ʻO ke ola ma nā wela kiʻekiʻe i ʻike ʻia in vitro me nā nanoparticles gula wela laser

Mahalo no kou kipa ʻana iā Nature.com. ʻO ka polokalamu kele pūnaewele āu e hoʻohana nei he kākoʻo CSS kaupalena. No ka ʻike maikaʻi loa, manaʻo mākou e hoʻohana i kahi polokalamu kele pūnaewele hou (a i ʻole e hoʻopau i ke ʻano Compatibility Mode ma Internet Explorer). I kēia manawa, e hōʻoia i ke kākoʻo mau ʻana, e hāʻawi mākou i ka pūnaewele me ka ʻole o nā ʻano a me JavaScript.
ʻO Thermophiles nā microorganism e ulu ana i nā wela kiʻekiʻe. Hiki i ke aʻo ʻana iā lākou ke hāʻawi i ka ʻike waiwai e pili ana i ke ʻano o ke ola i nā kūlana koʻikoʻi. Eia naʻe, paʻakikī ke hoʻokō i nā kūlana wela kiʻekiʻe me nā microscopes optical maʻamau. Ua ho'oholo 'ia kekahi mau mea i hana 'ia i ka home e pili ana i ka ho'omehana uila kū'ē kūloko, akā 'a'ohe mea ho'oponopono pā'oihana ma'alahi. Ma kēia pepa, hoʻolauna mākou i ka manaʻo o ka hoʻomehana laser microscale ma luna o ke kahua o ka microscope o ka nānā ʻana e hāʻawi i nā wela kiʻekiʻe no nā haʻawina thermophile aʻo ka mālama ʻana i ke kaiapuni o ka mea hoʻohana. Hiki ke hoʻokō ʻia ka hoʻomehana ʻana o ka microscale me ka hoʻohana ʻana i kahi substrate i uhi ʻia i ke gula nanoparticle ma ke ʻano he biocompatible a maikaʻi hoʻi. Kūkākūkā ʻia nā hopena e hiki mai ana o ka microscale fluid convection, cell retention, and centrifugal thermophoretic motion. Ua hōʻike ʻia ke ʻano hana ma nā ʻano ʻelua: (i) Geobacillus stearothermophilus, he huakini thermophilic ikaika e hana hou ana ma kahi o 65°C, a mākou i ʻike ai e ulu, ulu a ʻauʻau ma lalo o ka hoʻomehana microscale; (ii) Thiobacillus sp., he archaea hyperthermophilic maikaʻi loa. ma 80°C. Hāʻawi kēia hana i ke ala no ka nānā maʻalahi a me ka palekana o nā microorganism thermophilic me ka hoʻohana ʻana i nā mea hana microscopy hou a me ke kumukūʻai.
I loko o nā piliona makahiki, ua hoʻololi ke ola ma ka Honua e hoʻololi i kahi ākea o nā kūlana kaiapuni i manaʻo ʻia i kekahi manawa he koʻikoʻi loa mai ko mākou hiʻohiʻona kanaka. ʻO ka mea nui, ʻo kekahi mau microorganisms thermophilic (bacteria, archaea, fungi) i kapa ʻia ʻo thermophiles e ulu maikaʻi i ka pae wela mai 45 ° C a 122 ° C1, 2, 3, 4. Noho nā Thermophiles i nā ʻano kaiaola like ʻole, e like me nā puka makani hydrothermal kai hohonu, nā pūnāwai wela. a i ʻole nā ​​wahi pele. Ua loaʻa i kā lākou noiʻi ka hoihoi nui i nā makahiki i hala iho nei no nā kumu ʻelua. ʻO ka mua, hiki iā mākou ke aʻo mai iā lākou, no ka laʻana, pehea ka paʻa ʻana o nā thermophiles 5, 6, nā enzymes 7, 8 a me nā membrane 9 i kēlā mau wela kiʻekiʻe, a i ʻole pehea e hiki ai i nā thermophiles ke kū i nā pae kiʻekiʻe o ka radiation10. ʻO ka lua, ʻo ia ke kumu no ka nui o nā noi biotechnological koʻikoʻi1,11,12 e like me ka hana wahie13,14,15,16, chemical synthesis (dihydro, alcohols, methane, amino acids, etc.)17, biomining18 and thermostable biocatalysts7 ,11, 13. ʻO ka mea nui, ʻo ka polymerase chain reaction (PCR)19 i kaulana i kēia manawa e pili ana i kahi enzyme (Taq polymerase) i hoʻokaʻawale ʻia mai ka thermophilic bacterium Thermus aquaticus, kekahi o nā thermophiles mua i ʻike ʻia.
Eia nō naʻe, ʻaʻole maʻalahi ke aʻo ʻana i nā thermophiles a ʻaʻole hiki ke hoʻoponopono ʻia i loko o kekahi keʻena keʻena olaola. ʻO ka mea nui, ʻaʻole hiki ke nānā ʻia nā thermophiles ola i loko o ka vitro me kekahi microscope māmā maʻamau, ʻoiai me nā keʻena hoʻomehana i kūʻai ʻia, e helu pinepine ʻia no nā mahana ma lalo o 40°C. Mai ka 1990s, he mau pūʻulu noiʻi wale nō i hoʻolaʻa iā lākou iho i ka hoʻokomo ʻana i nā ʻōnaehana microscopy kiʻekiʻe (HTM). I ka makahiki 1994 Glukh et al. Ua hāpai ʻia ke keʻena mehana/hoʻoluʻu ma muli o ka hoʻohana ʻana i kahi cell Peltier e hoʻomalu i ka mahana o nā capillaries rectangular i pani ʻia e mālama i ka anaerobicity 20. Hiki ke hoʻomehana ʻia ka mea hana a hiki i 100 °C ma ka wikiwiki o 2 °C/s, hiki i nā mea kākau ke aʻo i ka motility o ka hyperthermophilic bacterium Thermotoga maritima21. I ka makahiki 1999 ʻo Horn et al. Ua hoʻomohala ʻia kahi mea like loa, e pili ana i ka hoʻohana ʻana i nā capillaries wela kūpono no ka microscopy kalepa e aʻo ai i ka mahele cell / pili. Ma hope o ka lōʻihi o ka hana ʻole ʻana, ua hoʻomaka hou ka ʻimi ʻana i nā HTM maikaʻi i ka makahiki 2012, ʻo ia hoʻi e pili ana i kahi pūʻulu pepa a ka hui Wirth i hoʻohana i kahi mea i haku ʻia e Horn et al. He ʻumikumamālima mau makahiki i hala aku nei, ua aʻo ʻia ka motility o ka nui o nā archaea, me nā hyperthermophiles, i nā mahana a hiki i 100 ° C me ka hoʻohana ʻana i nā capillaries wela23,24. Ua hoʻololi pū lākou i ka microscope kumu e loaʻa ai ka hoʻomehana wikiwiki (he mau minuke ma kahi o 35 mau minuke e hiki ai i ka mahana i hoʻonohonoho ʻia) a loaʻa i kahi gradient wela laina ʻoi aku ma mua o 2 knm ma waena o ka waena. Ua hoʻohana ʻia kēia mea hana hoʻoheheʻe wela (TGFD) no ke aʻo ʻana i ka neʻe ʻana o nā thermophile he nui i loko o nā ʻanuʻu wela ma nā mamao pili biologically 24, 25.
ʻO ka hoʻomehana ʻana i nā capillaries i pani ʻia ʻaʻole wale ke ala e nānā ai i nā thermophiles ola. Ma 2012, Kuwabara et al. Ua hoʻohana ʻia nā keʻena Pyrex i hoʻohana ʻia i ka home i hoʻopaʻa ʻia me ka mea hoʻopili pale wela (Super X2; Cemedine, Iapana). Ua kau ʻia nā laʻana ma luna o kahi pāhana hoʻomehana akaka (Micro Heat Plate, Kitazato Corporation, Iapana) hiki ke hoʻomehana a hiki i 110°C, akā ʻaʻole i manaʻo mua ʻia no ka bioimaging. Ua ʻike nā mea kākau i ka hoʻokaʻawale maikaʻi ʻana o nā bacteria thermophilic anaerobic (Thermosipho globiformans, manawa pālua 24 min) ma 65°C. Ma 2020, ʻo Pulshen et al. Ua hōʻike ʻia ka hoʻomehana maikaʻi ʻana o nā kīʻaha metala ʻoihana (AttofluorTM, Thermofisher) me ka hoʻohana ʻana i ʻelua mau mea hoʻomehana home: kahi poʻi a me kahi pae (PCR machine-inspired configuration). Hoʻopuka kēia hui i kahi mahana wai like ʻole a pale i ka evaporation a me ka condensation ma lalo o ka poʻi. ʻO ka hoʻohana ʻana i ka O-ring e pale i ka hoʻololi kinoea me ke kaiapuni. Ua hoʻohana ʻia kēia HTM, i kapa ʻia ʻo Sulfoscope, e kiʻi iā Sulfolobus acidocaldarius ma 75°C27.
ʻO kahi palena i ʻike ʻia o kēia mau ʻōnaehana a pau, ʻo ia ke kaupalena ʻana i ka hoʻohana ʻana i nā pahuhopu ea, ʻaʻole kūpono ka hoʻoinu ʻaila no kēlā ʻano wela kiʻekiʻe a no ke kiʻi ʻana ma o >1-mm mānoanoa o nā laʻana alohilohi. ʻO kahi palena i ʻike ʻia o kēia mau ʻōnaehana a pau, ʻo ia ke kaupalena ʻana i ka hoʻohana ʻana i nā pahuhopu ea, ʻaʻole kūpono ka hoʻoinu ʻaila no kēlā ʻano wela kiʻekiʻe a no ke kiʻi ʻana ma o >1-mm mānoanoa o nā laʻana alohilohi. Общепризнанным недостатком всех этих систем было ограничение на использование воздушных объективов, посколмьбужов, посколмибку ение в масло не подходило для такой высокой температуры и для визуализации через прозрачные образцы толщ1инмой. ʻO kahi hemahema i ʻike ʻia o kēia mau ʻōnaehana a pau, ʻo ia ka palena o ka hoʻohana ʻana i nā pahuhopu ea, no ka mea, ʻaʻole kūpono kēlā me kēia ʻaila immersion no kēlā ʻano wela kiʻekiʻe a no ka ʻike ʻana ma o nā laʻana alohilohi> 1 mm mānoanoa.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是他的适合这是是厚的透明样品成像。 ʻO ka palena i ʻike ʻia o kēia mau ʻōnaehana a pau, ʻo ia ka palena o ka hoʻohana ʻana i ke aniani i hoʻopaʻa ʻia i ka ea, no ka mea, ʻaʻole kūpono kēlā me kēia ʻaila immersion no ke kiʻi ʻana i nā laʻana alohilohi> 1 mm mānoanoa ma ia mau wela kiʻekiʻe. Общепризнанным недостатком всех этих систем является ограниченное использование воздушных объективов, лимурбов масло непригодно для таких высоких температур и визуализации через прозрачные образцы толщиной >1 мм. ʻO kahi drawback i ʻike ʻia o kēia mau ʻōnaehana āpau ʻo ia ka hoʻohana palena ʻana i nā lens lewa, ʻaʻole kūpono kēlā me kēia ʻaila immersion no nā wela kiʻekiʻe a me ka ʻike ʻana ma o nā laʻana alohilohi> 1 mm mānoanoa.I kēia mau lā, ua hāpai ʻia kēia palena e Charles-Orzag et al. 28, nāna i hoʻomohala i kahi mea ʻaʻole e hāʻawi hou i ka wela a puni ka ʻōnaehana o ka hoihoi, akā i loko o ke aniani uhi ponoʻī, i uhi ʻia me kahi ʻāpana aniani ʻeleʻele o kahi pale i hana ʻia me ITO (indium-tin oxide). Hiki ke hoʻomāhana ʻia ka poʻi a hiki i 75 °C ma o ke kau ʻana i kahi au uila ma o ka papa aniani. Eia nō naʻe, pono ka mea kākau e hoʻomehana i ka lens i ka pahuhopu, akā ʻaʻole ʻoi aku ma mua o 65 °C, i ʻole e hōʻino.
Hōʻike kēia mau hana i ka hoʻomohala ʻana o ka microscopy optical kiʻekiʻe kiʻekiʻe ʻaʻole i hoʻohana nui ʻia, koi pinepine i nā mea hana home, a loaʻa pinepine ʻia ma ke kumukūʻai o ka hoʻonā spatial, kahi pōʻino koʻikoʻi i hāʻawi ʻia ʻaʻole ʻoi aku ka nui o nā microorganism thermophilic ma mua o kekahi. micrometers. ʻO ka hoʻohaʻahaʻa ʻana i ka leo hoʻomehana ke kī i ka hoʻonā ʻana i ʻekolu pilikia o ka HTM: ʻino ka hoʻonā spatial, kiʻekiʻe thermal inertia i ka wā e wela ai ka ʻōnaehana, a me ka hoʻomehana pōʻino o nā mea hoʻopuni (ʻaila immersion, lens object… a i ʻole nā ​​lima o ka mea hoʻohana) i nā wela wela. ).
Ma kēia pepa, hoʻolauna mākou i kahi HTM no ka nānā ʻana i ka thermophile ʻaʻole i hoʻokumu ʻia i ka hoʻomehana resistive. Akā, ua hoʻokō mākou i ka hoʻomehana kūloko i loko o kahi palena palena o ke kahua o ka microscope o ka nānā ʻana ma o ka laser irradiation o kahi substrate e hoʻomāmā. ʻIke ʻia ka puʻunaue wela me ka hoʻohana ʻana i ka microscopy phase quantitative (QPM). Hōʻike ʻia ka maikaʻi o kēia ʻano hana e Geobacillus stearothermophilus, kahi huakini thermophilic motile e hana hou ma kahi o 65 ° C a he manawa pālua pōkole (ma kahi o 20 mau minuke), a me Sulfolobus shibatae, kahi hyperthermophile e ulu maikaʻi loa ma 80 ° C (archaea) e hoikeike. Ua ʻike ʻia ka nui o ka hoʻopiʻi maʻamau a me ka ʻauʻau ʻana ma ke ʻano he hana o ka mahana. ʻAʻole kaupalena ʻia kēia laser HTM (LA-HTM) e ka mānoanoa o ka uhi uhi a i ʻole ke ʻano o ka pahuhopu (ka ea a i ʻole ka ʻaila immersion). ʻAe kēia e hoʻohana i kekahi lens hoʻonā kiʻekiʻe ma ka mākeke. ʻAʻole hoʻi e pilikia i ka hoʻomehana lohi ma muli o ka inertia wela (loaʻa i ka hoʻomehana koke ma ka millisecond scale) a hoʻohana wale i nā mea kūʻai aku. ʻO nā hopohopo palekana hou wale nō e pili ana i ka hiki ʻana o nā kukuna laser ikaika (maʻamau a hiki i ka 100 mW) i loko o ka hāmeʻa a ma o nā maka paha, kahi e koi ai i nā goggles pale.
ʻO ke kumumanaʻo o LA-HTM ka hoʻohana ʻana i ka laser e hoʻomehana i ka hāpana ma ka ʻāina i loko o ke kahua o ka ʻike o ka microscope (Fig. 1a). No ka hana ʻana i kēia, pono e hoʻomaʻamaʻa ka hāpana. No ka hoʻohanaʻana i ka mana laser kūpono (ma lalo o 100 mW),ʻaʻole mākou i hilinaʻi i ka absorption o ka malamalama e ka wai, akā, ua hoʻonui artificially i ka absorption o ka hāpana ma ka uhiʻana i ka substrate me nā nanoparticles gula (Fig. 1c). ʻO ka hoʻomehana ʻana i nā nanoparticles gula me ka māmā he mea koʻikoʻi nui ia i ke kahua o nā plasmonics thermal, me nā noi i manaʻo ʻia i ka biomedicine, nanochemistry a i ʻole ka ʻohi ʻana o ka lā29,30,31. I nā makahiki i hala iho nei, ua hoʻohana mākou i kēia LA-HTM i nā haʻawina e pili ana i nā noi plasma thermal i ka physics, chemistry a me ka biology. ʻO ka pilikia nui me kēia ʻano hana, ʻo ia ka hōʻike ʻana i ka ʻaoʻao wela hope, no ka mea, ua kaupalena ʻia ka wela kiʻekiʻe i kahi ʻāpana microscale i loko o ka hāpana. Ua hōʻike mākou e hiki ke hoʻokō ʻia ka palapala ʻāina wela me ka interferometer shear transverse ʻehā-lōʻihi, kahi ala maʻalahi, kiʻekiʻe, a maʻalahi loa o ka microscopy māhele quantitative e pili ana i ka hoʻohana ʻana i nā gratings diffraction ʻelua-dimensional (i ʻike ʻia ʻo cross gratings) 33,34,35,36. Ua hōʻike ʻia ka hilinaʻi o kēia ʻenehana microscopy thermal, e pili ana i ka crossed grating wavefront microscopy (CGM), i loko o nā pepa he ʻumi i paʻi ʻia i nā makahiki he ʻumi i hala37,38,39,40,41,42,43.
Hoʻolālā o ka hoʻokomo ʻana i ka hoʻomehana laser parallel, ka hoʻohua ʻana a me ka microscope wela. b Laʻana geometry i loko o kahi keʻena AttofluorTM i loaʻa kahi uhi uhi i uhi ʻia me nā nanoparticles gula. c E nānā pono i ka hāpana (ʻaʻole i ka pālākiō). d e hōʻike ana i ka ʻikepili kukuna laser like ʻole a me (e) ka puʻunaue wela i hoʻohālikelike ʻia ma ka mokulele hāpana o nā nanoparticles gula. f he kiʻi kukuna laser annular kūpono no ka hoʻohua ʻana i kahi mahana like ʻole e like me ka mea i hōʻike ʻia ma ka hoʻohālikelike o ka hāʻawi ʻana i ka wela i hōʻike ʻia ma (g). ʻO ka pā unahi: 30 µm.
ʻO ka mea nui, ua hoʻokō mākou i ka hoʻomehana ʻana o nā pūnaewele mammalian me LA-HTM a me CGM a nānā i nā pane haʻalulu wela cellular ma ka laulā o 37-42 ° C, e hōʻike ana i ka hoʻohana ʻana o kēia ʻenehana i ke kiʻi kelepona ola hoʻokahi. Eia naʻe, ʻaʻole maopopo ka hoʻohana ʻana o LA-HTM i ke aʻo ʻana i nā microorganism i nā wela kiʻekiʻe, no ka mea, pono ia e makaʻala nui i ka hoʻohālikelike ʻia me nā cell mammalian: ʻo ka mea mua, ʻo ka hoʻomehana ʻana i ka lalo o ka waena i nā ʻumi degere (ma mua o kahi mau degere) ke alakaʻi. i kahi ʻoluʻolu wela kūpaʻa ikaika. hiki ke hana i ka convection wai 44, inā ʻaʻole i hoʻopili paʻa ʻia i ka substrate, hiki ke hoʻoulu i ka neʻe ʻana a me ka hui ʻana o ka bacteria. Hiki ke hoʻopau ʻia kēia convection ma ka hoʻemi ʻana i ka mānoanoa o ka papa wai. No kēia kumu, i loko o nā hoʻokolohua a pau i hōʻike ʻia ma lalo nei, ua kau ʻia nā mea hoʻokuʻu bacteria ma waena o ʻelua mau uhi uhi ma kahi o 15 µm mānoanoa i waiho ʻia i loko o kahi kīʻaha metala (AttofluorTM, Thermofisher, Fig. 1b,c). Ma ke kumu, hiki ke pale ʻia ka convection inā ʻoi aku ka liʻiliʻi o ka mānoanoa o ka wai ma mua o ka nui o ka beam o ka laser wela. ʻO ka lua, ʻo ka hana ʻana i kahi geometry liʻiliʻi hiki ke hoʻopau i nā mea ola aerobic (e ʻike i ke kiʻi S2). Hiki ke pale ʻia kēia pilikia ma o ka hoʻohana ʻana i kahi substrate hiki ke paʻa i ka oxygen (a i ʻole kekahi kinoea koʻikoʻi ʻē aʻe), ma ka waiho ʻana i nā ʻōhū ea i paʻa i loko o ka uhi uhi, a i ʻole ma ka ʻeli ʻana i nā lua ma ka uhi uhi luna (e nānā i ka Fig. S1) 45 . Ma kēia haʻawina, ua koho mākou i ka hopena hope (Nā Kiʻi 1b a me S1). ʻO ka hope, ʻaʻole hāʻawi ka hoʻomehana laser i ka hāʻawi like ʻana o ka mahana. ʻOiai ma ka ikaika like o ke kukuna laser (Fig. 1d), ʻaʻole like ka puʻunaue wela, akā ua like me ka puʻunaue Gaussian ma muli o ka hoʻoheheʻe wela (Fig. 1e). Aia ka pahuhopu e hoʻokumu i nā mahana kūpono ma ke kahua o ka nānā ʻana no ke aʻo ʻana i nā ʻōnaehana olaola, ʻaʻole kūpono nā ʻaoʻao like ʻole a hiki ke alakaʻi i ka neʻe thermophoretic o ka bacteria inā ʻaʻole lākou e pili i ka substrate (e nānā i ka Fig. S3, S4)39. No kēia hopena, ua hoʻohana mākou i kahi modulator māmā spatial (SLM) e hana i ka kukuna laser infrared e like me ke ʻano o ke apo (Fig. 1f) i loko o ka mokulele o ka hāpana e hoʻokō i ka hāʻawi ʻana i ka wela kūpono i loko o kahi wahi geometric i hāʻawi ʻia. ʻoiai ka hoʻopuehu wela (Fig. 1d) 39 , 42, 46. E kau i ka uhi uhi ma luna o kahi pā metala (Figure 1b) i pale aku i ka hoʻoheheʻe ʻana o ka mea ʻenehana a nānā i kekahi mau lā liʻiliʻi. No ka mea ʻaʻole i hoʻopaʻa ʻia kēia uhi uhi luna, hiki ke maʻalahi ke hoʻohui ʻia i kēlā me kēia manawa inā pono.
No ka hōʻike ʻana i ka hana ʻana o LA-HTM a hōʻike i kona kūpono i ka noiʻi thermophilic, ua aʻo mākou i ka bacteria aerobic Geobacillus stearothermophilus, nona ka mahana ulu maikaʻi loa ma kahi o 60-65°C. Loaʻa i ka bacteria ka flagella a me ka hiki ke ʻauʻau, e hāʻawi ana i kahi hōʻailona ʻē aʻe o ka hana kelepona maʻamau.
ʻO nā laʻana (Fig. 1b) i hoʻokomo muaʻia ma 60 ° C no hoʻokahi hola a laila hoʻokomoʻia i loko o kahi mea hoʻohālike LA-HTM. He koho kēia pre-incubation, akā pono nō naʻe, no nā kumu ʻelua: ʻO ka mua, ke hoʻāla ʻia ka laser, e ulu koke a puʻunaue nā cell (e nānā i ke kiʻiʻoniʻoni M1 i nā Mea Hoʻohui). Me ka ʻole pre-incubation, hoʻopaneʻe pinepine ʻia ka ulu ʻana o ka maʻi bacteria ma kahi o 40 mau minuke i kēlā me kēia manawa i hoʻomehana ʻia kahi wahi nānā hou ma ka hāpana. ʻO ka lua, ʻo ka 1 hola pre-incubation i hāpai i ka hoʻopili ʻana o ka bacteria i ka uhi uhi, e pale ana i ka neʻe ʻana o nā cell mai ke kahua o ka ʻike ma muli o ka thermophoresis i ka wā i hoʻāla ʻia ai ka laser (e nānā i ke kiʻiʻoniʻoni M2 i nā Mea Hoʻohui). ʻO Thermophoresis ka neʻe ʻana o nā ʻāpana a i ʻole nā ​​molekole ma kahi ʻanuʻu wela, maʻamau mai ka wela a hiki i ke anu, a ʻaʻole ʻokoʻa ka bacteria43,47. Hoʻopau ʻia kēia hopena maikaʻi ʻole ma luna o kahi wahi i hāʻawi ʻia ma o ka hoʻohana ʻana iā SLM e hana i ka kukuna laser a loaʻa i kahi puʻu wela palahalaha.
Ma ka fig. Hōʻike ka Figure 2 i ka mahele wela i ana ʻia e CGM i loaʻa ma ka hoʻomālamalama ʻana i kahi pani aniani i uhi ʻia me nā nanoparticles gula me kahi kukuna laser annular (Fig. 1f). Ua ʻike ʻia ka puʻunaue wela pālahalaha ma luna o ka wahi holoʻokoʻa i uhi ʻia e ka kukuna laser. Ua hoʻonohonoho ʻia kēia wahi i 65 ° C, ʻo ka mahana ulu maikaʻi loa. Ma waho o kēia māhele, hā'ule maoli ka pō'ai wela i \(1/r\) (kahi o \(r\) ka ho'onohonoho radial).
he palapala ʻāina wela o nā ana CGM i loaʻa ma ka hoʻohana ʻana i ka kukuna laser annular e hoʻomālamalama i kahi papa o nā nanoparticles gula no ka loaʻa ʻana o kahi kiʻi wela pālahalaha ma luna o kahi pōʻai. b Isotherm o ka palapala 'āina wela (a). Hōʻike ʻia ka contour o ka kukuna laser e kahi pōʻai kiko hina hina. Ua hana hou ʻia ka hoʻokolohua ʻelua (e ʻike i nā Mea Hoʻohui, Kiʻi S4).
Ua nānā ʻia ka viability o nā cell bacteria no kekahi mau hola me ka hoʻohana ʻana i ka LA-HTM. Ma ka fig. Hōʻike ka 3 i ka manawa manawa no nā kiʻi ʻehā i lawe ʻia mai kahi kiʻiʻoniʻoni 3 hola 20 minuke (Kiʻiʻoniʻoni M3, ʻIke Hoʻohui). Ua ʻike ʻia ka nui o nā maʻi bacteria i loko o ka ʻāpana pōʻai i wehewehe ʻia e ka laser kahi i ʻoi aku ka maikaʻi o ka mahana, kokoke i 65 ° C. I ka hoʻokaʻawale ʻana, ua hoʻemi nui ʻia ka ulu ʻana o ke kelepona i ka wā i hāʻule ai ka mahana ma lalo o 50 ° C no 10 mau kekona.
ʻO nā kiʻi hohonu o G. stearothermophilus bacteria e ulu ana ma hope o ka hoʻomehana laser i nā manawa like ʻole, (a) t = 0 min, (b) 1 h 10 min, (c) 2 h 20 min, (d) 3 h 20 min, ma waho o 200 Wehe ʻia mai kahi kiʻiʻoniʻoni hoʻokahi minuke (M3 kiʻiʻoniʻoni i hāʻawi ʻia ma ka ʻIke Hoʻohui) i hoʻopaʻa ʻia ma ka palapala ʻāina wela. Huli ka laser i ka manawa \(t=0\). Ua hoʻohui ʻia nā isotherms i ke kiʻi ikaika.
No ka hoʻonui hou ʻana i ka ulu ʻana o ke kelepona a me kona hilinaʻi ʻana i ka mahana, ua ana mākou i ka piʻi ʻana o ka biomass o nā kolone like ʻole o ka bacteria i hoʻokaʻawale mua ʻia i ke kahua ʻike Movie M3 (Fig. 4). Hōʻike ʻia ka hua bacteria makua i koho ʻia ma ka hoʻomaka ʻana o ka hoʻokumu ʻana o ka mini colony forming unit (mCFU) ma ke Kiʻi S6. Ua lawe ʻia nā ana nuipaʻa maloʻo me kahi kāmela CGM 48 i hoʻohana ʻia no ka palapala ʻana i ka māhele wela. ʻO ka hiki o ka CGM ke ana i ke kaumaha maloʻo a me ka mahana ka ikaika o ka LA-HTM. E like me ka mea i manaʻo ʻia, ʻoi aku ka wikiwiki o ka ulu ʻana o ka maʻi bacteria (Fig. 4a). E like me ka mea i hōʻike ʻia ma ka ʻāpana semi-log ma Fig. 4b, ʻo ka ulu ʻana ma nā wela a pau e hahai ana i ka ulu hoʻonui, kahi e hoʻohana ai ka ʻikepili i ka hana exponential \(m={m}_{0}{10}^{t/\ tau }+ {{ \mbox{cst}}}\), kahi \(\tau {{{{{\rm{log}}}}}}}2\) – ka manawa hanau (a i ole ka manawa pālua), \(g =1/ \tau\) – ka ulu ʻana (ka helu o nā māhele i kēlā me kēia ʻāpana manawa ). Ma ka fig. Hōʻike ka 4c i ka nui o ka ulu ʻana a me ka manawa hana ma ke ʻano he hana o ka mahana. Hōʻike ʻia nā mCFU e ulu wikiwiki ana e ka saturation o ka ulu ʻana ma hope o ʻelua mau hola, kahi ʻano i manaʻo ʻia ma muli o ke kiʻekiʻe o ka maʻi bacterial kiʻekiʻe (e like me ka pae paʻa i nā moʻomeheu wai kahiko). ʻO ke ʻano maʻamau \(g\hema(T\ʻākau)\) (Fig. 4c) e pili ana i ka pihi ʻelua-phase i manaʻo ʻia no G. stearothermophilus me ka ulu ulu maikaʻi loa ma kahi o 60-65°C. Hoʻohālikelike i ka ʻikepili me ka hoʻohana ʻana i ke kumu hoʻohālike cardinal (Figure S5)49 kahi \(\left({{G}_{0}{;\;T}}_{{\min }};{T}_{{opt} } ;{T}_{{\max}}\'ākau)\) = (0.70 ± 0.2; 40 ± 4; 65 ± 1.6; 67 ± 3) °C, e like me nā waiwai ʻē aʻe i hōʻike ʻia ma ka palapala49. ʻOiai hiki ke hana hou ʻia nā ʻāpana pili i ka mahana, ʻokoʻa paha ka nui o ka ulu ʻana o \({G}_{0}\) mai kekahi hoʻokolohua a i kekahi (e nānā i nā kiʻi S7-S9 a me ke kiʻiʻoniʻoni M4). He ʻokoʻa i nā ʻāpana hoʻonohonoho wela, pono e like me ke ao holoʻokoʻa, ʻo ka nui o ka ulu ʻana e pili ana i nā waiwai o ka mea waena (loaʻa nā meaʻai, ʻike oxygen) i loko o ka microscale geometry i ʻike ʻia.
he ulu microbial ma nā wela like ʻole. mCFU: Nā ʻĀpana Hoʻokumu ʻana i nā ʻāpana liʻiliʻi. Loaʻa ka ʻikepili mai kahi wikiō o kahi huakini hoʻokahi e ulu ana i kahi ʻanuʻu wela (kiʻiʻoniʻoni M3). b E like me (a), ka pālākiō semi-logarithmic. c Ka helu o ka ulu\(\tau\) a me ka manawa hanau\(g\) mai ka ho'iho'i laina laina (b). Nā pahu kuhi hewa: ka pae wela ma luna o nā mCFU i hoʻonui i ke kahua o ka ʻike i ka wā ulu. Paʻa hewa kū pololei: kuhi hewa maʻamau.
Ma waho aʻe o ka ulu maʻamau, lele ʻia kekahi mau bacteria i kekahi manawa i ka wā hoʻomehana laser, kahi hana i manaʻo ʻia no ka bacteria me ka flagella. Hōʻike ka kiʻiʻoniʻoni M5 i nā ʻike hou aku i nā hana ʻauʻau. I loko o kēia hoʻokolohua, ua hoʻohana ʻia ka pāhawewe laser like ʻole e hana i kahi gradient wela, e like me ka hōʻike ʻana ma nā Kiʻi 1d, e a me S3. Hōʻike ka Kiʻi 5 i ʻelua mau kaʻina kiʻi i koho ʻia mai ka kiʻiʻoniʻoni M5 e hōʻike ana i ka neʻe ʻana o ka hua bacteria hoʻokahi a ʻo nā hua bacteria ʻē aʻe e noho ʻole.
Hōʻike nā papa manawa ʻelua (a) a me (b) i ka ʻauʻau ʻana o ʻelua bacteria like ʻole i kaha ʻia me nā pōʻai kiko. Ua unuhi ʻia nā kiʻi mai ka kiʻiʻoniʻoni M5 (i hāʻawi ʻia ma ke ʻano he mea hoʻohui).
I ka hihia o G. stearothermophilus, ua hoʻomaka ka neʻe ikaika o ka bacteria (Fig. 5) i kekahi mau kekona ma hope o ka hoʻāla ʻia ʻana o ka kukuna laser. Hoʻomaopopo kēia ʻike i ka pane kino o kēia microorganism thermophilic i ka piʻi ʻana o ka mahana, e like me ka mea i ʻike mua ʻia e Mora et al. 24 . Hiki ke ʻimi hou ʻia ke kumuhana o ka motility bacteria a me ka thermotaxis me ka hoʻohana ʻana i ka LA-HTM.
ʻAʻole pono e huikau ka ʻauʻau microbial me nā ʻano ʻoni kino ʻē aʻe, ʻo ia hoʻi (i) ʻO ka neʻe Brownian, ka mea i ʻike ʻia he neʻe huikau me ke kuhikuhi ʻole ʻia, (ii) convection 50 a me thermophoresis 43, i loko o ka neʻe maʻamau o ka neʻe ʻana ma kahi mahana. gradient.
ʻIke ʻia ʻo G. stearothermophilus no kona hiki ke hana i nā spores kūpaʻa loa (spore formation) i ka wā e ʻike ʻia ai i nā kūlana kaiapuni ʻino ma ke ʻano he pale. Ke lilo hou nā kūlana kaiapuni, ulu nā spores, hana i nā sela ola a hoʻomaka hou i ka ulu. ʻOiai ua ʻike ʻia kēia kaʻina hana sporulation/germination, ʻaʻole i ʻike ʻia i ka manawa maoli. Ke hoʻohana nei i ka LA-HTM, hōʻike mākou ma aneʻi i ka ʻike mua ʻana o nā hanana germination ma G. stearothermophilus.
Ma ka fig. Hōʻike ka 6a i nā kiʻi o ka hohonu hohonu (OT) i loaʻa me ka hoʻohana ʻana i kahi CGM o 13 spores. No ka manawa hōʻiliʻili holoʻokoʻa (15 h 6 min, \(t=0\) - ka hoʻomaka ʻana o ka hoʻomehana laser), 4 mai loko o 13 spores i hoʻoulu ʻia, ma nā helu manawa hou \(t=2\) h, \( 3\ ) h \(10 \)', \(9\) h \(40\)' a me \(11\) h \(30\)'. ʻOiai hoʻokahi wale nō o kēia mau hanana i hōʻike ʻia ma ka Figure 6, hiki ke ʻike ʻia nā hanana germination 4 i ka kiʻiʻoniʻoni M6 i ka mea hoʻohui. ʻO ka mea e mahalo ai, ʻike ʻia ka germination ma ke ʻano maʻamau: ʻaʻole ʻo nā spores a pau e ulu a ʻaʻole e ulu i ka manawa like, ʻoiai nā loli like i nā kūlana kaiapuni.
he 8 mau kiʻi OT (ka ʻaila immersion, 60x, 1.25 NA pahuhopu) a me (b) biomass evolution o G. stearothermophilus aggregates. c (b) Kakau ʻia ma ka pālākiō semi-log e hōʻike i ka linearity o ka ulu ulu (laina kaha).
Ma ka fig. Hōʻike ka 6b,c i ka biomass o nā heluna cell ma ke kahua o ka nānā ʻana ma ke ʻano he hana o ka manawa ma luna o ka manawa holoʻokoʻa o ka hōʻiliʻili ʻikepili. ʻO ka pohō wikiwiki o ka nui maloʻo i ʻike ʻia ma \(t=5\)h ma ka fig. 6b, c, ma muli o ka puka ʻana o kekahi mau cell mai ke kahua ʻike. ʻO ka ulu nui o kēia mau hanana ʻehā he \(0.77\pm 0.1\) h-1. ʻOi aku ka kiʻekiʻe o kēia waiwai ma mua o ka ulu ulu e pili ana i ka Figure 3. 3 a me 4, kahi e ulu maʻamau ai nā cell. ʻAʻole maopopo ke kumu o ka piʻi ʻana o ka ulu ʻana o G. stearothermophilus mai nā spores, akā ke hōʻike nei kēia mau ana i ka hoihoi o LA-HTM a hana ma ka pae kelepona hoʻokahi (aiʻole ma ka pae mCFU hoʻokahi) e aʻo hou e pili ana i ka dynamics o ke ola cell. .
No ka hōʻike hou aku i ka versatility o LA-HTM a me kāna hana ma nā wela kiʻekiʻe, ua nānā mākou i ka ulu ʻana o Sulfolobus shibatae, kahi hyperthermophilic acidophilic archaea me kahi mahana ulu maikaʻi loa o 80 ° C51. Hoʻohālikelike ʻia me G. stearothermophilus, ʻokoʻa loa kēia mau archaea, e like me 1 micron spheres (cocci) ma mua o nā lāʻau elongated (bacilli).
Aia ka kiʻi 7a i nā kiʻi hohonu optical sequential o S. shibate mCFU i loaʻa me ka hoʻohana ʻana iā CGM (e nānā i ke kiʻi hiʻohiʻona M7 ma nā Mea Hoʻohui). Ke ulu nei kēia mCFU ma kahi o 73 ° C, ma lalo o ka mahana maikaʻi loa o 80 ° C, akā i loko o ka pae wela no ka ulu ikaika. Ua ʻike mākou i nā hanana fission he nui i hoʻohālikelike ʻia nā mCFU me nā micrograpes o archaea ma hope o kekahi mau hola. Mai kēia mau kiʻi OT, ua ana ʻia ka biomass mCFU i ka manawa a hōʻike ʻia ma ka Figure 7b. ʻO ka mea hoihoi, ua hōʻike nā S. shibate mCFUs i ka ulu laina ma mua o ka ulu ʻana o ka exponential i ʻike ʻia me G. stearothermophilus mCFUs. He kūkākūkā lōʻihi 52 e pili ana i ke ʻano o ka ulu ʻana o ke kelepona: ʻoiai ʻo kekahi mau haʻawina e hōʻike ana i ka ulu ʻana o nā microbes i kūlike me ko lākou nui (exponential growth), ʻo kekahi e hōʻike i ka helu mau (ka ulu laina a i ʻole bilinear). E like me ka wehewehe ʻana e Tzur et al.53, ʻo ka hoʻokaʻawale ʻana ma waena o ka ulu ʻana o ka exponential a me (bi) linear e pono ai ka pololei o <6% i nā ana biomass, ʻaʻole hiki ke loaʻa i ka hapa nui o nā ʻenehana QPM, ʻoiai e pili ana i ka interferometry. E like me ka wehewehe ʻana e Tzur et al.53, ʻo ka hoʻokaʻawale ʻana ma waena o ka ulu ʻana o ka exponential a me (bi) linear e pono ai ka pololei o <6% i nā ana biomass, ʻaʻole hiki ke loaʻa i ka hapa nui o nā ʻenehana QPM, ʻoiai e pili ana i ka interferometry. Как объяснили Цур и др.53, различение экспоненциального и (би)линейного роста требует точности <6% в избеиостих для большинства методов QPM, даже с использованием интерферометрии. E like me ka wehewehe ʻana e Zur et al.53, ʻo ka hoʻokaʻawale ʻana ma waena o ka ulu exponential a me (bi) laina pono <6% pololei i nā ana biomass, ʻaʻole hiki ke loaʻa no ka hapa nui o nā ʻano QPM, ʻoiai me ka hoʻohana ʻana i ka interferometry.E like me ka wehewehe ʻana e Zur et al. 53, ʻo ka hoʻokaʻawale ʻana ma waena o ka ulu exponential a me (bi) laina pono e emi iho ma mua o 6% pololei i nā ana biomass, ʻaʻole hiki ke loaʻa no ka hapa nui o nā ala QPM, ʻoiai ke hoʻohana ʻia ka interferometry. Loaʻa iā CGM kēia pololei me ka pololei sub-pg i nā ana biomass36,48.
he 6 mau kiʻi OT (ka ʻaila immersion, 60x, NA pahuhopu 1.25) a me (b) micro-CFU biomass evolution i ana ʻia me CGM. E nānā i ke kiʻiʻoniʻoni M7 no ka ʻike hou aku.
ʻAʻole i manaʻo ʻia ka ulu laina kūpono o S. shibate a ʻaʻole i hōʻike ʻia. Eia nō naʻe, manaʻo ʻia ka ulu ʻana o ka exponential, ma ka liʻiliʻi loa, no ka mea, i ka hala ʻana o ka manawa, pono e puka mai nā māhele he nui o 2, 4, 8, 16 ... Manaʻo mākou ʻo ka ulu laina ma muli o ka pale ʻana i ke kelepona ma muli o ka paʻa ʻana o ke kelepona paʻa, e like me ka lohi ʻana o ka ulu ʻana o ka cell a hiki i kahi kūlana dormant ke kiʻekiʻe loa ke kiʻekiʻe o ka cell.
Hoʻopau mākou ma ke kūkākūkā ʻana i kēia mau wahi ʻelima o ka hoihoi: hoʻemi i ka leo hoʻomehana, hoʻemi i ka inertia thermal, hoihoi i nā nanoparticles gula, hoihoi i ka microscopy phase quantitative, a me kahi pae wela hiki ke hoʻohana ʻia i LA-HTM.
Hoʻohālikelike ʻia me ka hoʻomehana resistive, ʻo ka hoʻomehana laser i hoʻohana ʻia no ka hoʻomohala ʻana i ka HTM e hāʻawi i nā pono he nui, a mākou e hōʻike nei i kēia haʻawina. ʻO ka mea kūikawā, i loko o ka media wai ma ke kahua o ka nānā ʻana o ka microscope, mālama ʻia ka leo hoʻomehana i loko o kahi liʻiliʻi (10 μm) 3 mau puke. Ma kēia ʻano, ʻo nā microbes i ʻike ʻia e ʻeleu ana, ʻoiai ʻo nā bacteria ʻē aʻe e moe ana a hiki ke hoʻohana ʻia no ke aʻo hou ʻana i ka hāpana - ʻaʻohe pono e hoʻololi i ka hāpana i kēlā me kēia manawa e pono ke nānā ʻia kahi wela hou. Eia hou, hiki i ka microscale heating ke nānā pono i nā ʻano wela nui: Ua loaʻa mai ke kiʻi 4c mai kahi kiʻiʻoniʻoni 3-hola (Movie M3), ʻo ia ka mea e koi pinepine ai i ka hoʻomākaukau ʻana a me ka nānā ʻana i kekahi mau laʻana - hoʻokahi no kēlā me kēia o nā laʻana e aʻo ʻia. ʻO y ka mahana e hōʻike ana i ka helu o nā lā o ka hoʻokolohua. ʻO ka hoʻohaʻahaʻa ʻana i ka leo wela e mālama pū i nā ʻāpana optical a puni o ka microscope, ʻoi aku ka nui o ka lens, ma ka lumi wela, kahi pilikia nui i alo i ke kaiāulu a hiki i kēia manawa. Hiki ke hoʻohana ʻia ka LA-HTM me kekahi lens, me nā lens immersion aila, a e noho ma ka lumi wela me ka wela loa o ke kahua o ka nānā. ʻO ka palena nui o ke ʻano hoʻomehana laser a mākou e hōʻike nei i kēia noiʻi ʻana ʻo nā cell i pili ʻole a lana paha i mamao loa mai ke kahua o ka ʻike a paʻakikī ke aʻo ʻana. Hiki ke hoʻohana i nā lens hoʻonui haʻahaʻa no ka piʻi ʻana o ka wela ma mua o kahi mau haneli microns. Hoʻopili ʻia kēia akahele me ka emi ʻana o ka hoʻonā spatial, akā inā ʻo ka pahuhopu e aʻo i ka neʻe ʻana o nā microorganisms, ʻaʻole koi ʻia ka hoʻonā spatial kiʻekiʻe.
ʻO ka pālākiō manawa no ka hoʻomehana (a me ka hoʻomaha) o ka ʻōnaehana \({{{{\rm{\tau }}}}}}}}}_{{{\mbox{D}}}}\) pili i kona nui , e like me ke kanawai \({{{({\rm{\tau }}}}}}}}_{{{\mbox{D}}}}={L}^{2}/D\), kahi \ (L\ ) 'o ia ka nui hi'ona o ke kumu wela ('o ke anawaena o ka kukuna laser i loko o kā mākou ha'awina 'o \(L\ ma kahi o 100\) μm), \(D\) 'o ia ka diffusivity wela o ke kaiapuni (awelika i kā mākou. hihia, aniani a me ka wai Laki hoʻopuehu\(D\ ma kahi o 2\fold {10}^{-7}\) m2/s no laila, i kēia haʻawina, pane manawa o ke kauoha o 50 ms, ie, quasi-instantaneous). Hiki ke mana'o 'ia ka ho'ololi 'ana o ka mahana i ka manawa o ka ho'okolohua 'ana, akā, hiki ke 'ike 'ia i ka manawa pololei \(t=0\).
Hoʻohana ʻia kā mākou ʻano hana i manaʻo ʻia i kekahi substrate e hoʻomoʻi i ka māmā (no ka laʻana, nā laʻana kalepa me ka uhi ʻana o ITO). Eia nō naʻe, hiki i nā nanoparticles gula ke hāʻawi i ka absorption kiʻekiʻe i ka infrared a me ka absorption haʻahaʻa i ka laulā ʻike ʻia, ʻo nā hiʻohiʻona hope o ka hoihoi no ka nānā ʻana i ka optical i ka laulā ʻike ʻia, ʻoi aku ka hoʻohana ʻana i ka fluorescence. Eia kekahi, biocompatible ke gula, chemically inert, optical density hiki ke hoʻololi ʻia mai 530 nm a kokoke i infrared, a maʻalahi ka hoʻomākaukau ʻana o ka laʻana.
ʻAʻole hiki i ka transverse grating wavefront microscopy (CGM) ʻaʻole wale ka palapala ʻāina wela ma ka microscale, akā me ka nānā ʻana i ka biomass, e hoʻohana pono ai (inā ʻaʻole pono) i hui pū me LA-HTM. I loko o nā makahiki he umi i hala iho nei, ua hoʻomohala ʻia nā ʻenehana microscopy ʻē aʻe, ʻoi aku ka nui o ka bioimaging, a ʻo ka hapa nui o lākou e koi i ka hoʻohana ʻana i nā probes fluorescent wela-sensitive54,55. Eia nō naʻe, ua ʻāhewa ʻia kēia mau ʻano a ua ana kekahi mau hōʻike i nā loli wela kūpono ʻole i loko o nā cell, ma muli paha o ka hilinaʻi ʻana o ka fluorescence i nā kumu he nui ʻē aʻe ma mua o ka mahana. Eia kekahi, ʻaʻole paʻa ka hapa nui o nā ʻimi fluorescent i nā wela kiʻekiʻe. No laila, hōʻike ʻo QPM a me CGM i kahi ʻenehana microscopy wela kūpono no ke aʻo ʻana i ke ola ma nā wela kiʻekiʻe me ka hoʻohana ʻana i ka microscopy optical.
Hōʻike nā haʻawina o S. shibate, e ola maikaʻi ana ma 80 ° C, hiki ke hoʻohana ʻia ka LA-HTM e aʻo i nā hyperthermophiles, ʻaʻole nā ​​thermophiles maʻalahi. Ma ke kumu, ʻaʻohe palena i ka laulā o nā mahana e hiki ke loaʻa me ka hoʻohana ʻana i ka LA-HTM, a hiki i nā mahana ma luna o 100 ° C ke hiki i ke kaomi ʻana me ka paila ʻole, e like me ka hōʻike ʻana e kā mākou hui o 38 i nā noi kemika hydrothermal ma ka lewa. kaomi A. Hoʻohana ʻia kahi laser no ka hoʻomehana ʻana i nā nanoparticles gula 40 ma ke ʻano like. No laila, hiki i ka LA-HTM ke hoʻohana ʻia e nānā i nā hyperthermophiles i loaʻa ʻole me ka microscopy optical kiʻekiʻe kiʻekiʻe ma lalo o nā kūlana maʻamau (ʻo ia hoʻi ma lalo o ke koʻikoʻi o ke kaiapuni).
Ua hana ʻia nā hoʻokolohua a pau me ka microscope homemade, me ka hoʻomālamalama ʻo Köhler (me LED, M625L3, Thorlabs, 700 mW), mea paʻa specimen me ka manual xy movement, nā pahuhopu (Olympus, 60x, 0.7 NA, ea, LUCPlanFLN60X a i ʻole 60x, 1.25 NA, ʻaila. , UPLFLN60XOI), pahupaʻi CGM (QLSI cross grating, 39 µm pitch, 0.87 mm mai Andor Zyla camera sensor) e hāʻawi i ka ikaika a me ke kiʻi nalu, a me ka pahu pahu sCMOS (ORCA Flash 4.0 V3, 16-bit mode, mai Hamamatsu) e hoʻopaʻa i ka ʻikepili i hōʻike ʻia ma ke Kiʻi 5 (ʻauʻau bacteria). ʻO ka dichroic beam splitter he 749 nm BrightLine edge (Semrock, FF749-SDi01). ʻO ka kānana ma ke alo o ke kāmela he kānana pōkole pōkole 694 (FF02-694/SP-25, Semrock). Titanium sapphire laser (Laser Verdi G10, 532 nm, 10 W, pumped tsunami laser cavity, Spectra-Physics ma Fig. 2-5, pani hou ia e ka Millenia laser, Spectraphysics 10 W, pumped Mira laser cavity, Coherent, no Fig. 2 -5). 6 a me 7) ua hoʻonohonoho ʻia i ka lōʻihi nalu \({{{({\rm{\lambda }}}}}}}=800\) nm, e pili ana i ka plasmon resonance spectrum o nā nanoparticles gula. 1152 pixels) i kūʻai ʻia mai Meadowlark Optics Ua helu ʻia nā hologram me ka Gerchberg-Saxton algorithm e like me ka mea i wehewehe ʻia ma ka loulou.
ʻO ka cross grating wavefront microscopy (CGM) kahi ʻenehana microscopy optical e pili ana i ka hoʻohui ʻana i kahi ʻāpana diffraction grating ʻelua (ʻike ʻia hoʻi ʻo cross grating) ma kahi mamao o hoʻokahi millimeter mai ka mea ʻike kamepiula maʻamau. ʻO ka laʻana maʻamau o kahi CGM a mākou i hoʻohana ai i kēia haʻawina, ua kapa ʻia ʻo ka interferometer transverse shift ʻehā-lōʻihi (QLSI), kahi i loaʻa ai i ka cross-grating kahi hiʻohiʻona kaha/phase checkerboard i hoʻokomo ʻia a patent ʻia e Primot et al. i ka makahiki 200034. Hoʻokumu nā laina kuʻikuʻi kū a me ke ākea i nā aka e like me ka grid ma luna o ka mea ʻike, hiki ke hoʻololi ʻia ka distortion i ka manawa maoli no ka loaʻa ʻana o ka distortion wavefront (a i ʻole profile phase like) o ke kukui hanana. Ke hoʻohana ʻia ma kahi microscope, hiki i kahi pahupaʻikiʻi CGM ke hōʻike i ka ʻokoʻa o ke ala ʻokoʻa o kahi mea i kiʻi ʻia, ʻike ʻia hoʻi ʻo ka hohonu hohonu (OT), me ka ʻike ma ke kauoha o nanometers36. Ma kekahi ana CGM, i mea e hoopau ai i na hemahema o na mea opua a i ole na kukuna, pono e lawe a unuhiia mai kekahi kii ma hope mai.
Hana ʻia ka microscopy me ka hoʻohana ʻana i kahi kāmela CGM e like me ka mea i wehewehe ʻia ma ka kuhikuhi. 32. I ka pōkole, hoʻololi ka hoʻomehana ʻana i ka wai i kona ʻano kuhikuhi refractive, e hana ana i kahi hopena lens wela e hoʻohuli i ke kukui hanana. Hoʻopili ʻia kēia ʻāwili nalu e ka CGM a hana ʻia me ka hoʻohana ʻana i kahi algorithm deconvolution e loaʻa ai kahi mahele wela ʻekolu-dimensional i loko o ka wai. Inā māhele like ʻia nā nanoparticles gula a puni ka hāpana, hiki ke hana ʻia ka palapala ʻāina wela ma nā wahi bacteria-free e hana i nā kiʻi ʻoi aku ka maikaʻi, ʻo ia kā mākou e hana ai i kekahi manawa. Ua kiʻi ʻia ke kiʻi CGM me ka hoʻomehana ʻole (me ka laser off) a ma hope i hopu ʻia ma ka wahi like i ke kiʻi me ka laser on.
Hoʻokō ʻia ke ana nuipa maloʻo me ka hoʻohana ʻana i ka pahu pahu CGM like i hoʻohana ʻia no ke kiʻi wela. Ua loaʻa nā kiʻi kuhikuhi CGM ma ka neʻe wikiwiki ʻana i ka hāpana ma x a me y i ka wā o ka hoʻolaha ʻana ma ke ʻano o ka hoʻohālikelike ʻana i kekahi inhomogeneity i ka OT ma muli o ka loaʻa ʻana o ka bacteria. Mai nā kiʻi OT o ka bacteria, ua kiʻi ʻia ko lākou biomass me ka hoʻohana ʻana i kahi hui o nā kiʻi ma luna o nā wahi i koho ʻia me ka hoʻohana ʻana i ka algorithm segmentation homemade o Matlab (e nānā i ka ʻāpana "Numerical code"), ma hope o ke kaʻina hana i wehewehe ʻia ma ref. 48. I ka pōkole, hoʻohana mākou i ka pilina \(m={\alpha}^{-1}\iint {{\mbox{OT}}}\left(x,y\right){{\mbox{d}} } x{{\mbox{d}}}y\), kahi o \({{\mbox{OT}}}\hema(x,y\'ākau)\) ke kiʻi hohonu opua, \(m\) ka paona maloo a me \({{{{\rm{\alpha }}}}}}}\) he mau. Ua koho mākou i \({{{{\rm{\alpha))))))=0.18\) µm3/pg, he mea maʻamau no nā pūnaewele ola.
Ua hoʻokomo ʻia kahi pepa uhi 25 mm ke anawaena a me 150 µm mānoanoa i uhi ʻia me nā nanoparticles gula i loko o kahi keʻena AttofluorTM (Thermofisher) me nā nanoparticles gula e kū pono ana i luna. Ua mālama mua ʻia ʻo Geobacillus stearothermophilus i ka pō ma ke kikowaena LB (200 rpm, 60°C) ma mua o kēlā me kēia lā o nā hoʻokolohua. ʻO kahi hāʻule o 5 µl o kahi hoʻokuʻu o G. stearothermophilus me kahi optical density (OD) o 0.3 a i 0.5 i kau ʻia ma luna o kahi pepa uhi me nā nanoparticles gula. A laila, ua hoʻokuʻu ʻia kahi pahu uhi puni 18 mm ka anawaena me kahi puka 5 mm i ke anawaena ma ke kikowaena, a ua hoʻopili pinepine ʻia ka 5 μl o ka hoʻokuʻu ʻana i ka bacteria me ka ʻano optical like i ke kikowaena o ka lua. Ua hoʻomākaukau ʻia nā pūnāwai ma nā uhi uhi e like me ke kaʻina hana i wehewehe ʻia ma ref. 45 (e nānā i ka ʻike hou aku no ka ʻike hou aku). A laila e hoʻohui i ka 1 ml o LB medium i ka uhi uhi e pale ai i ka maloʻo ʻana o ka wai. Hoʻokomo ʻia ka uhi uhi hope ma luna o ka poʻi paʻa o ke keʻena Attofluor™ e pale ai i ka hoʻoheheʻe ʻana o ka mea i ka wā o ka incubation. No nā hoʻokolohua germination, ua hoʻohana mākou i nā spores, ka mea, ma hope o nā hoʻokolohua maʻamau, i kekahi manawa uhi i ka uhi uhi o luna. Ua hoʻohana ʻia kahi ʻano like no ka loaʻa ʻana o Sulfolobus shibatae. ʻEkolu mau lā (200 rpm, 75 ° C) o ka mahi mua ʻana o Thiobacillus serrata i lawe ʻia ma waena o 182 (DSMZ).
Ua hoʻomākaukau ʻia nā laʻana o nā nanoparticles gula e ka micellar block copolymer lithography. Ua wehewehe kikoʻī ʻia kēia kaʻina hana ma Chap. 60. ʻO ka pōkole, ua hui pū ʻia nā micelles e hoʻopili ana i nā ion gula ma ka hui ʻana i ka copolymer me ka HAuCl4 i toluene. Hoʻokomo ʻia nā uhi uhi i hoʻomaʻemaʻe ʻia i loko o ka hopena a mālama ʻia me ka UV irradiation i mua o kahi mea hoʻohaʻahaʻa e loaʻa ai nā hua gula. ʻO ka hope, ua ulu ʻia nā hua gula ma ka hoʻopili ʻana i kahi uhi uhi me ka wai wai o KAuCl4 a me ka ethanolamine no 16 mau minuke, a ua hopena i kahi hoʻonohonoho quasi-periodic a kūlike loa o nā nanoparticles gula non-spherical i loko o ka infrared kokoke.
No ka hoʻololi ʻana i nā interferograms i nā kiʻi OT, ua hoʻohana mākou i kahi algorithm homemade, e like me ka kikoʻī ma ka loulou. 33 a loaʻa ma ke ʻano he pūʻulu Matlab i loko o ka waihona waihona lehulehu: https://github.com/baffou/CGMprocess. Hiki i ka pūʻolo ke helu i ka ikaika a me nā kiʻi OT e pili ana i nā interferograms i hoʻopaʻa ʻia (me nā kiʻi kuhikuhi) a me nā mamao o nā kāmela.
No ka helu ʻana i ke kumu hoʻohālike i hoʻohana ʻia i ka SLM no ka loaʻa ʻana o kahi kiʻi wela i hāʻawi ʻia, ua hoʻohana mākou i kahi algorithm39,42 homemade i kūkulu mua ʻia i loaʻa i loko o kēia waihona lehulehu: https://github.com/baffou/SLM_temperatureShaping. ʻO ka mea hoʻokomo ke kahua wela i makemake ʻia, hiki ke hoʻonohonoho ʻia ma ke kikohoʻe a i ʻole ma ke kiʻi bmp monochrome.
No ka hoʻokaʻawale ʻana i nā cell a ana i ko lākou kaumaha maloʻo, ua hoʻohana mākou i kā mākou Matlab algorithm i paʻi ʻia ma kēia waihona lehulehu: https://github.com/baffou/CGM_magicWandSegmentation. Ma kēlā me kēia kiʻi, pono ka mea hoʻohana e kaomi i ka bacteria a i ʻole mCFU o ka hoihoi, e hoʻololi i ka ʻike wand, a hōʻoia i ke koho.
No ka ʻike hou aku e pili ana i ka hoʻolālā haʻawina, e ʻike i ka abstract Nature Research Report i pili i kēia ʻatikala.
Loaʻa nā ʻikepili e kākoʻo ana i nā hopena o kēia noiʻi mai nā mea kākau ma muli o ke noi kūpono.
ʻO ke kumu kumu i hoʻohana ʻia ma kēia noiʻi ʻana ua kikoʻī ʻia ma ka ʻāpana Methods, a hiki ke hoʻoiho ʻia nā mana debug mai https://github.com/baffou/ ma kēia mau waihona: SLM_temperatureShaping, CGMprocess, a me CGM_magicWandSegmentation.
Mehta, R., Singhal, P., Singh, H., Damle, D. & Sharma, AK Insight i nā thermophiles a me kā lākou mau noi ākea ākea. Mehta, R., Singhal, P., Singh, H., Damle, D. & Sharma, AK Insight i nā thermophiles a me kā lākou mau noi ākea ākea.Mehta, R., Singhal, P., Singh, H., Damle, D. a me Sharma, AK Nānā o nā thermophiles a me kā lākou noi ākea. 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. a me Sharma AK ʻO ka ʻike hohonu o nā thermophiles a me kahi ākea o nā noi.3 Biotechnology 6, 81 (2016).