwa chosinthira chitsanzo nthawi iliyonse kutentha kwatsopano kukufunika kufufuzidwa. Kuonjezera apo, kutentha kwa microscale kumalola kufufuza kwachindunji kwa kutentha kwakukulu: Chithunzi cha 4c chinapezedwa kuchokera ku filimu ya maola atatu (Movie M3), yomwe nthawi zambiri imafuna kukonzekera ndi kufufuza zitsanzo zingapo - imodzi mwa zitsanzo zomwe zimaphunzira. y ndi kutentha komwe kumayimira chiwerengero cha masiku akuyesa. Kuchepetsa kutentha kwa kutentha kumasunganso mbali zonse zozungulira za microscope, makamaka lens cholinga, kutentha kwa chipinda, chomwe chakhala vuto lalikulu lomwe anthu ammudzi akukumana nawo mpaka pano. LA-HTM ikhoza kugwiritsidwa ntchito ndi lens iliyonse, kuphatikizapo ma lens omiza mafuta, ndipo imakhalabe kutentha ngakhale kutentha kwakukulu m'munda wowonera. Cholepheretsa chachikulu cha njira yotenthetsera ya laser yomwe timafotokoza mu phunziroli ndikuti maselo omwe samamamatira kapena kuyandama amatha kukhala kutali ndi momwe amawonera komanso ovuta kuphunzira. Njira yothanirana ndi vutoli ingakhale kugwiritsa ntchito magalasi okulira otsika kuti akwaniritse kutentha kwakukulu kopitilira ma microns mazana angapo. Chenjezoli likutsatizana ndi kuchepa kwa malo, koma ngati cholinga ndi kuphunzira kayendedwe ka tizilombo toyambitsa matenda, kusamvana kwakukulu kwa malo sikofunikira.
Nthawi yotenthetsera (ndi kuziziritsa) dongosolo \({{{{\rm{\tau }}}}}}}}_{{{\mbox{D}}}}}\) zimatengera kukula kwake , molingana ndi lamulo \({{({\rm{\tau }}}}}}}_{{{\mbox{D}}}}}={L}^{2}/D\), kuti \ (L\ ) ndi kukula kwa gwero la kutentha (m'mimba mwake mwa laser mtengo mu phunziro lathu ndi \ ( L \ pafupifupi 100 \) μm ), \ (D \) ndi kutentha kwachilengedwe kwa chilengedwe (chiwerengero chathu. Mlandu, galasi ndi madzi Diffusion rate\(D\ about 2\fold {10}^{-7}\) m2/s Choncho, mu phunziro ili, mayankho a nthawi ya 50 ms, mwachitsanzo, quasi-instantaneous). Kusintha kwa kutentha, kungathe kuyembekezera kukhazikitsidwa kwa nthawi yomweyo kwa kutentha sikungofupikitsa nthawi yoyesera, komanso kumalola nthawi yeniyeni \ (t = 0 \) pa phunziro lililonse lamphamvu la zotsatira za kutentha.
Njira yathu yomwe tikufuna imagwira ntchito pagawo lililonse loyamwa kuwala (mwachitsanzo, zitsanzo zamalonda zokhala ndi zokutira za ITO). Komabe, ma nanoparticles a golide amatha kupereka kuyamwa kwakukulu mu infuraredi ndi kutsika kotsika mumtundu wowoneka, mawonekedwe omaliza omwe ali ndi chidwi kuti awonetsetse bwino pamawonekedwe owoneka, makamaka pogwiritsa ntchito fulorosenti. Kuphatikiza apo, golidi ndi biocompatible, inert yamankhwala, kachulukidwe ka kuwala kumatha kusinthidwa kuchoka pa 530 nm kupita kufupi ndi infrared, ndipo kukonzekera kwachitsanzo ndikosavuta komanso kopanda ndalama29.
Transverse grating wavefront microscopy (CGM) imalola osati kungojambula kutentha pa microscale, komanso kuwunika kwa biomass, kumapangitsa kuti ikhale yothandiza (ngati sikofunikira) kuphatikiza ndi LA-HTM. M'zaka khumi zapitazi, njira zina zowonetsera kutentha kwa microscopy zapangidwa, makamaka pankhani ya bioimaging, ndipo zambiri zimafuna kugwiritsa ntchito ma probes54,55 a fulorosenti osamva kutentha. Komabe, njirazi zatsutsidwa ndipo malipoti ena ayesa kusintha kosasinthika kwa kutentha mkati mwa maselo, mwinamwake chifukwa chakuti fulorosenti imadalira zinthu zambiri osati kutentha. Kuphatikiza apo, ma probe ambiri a fulorosenti amakhala osakhazikika pa kutentha kwakukulu. Chifukwa chake, QPM makamaka CGM imayimira njira yabwino yowonera ma microscope kuti muphunzire za moyo pa kutentha kwambiri pogwiritsa ntchito ma microscope.
Kafukufuku wa S. shibatae, omwe amakhala bwino pa 80 ° C, amasonyeza kuti LA-HTM ingagwiritsidwe ntchito pophunzira ma hyperthermophiles, osati thermophiles wamba. Kwenikweni, palibe malire a kutentha komwe kungathe kufika pogwiritsa ntchito LA-HTM, ndipo ngakhale kutentha pamwamba pa 100 ° C kumatha kufika pamtunda wamlengalenga popanda kuwira, monga momwe gulu lathu la 38 likusonyezera mu ntchito za hydrothermal chemistry pamlengalenga. pressure A. Laser imagwiritsidwa ntchito potenthetsa nanoparticles golide 40 mwanjira yomweyo. Chifukwa chake, LA-HTM imatha kugwiritsidwa ntchito kuyang'ana ma hyperthermophiles omwe anali asanakhalepo ndi ma microscope omwe ali ndi mawonekedwe apamwamba kwambiri pamikhalidwe yokhazikika (ie pansi pa zovuta zachilengedwe).
Zoyeserera zonse zidachitika pogwiritsa ntchito maikulosikopu opangira tokha, kuphatikiza kuwunikira kwa Köhler (ndi LED, M625L3, Thorlabs, 700 mW), chotengera chazithunzi chokhala ndi mayendedwe amanja a xy, zolinga (Olympus, 60x, 0.7 NA, mpweya, LUCPlanFLN60X kapena 60x, NA25, O1. , UPLFLN60XOI), kamera ya CGM (QLSI cross grating, 39 µm pitch, 0.87 mm kuchokera ku Andor Zyla camera sensor) kuti ipereke chithunzithunzi champhamvu komanso chakutsogolo, ndi kamera ya sCMOS (ORCA Flash 4.0 V3, 16-bit mode, kuchokera ku Hamamatsu) kujambula zomwe zikuwonetsedwa mu Chithunzi 5 (kusambira kwa bakiteriya). Dichroic beam splitter ndi 749 nm BrightLine m'mphepete (Semrock, FF749-SDi01). Zosefera kutsogolo kwa kamera ndi 694 short pass fyuluta (FF02-694/SP-25, Semrock). Titanium safiro laser (Laser Verdi G10, 532 nm, 10 W, pumped tsunami laser cavity, Spectra-Physics mu Fig. 2-5, m'malo mwake ndi Millenia laser, Spectraphysics 10 W, kupopera Mira laser cavity, Coherent, kwa Mkuyu. -5). 6 ndi 7) adayikidwa ku kutalika kwa mawonekedwe \({{({\rm{\lambda}}}}}}}}=800\) nm, yomwe imagwirizana ndi plasmon resonance sipekitiramu ya nanoparticles golide. 1152 pixels) adagulidwa kuchokera ku Meadowlark Optics Mahologalamu adawerengedwa pogwiritsa ntchito algorithm ya Gerchberg-Saxton monga momwe 39 yafotokozera.
Cross grating wavefront microscopy (CGM) ndi njira yowonera ma microscopy yotengera kuphatikizira kagayidwe kamitundu iwiri (yomwe imadziwikanso kuti cross grating) pamtunda wa milimita imodzi kuchokera pa sensa wamba ya kamera. Chitsanzo chofala kwambiri cha CGM chomwe tagwiritsa ntchito mu phunziroli chimatchedwa interferometer yawavelength transverse shift interferometer (QLSI), pomwe kukwera pamtanda kumakhala ndi mawonekedwe a intensity/phase checkerboard oyambitsidwa ndi ovomerezeka ndi Primot et al. mu 200034. Mizere yowongoka ndi yopingasa imapanga mithunzi yofanana ndi gridi pa sensa, kupotoza kwake komwe kungathe kuwerengedwera mu nthawi yeniyeni kuti apeze kusokonezeka kwa mawonekedwe a kuwala (kapena gawo lofanana) la kuwala kwa chochitikacho. Ikagwiritsidwa ntchito pa microscope, kamera ya CGM imatha kuwonetsa kusiyana kwa njira ya chinthu chojambulidwa, chomwe chimatchedwanso kuzama kwa kuwala (OT), yokhala ndi chidwi pa dongosolo la nanometers36. Muyeso iliyonse ya CGM, kuti athetse vuto lililonse muzinthu zowoneka bwino kapena matabwa, chithunzi choyambirira cha OT chiyenera kutengedwa ndikuchotsedwa pazithunzi zilizonse zotsatila.
Kutentha kwa microscopy kunachitika pogwiritsa ntchito kamera ya CGM monga momwe tafotokozera muzofotokozera. 32. Mwachidule, kutentha kwamadzimadzi kumasintha index yake ya refractive, kupanga ma lens otentha omwe amasokoneza mtengowo. Kupotoza kwa mafundewa kumayesedwa ndi CGM ndikukonzedwa pogwiritsa ntchito deconvolution algorithm kuti mupeze magawo atatu a kutentha kwapakati pamadzi. Ngati ma nanoparticles agolide akugawidwa mofanana mu chitsanzo chonse, mapu a kutentha amatha kuchitidwa m'madera opanda mabakiteriya kuti apange zithunzi zabwino, zomwe nthawi zina timachita. Chithunzi cha CGM chinapezedwa popanda kutenthedwa (chozimitsa laser) ndipo kenako chinajambulidwa pamalo omwewo pachithunzichi ndi laser.
Muyeso wowuma umatheka pogwiritsa ntchito kamera ya CGM yomwe imagwiritsidwa ntchito pojambula kutentha. Zithunzi zofotokozera za CGM zidapezedwa ndikusuntha mwachangu zitsanzo mu x ndi y panthawi yowonekera ngati njira yochepetsera kusagwirizana kulikonse mu OT chifukwa cha kukhalapo kwa mabakiteriya. Kuchokera pazithunzi za mabakiteriya a OT, ma biomass awo adapezedwa pogwiritsa ntchito zithunzi zambiri m'malo omwe adasankhidwa pogwiritsa ntchito gawo lopanga la Matlab (onani gawo la "Numerical code"), kutsatira njira yomwe yafotokozedwa mu ref. 48. Mwachidule, timagwiritsa ntchito chiyanjano \(m={\alpha}^{-1}\iint {{\mbox{OT}}}\left(x,y\right){{\mbox{d}} } x{{\mbox{d}}}}y\), pomwe \({{\mbox{OT}}}\left(x,y\right)\) ndi chithunzi chakuya, \(m\) ndi kulemera kouma ndipo \({{{{\rm{\ alpha }}}}}}}\) kumakhala kosasintha. Tidasankha \({{{{\rm{\alpha)))))))=0.18\) µm3/pg, chomwe ndi chokhazikika cha maselo amoyo.
Chophimba chophimba 25 mm m'mimba mwake ndi 150 µm wandiweyani wokutidwa ndi nanoparticles wagolide chinayikidwa mu chipinda cha AttofluorTM (Thermofisher) ndi nanoparticles zagolide zomwe zikuyang'ana mmwamba. Geobacillus stearothermophilus idakonzedwa usiku wonse mu LB sing'anga (200 rpm, 60 ° C) tsiku lililonse la zoyeserera lisanakwane. Dontho la 5 µl la kuyimitsidwa kwa G. stearothermophilus ndi kachulukidwe kakang'ono (OD) ka 0.3 mpaka 0.5 adayikidwa pa chivundikiro chokhala ndi nanoparticles zagolide. Kenako, chivundikiro chozungulira cha 18 mm m'mimba mwake chokhala ndi dzenje la 5 mm m'mimba mwake chapakati chinagwetsedwa padontho, ndipo 5 μl ya kuyimitsidwa kwa bakiteriya yokhala ndi kachulukidwe kofananako kamene kamagwiritsidwa ntchito mobwerezabwereza pakati pa dzenje. Zitsime zomwe zili pazivundikiro zidakonzedwa motsatira ndondomeko yomwe yafotokozedwa mu ref. 45 (onani Zowonjezera Zowonjezera kuti mudziwe zambiri). Kenako onjezerani 1 ml ya LB sing'anga pa chivundikiro kuti madzi asanjike kuti asawume. Chophimba chomaliza chimayikidwa pamwamba pa chivindikiro chotsekedwa cha chipinda cha Attofluor™ kuti ateteze kuphulika kwa sing'anga panthawi yoyamwitsa. Poyesera kumera, tidagwiritsa ntchito spores, zomwe, pambuyo poyeserera wamba, nthawi zina zimaphimba chivundikiro chapamwamba. Njira yofananayi inagwiritsidwa ntchito kupeza Sulfolobus shibatae. Masiku atatu (200 rpm, 75 ° C) a kulima koyambirira kwa Thiobacillus serrata kunachitika pakati pa 182 (DSMZ).
Zitsanzo za nanoparticles zagolide zidakonzedwa ndi micellar block copolymer lithography. Ndondomekoyi ikufotokozedwa mwatsatanetsatane mu Chap. 60. Mwachidule, micelles encapsulating ayoni golide anapangidwa mwa kusakaniza copolymer ndi HAuCl4 mu toluene. Zophimba zotsukidwazo zidamizidwa mumtsuko ndikuwaza ndi kuwala kwa UV pamaso pa chochepetsera kuti mupeze mbewu zagolide. Pomaliza, njere zagolide zidakulitsidwa polumikizana ndi chivundikiro chokhala ndi njira yamadzi ya KAuCl4 ndi ethanolamine kwa mphindi 16, zomwe zidapangitsa kuti ma nanoparticles agolide asakhale ozungulira komanso ofanana kwambiri.
Kuti tisinthe ma interferograms kukhala zithunzi za OT, tidagwiritsa ntchito algorithm yodzipangira tokha, monga tafotokozera mu ulalo. 33 ndipo likupezeka ngati phukusi la Matlab m'malo otsatirawa: https://github.com/baffou/CGMprocess. Phukusili limatha kuwerengera kukula ndi zithunzi za OT kutengera ma interferogram ojambulidwa (kuphatikiza zithunzi zofananira) ndi mtunda wamakamera.
Kuti tiwerengere gawo lomwe likugwiritsidwa ntchito ku SLM kuti tipeze mbiri ya kutentha, tidagwiritsa ntchito algorithm39,42 yopangidwa kunyumba yomwe imapezeka m'malo otsatirawa: https://github.com/baffou/SLM_temperatureShaping. Cholowetsacho ndi gawo la kutentha lomwe mukufuna, lomwe lingathe kukhazikitsidwa ndi digito kapena kudzera pa chithunzi cha monochrome bmp.
Kuti tigawane ma cell ndikuyesa kulemera kwawo, tidagwiritsa ntchito algorithm yathu ya Matlab yosindikizidwa m'malo otsatirawa: https://github.com/baffou/CGM_magicWandSegmentation. Pa chithunzi chilichonse, wogwiritsa ntchitoyo ayenera kudina mabakiteriya kapena mCFU wokonda, kusintha kukhudzika kwa wand, ndikutsimikizira kusankha.
Kuti mumve zambiri pamapangidwe amaphunziro, onani Nature Research Report abstract yolumikizidwa ndi nkhaniyi.
Deta yochirikiza zotsatira za kafukufukuyu ikupezeka kuchokera kwa olemba omwe afunsidwa ngati akufuna.
Khodi yomwe yagwiritsidwa ntchito mu kafukufukuyu yafotokozedwa mwatsatanetsatane mu gawo la Njira, ndipo zomasulira zitha kutsitsidwa kuchokera ku https://github.com/baffou/ m'malo otsatirawa: SLM_temperatureShaping, CGMprocess, ndi CGM_magicWandSegmentation.
Mehta, R., Singhal, P., Singh, H., Damle, D. & Sharma, AK Insight into thermophiles ndi kugwiritsa ntchito kwawo kosiyanasiyana. Mehta, R., Singhal, P., Singh, H., Damle, D. & Sharma, AK Insight into thermophiles ndi kugwiritsa ntchito kwawo kosiyanasiyana.Mehta, R., Singhal, P., Singh, H., Damle, D. ndi Sharma, AK Overview of thermophiles and their wide application. 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. ndi Sharma AK Kumvetsetsa kwakukulu kwa thermophiles ndi ntchito zosiyanasiyana.3 Biotechnology 6, 81 (2016).
Nthawi yotumiza: Sep-26-2022