Fluorescence microscopy: izimiso indlela

Absorption futhi kabusha ekushayweni ukukhanya okwengeziwe uketshezi wezinto ezingaphili futhi organic kuwumphumela phosphorescence noma fluorescence. Umehluko phakathi imihlola ubude isikhawu phakathi ukumuncwa futhi ekushayweni ekucucuzeni ukukhanya. Lapho fluorescence kilelihlelo zenzeka cishe ngasikhathi sinye, kanti phosphorescence - nabathile kokulibala.

Imininingwane zomlando

Ngo 1852, usosayensi waseBrithani Stokes, owokuqala owachaza lesi fluorescence. Wethula i-term entsha ngenxa ukucwaninga nge fluorspar, okuyinto zikhiphela elibomvu ngaphansi kwe-ultraviolet. Stokes waphawula buyinto ezithakazelisayo. Litholakala ukuthi nombono we emisebeni Fluorescent uhlale mkhulu kunezinhliziyo ukugeleza ukukhanya excitation.

Ukuqinisekisa umbono ekhulwini le-19 ziye zaba khona ucwaningo. Babonisa ukuthi ezihlukahlukene amasampula fluoresce bedle zokukhanya oku-ultraviolet. Phakathi kwezinto, phakathi kwezinye izinto, zinhlayiya kwaba, inhlaka, amaminerali, asinayo i-chlorophyll, izidakamizwa ongahluziwe, compounds wezinto ezingaphili, amavithamini, amafutha. I Ukulisebenzisa ngokuqondile kanjena yokudaya ukuhlolwa yemvelo waqala kuphela ngo-1930

Fluorescence microscopy: incazelo

Ezinye izimpahla ezisetshenziselwa Engxenyeni yokuqala izifundo kwekhulu lama-20 embukisweni ngokucacile eliphezulu. стал важнейшим инструментом и в биомедицинских, и в биологических исследованиях. Ngenxa ukusebenza, okungase awutholakali ngokumadanisa izindlela, indlela fluorescence microscopy isibe ithuluzi ebalulekile ocwaningweni bezokwelapha nezinto eziphilayo. Ngokulinganayo Imiphumela ebalulekile eyatholakala, futhi izinto.

? Ziyini izinzuzo owenza indlela fluorescence microscopy? Usebenzisa amathuluzi amasha isibe kungenzeka nokukhetha cell ethize kakhulu izingxenye submicroscopic. Fluorescence microscopy kungathola molecule elilodwa. A ezihlukahlukene udayi litfuba izinto eziningi ngesikhathi esisodwa. Naphezu elinganiselwe isinqumo kwezendawo umkhawulo umumo womsebenzi we imishini, okuyinto yena, kuncike izakhiwo ezithile isampuli, ukuhlonza zishintshe ngezansi alolu ubuye ngempela kungenzeka. amasampula abahlukahlukene ngemva ngemisebe umbukiso autofluorescence. Lesi simo esiyingqayizivele sibangelwa kabanzi lisetshenziswe petrology, ezesayensi yezitshalo embonini Semiconductor.

Izici

Tadisha isilwane izicubu noma abangelwa ngokuvamile eziyinkimbinkimbi noma ayinawo amandla noma enamandla kakhulu autofluorescence unspecific. Nokho, ukubaluleka ezifundweni ozuza isingeniso etincenyeni impahla yavusa ngesikhathi wavelength ethize futhi likhipha adingekayo ukukhanya ukugeleza umfutho. Fluorochromes ukwenza njenge udayi uyakwazi ngokuzimela enamathiselwe izakhiwo (ezibonwayo kumbe ezingabonwayo). Ngakho banalo selectivity eliphezulu elitsheni, futhi isivuno quantum.

стала широко применяться с появлением естественных и синтетических красителей. Fluorescence microscopy iye kabanzi kusukela obhekwe udayi yemvelo kanye zokwenziwa. Alihlutha amaphrofayli ezithile ukushuba ekushayweni futhi excitation futhi ziqondiswe Okuqondiwe ethize begazi.

Ukukhomba molecule ngabanye

Kaningi, izimo ezikahle, ungabhalisa ehlukile isici kukhanya. Ngenxa yalesi, phakathi kwezinye izinto, kubalulekile ukuhlinzeka umsindo ngokwanele eliphansi idetector futhi ngemuva optical. i-molecule Fluorescein yokuhluleka ngenxa photobleaching engase ikhiphe kuze kufinyelele ayizinkulungwane. 300 izinhlayiyana eziningi zokukhanya. Ku-20% iqoqo nekusebenta inqubo futhi kungaba ukubhalisa nabo isamba lezingalo eziyizinkulungwane ezingu-60.

, основанная на лавинных фотодиодах или электронном умножении, позволяла исследователям наблюдать поведение отдельных молекул на протяжении секунд, а в ряде случаев и минут. Fluorescence microscopy esekelwe photodiodes ngendathane nofana ukubuyabuyelela electronic, wavumela abacwaningi ukuba bagcine ukuziphatha molecule umuntu ngokusebenzisa yesibili, futhi kwezinye izimo ngisho imizuzu.

eyinkimbinkimbi

A okumqoka esivuna nokucindezela optical umsindo omkhulu ovela ngemuva. Ngenxa yokuthi abaningi ezisetshenziswa umklamo okusefa lens abonise abanye autofluorescence, imizamo ososayensi kusaqalwa zazibheka ekukhiqizeni izakhi kokuba fluorescence ongaphakeme. Nokho, ucwaningo okwalandela sezishiye iziphetho entsha. , основанная на полном внутреннем отражении, позволяет достичь низкого фона и высокоинтенсивного возбуждающего светового потока. Ikakhulukazi, kwatholakala ukuthi fluorescence microscopy, esekelwe kuveza Imininingwane yangaphakathi, ivumela ukuba kuzuzwe isizinda ongaphakeme futhi umfutho high excitation ukukhanya.

indlela

, основанной на полном внутреннем отражении, заключаются в использовании быстрозатухающей или нераспространяющейся волны. Izimiso fluorescence microscopy, esekelwe kuveza Imininingwane yangaphakathi ukusetshenziswa wave evanescent noma evanescent. Kwenzeka emngceleni phakathi kwabezindaba nge ezahlukene indices yezokubona. Kulokhu, ugongolo ukukhanya sidlula iphrizimu. It has a high yezokubona Inkomba ipharamitha.

Iphrizimu eseduze isixazululo aqueous noma ingilazi nge ipharamitha ongaphakeme. Uma umsebe wokukhanya sibhekiswe ke engela ukuthi kubalulekile ngaphezulu, ugongolo ngokuphelele kubonakala kusukela esibonakalayo. Lesi simo esiyingqayizivele sibangelwa yena, kubangela amagagasi nonpropagating. Ngamanye amazwi, lo elakhiwe ensimini kagesi okuyinto ingena phakathi i-medium ehambisana ephansi yezokubona Inkomba ipharamitha ibanga esingaphansi 200 nanometer.

I evanescent wave Amandla okukhanya kwakwanele ukuba bajabule fluorophores. Nokho, ngenxa ukujula yayo encane kakhulu, umthamo yayo kuyoba encane kakhulu. Imiphumela iba yinhlanganisela yokukhandleka isizinda okusezingeni eliphansi.

ukuguqulwa

Fluorescence microscopy kusekelwe kuveza Imininingwane yangaphakathi, ezingase zithathwe nge EPI-ukukhanya. Lokhu kudinga lens nge high beso zezinombolo (okungenani 1.4, kodwa kuyatuseka ukuthi wafika 1.45-1.6), futhi kancane esikhanyayo apharathasi ensimini. Lesi sakamuva kufinyelelwa nge usayizi encane indawo. Ukuze ukufana okukhulu usebenzisa indandatho mncane, iyiphi ivinjelwe ingxenye ukusakaza. Ukuze engela ezibucayi, ngemva kwalokho kukhona kuveza Imininingwane, sidinga ezingeni lelisetulu kwalungiswa izinkunga zokubona medium ukucwiliswa in lens kanye ikhava ingilazi microscope.