I-microscopy ye-Fluorescence: imigaqo yendlela

Ukuxhamla kunye nokuhlaziywa kombane ngokugqithisileyo kunye nemithombo yezolimo kubangelwa yi-phosphorescence okanye i-fluorescence. Ukwahlukana phakathi kwezinto eziqhambukayo kubandakanya ubude bexesha eliphakathi kokungena kokukhanya kunye nokukhutshwa komkhuhlane. Ngaphantsi kwe-fluorescence, ezi nkqubo zenzeka phantse, kunye ne-phosphorescence, kunye nokulibaziseka.

Umlando

Ngomnyaka we-1852 isazi senzululwazi yaseStrans Stokes saqala ukuchaza ukukhanya kwe-fluorescence. Wazisa ixeshana elitsha ngenxa yezinto ezenziwayo kunye ne- fluorspar, ebonisa ukukhanya okubomvu phantsi kwefuthe le-ultraviolet. I-Stokes yathetha into ekhangayo. Wafumanisa ukuba i-longueth longth with radiorescent radiation ihlala ikhulu kuneleyo ye-excitation light flux.

Ukuqinisekisa i-hypothesis ekhulwini le-19, ezininzi iinzame zenziwa. Bakhombisa ukuba iintlobo zeesampulu zifake i-fluoresce phantsi kwesenzo se-ultraviolet. Phakathi kwezinye izixhobo, phakathi kwezinye izinto, kwakukhona iikristal, i-resins, amaminerali, i-chlorophyll, izinto eziluncedo zokwelapha, ama-inorganic compounds, iivithamini, ii-oyile. Ukusetyenziswa ngokuthe ngqo kwameyta yokwenza uhlalutyo lwe-biological kuqale ngo-1930.

I-microscopy ye-Fluorescence: inkcazo

Ezinye zezinto ezisetyenziswe kwizifundo zesahlulo sokuqala sekhulu lama-20 zinemeko ephezulu. стал важнейшим инструментом и в биомедицинских, и в биологических исследованиях. Ngenxa yezalathisi ezingenakufezwa ngeendlela ezahlukeneyo, indlela yokwenza i-microscopy ye-fluorescence ibe yinto ebalulekileyo kuzo zombini uphando lwezinto eziphilayo. Iziphumo ezifumeneyo zazingabalulekanga kakhulu kwizixhobo zesayensi.

? Ziziphi iingenelo ze-microscopy ye-fluorescence ? Ngokuncediswa kwezinto ezintsha zenzeke ukuba zihlukanise iiseli ezikhethekileyo kunye neengxenye ezincinci. I-microscope ye-fluorescent iyakwazi ukubona iamolekyu nganye. Iindidi ezahlukeneyo zeetayiti zivumela ukuchongwa kwezinto eziliqela ngexesha elifanayo. Nangona isisombululo esincinci sendawo eso sixhobo se-diffraction, esele sithembele kwiipropati ezithile zeesampuli, kuyafumaneka kwakhona ukubona iamolekyu ngaphantsi kweli nqanaba. Iisampuli ezahlukahlukeneyo emva kokuba i-irradiation ibonise i-autofluorescence. Le ngxaki iyasetyenziswa ngokubanzi kwi-petroleji, i-botany, i-semiconductor industry.

Izixhobo

Ukufunda izicubu zezilwanyana okanye i-pathogenic microorganisms kudla ngokuxakeka okanye kunobuthakathaka okanye obunamandla obukhulu be-auto fluorescence. Nangona kunjalo, ukubaluleka kwezi zifundo kukungeniswa kwizinto eziphathekayo ezixhamle kwiqondo elithile kunye nokuhambisa ukukhanya okubalulekileyo kokufuna ngamandla. I-Fluorochromes isebenza njengedayi ekwazi ukuzibamba kwizakhiwo (ezingabonakaliyo okanye ezibonakalayo). Ngexesha elifanayo, zikhethe kakhulu ngokubhekiselele kwiithagethi kunye nokuvunwa kwe-quantum.

стала широко применяться с появлением естественных и синтетических красителей. I-microscopy ye-Fluorescence isetyenziswa ngokubanzi kunye nokuza kweedayi zendalo kunye nokwenziwa. Babeneenkcukacha ezichanekileyo zokukhupha nokukhutshulwa kwaye zajoliswe kwiinjongo ezithile zezinto eziphilayo.

Ukuchongwa kwamakhemleksi ngamnye

Ngokuqhelekileyo, phantsi kweemeko ezifanelekileyo, kunokwenzeka ukuba ubhalise ukukhanya kwento ethile. Ukwenza oku, phakathi kwezinye izinto, kuyimfuneko ukuqinisekisa ukuvakala kwengxolo ephantsi kunye nemvelaphi ebonakalayo. I-moleculecule fluorescein ngaphambi kokutshatyalaliswa ngenxa ye-photobleaching ingavela kuma-photons ayi-300. Kwi-20% yokuqokelela nokusebenza kwenkqubo, banokubhaliswa kwisixa samawaka angama-60.

, основанная на лавинных фотодиодах или электронном умножении, позволяла исследователям наблюдать поведение отдельных молекул на протяжении секунд, а в ряде случаев и минут. I-microscopy ye-Fluorescence , esekelwe kwi-photodiode ye-avalanche okanye ukuphindaphinda kwe-electron, abaphengululi bavumelekile ukuba bagcine ukuziphatha kwama-molecule ngamanye imizuzu, kwaye kwezinye iimeko, imizuzu.

Bunzima

Ingxaki ebalulekileyo kukunciphisa ingxolo evela kwimvelaphi ebonakalayo. Ngenxa yokuba ezininzi izinto ezisetyenziselwa ukwenziwa kwezihlunu kunye neelensi zibonakalisa ukuzenzekela ngokuzenzekelayo, imizamo yenzululwazi kwimigangatho yokuqala ibhekiselele kwimveliso yamacandelo ane-fluorescence ephantsi. Nangona kunjalo, uvavanyo olwalandelayo lukhokelela kwizigqibo ezintsha. , основанная на полном внутреннем отражении, позволяет достичь низкого фона и высокоинтенсивного возбуждающего светового потока. Ngokukodwa, kwafunyanwa ukuba i-microscopy ye-fluorescence , ngokusekelwe kwingcamango yangaphakathi, ifikelela kwimvelaphi ephantsi kunye nokukhanya okunamandla okukhulu.

Indlela

, основанной на полном внутреннем отражении, заключаются в использовании быстрозатухающей или нераспространяющейся волны. Imigaqo ye-microscopy ye-fluorescence , ngokusekelwe kwingcamango yangaphakathi yangaphakathi, iqulethwe ukusetyenziswa kokutshatyalaliswa ngokukhawuleza okanye okungabonakaliyo. Kuvela kumda weendaba kunye nama-indices ahlukeneyo . Kule meko, isibonda sokukhanya sidlula kwi-prism. Inokhenketho oluphezulu lokukhupha.

I-prism iqhotyoshelwe kwisisombululo esinamandla okanye iglasi eneeperitha ephantsi. Ukuba umbane wokukhanya ukhankanywe kuwo ngecala elona likhulu kuneyona nto ibalulekileyo, i-boram ibonakaliswe ngokupheleleyo kwi-interface. Le nto ibangela ukuba kungabonakali. Ngamanye amagama, intsimi ye-electromagnetic eyenziwa yongena phakathi kunye nenani elincinci lokukhangela ibanga elingaphantsi kwama-nanometer angama-200.

Ngomatshini ongeyena, ubukhulu bokukhanya buya kukwanela ukukhuthaza ama-fluorophores. Nangona kunjalo, ngenxa yobunzulu bayo obunzima kakhulu, umthamo wayo uya kuba mncinci kakhulu. Ngenxa yoko, isiqalo esisezantsi sivela.

U lungiso

I-microscopy ye-Fluorescence, esekelwe kwingcamango yangaphakathi yangaphakathi, inokuzaliswa ngoncedo lwe-epi-illumination. Oku kufuna iilensi ngokukhutshwa kwamanani amaninzi (ubuncinane be-1.4, kodwa kuyinqweneleka ukuba ifike kwi-1.45-1.6), kunye nensimu ekhanyisiweyo yeso sixhobo. Olu phuhliso lufezekiswa ngokusebenzisa indawo yobukhulu obukhulu. Ukuze ulungelelwano olukhulu, isalathiso esincinci sisetyenzisiweyo, ngaluphi udidi lokungqubuzana oluvaliweyo. Ukufumana i-angle engundoqo, emva koko kubonakaliswe ukugqitywa okupheleleyo, inqanaba eliphezulu lokuchithwa kwemidiza yokucwilisa kwiilensi kunye neklasi yokugubungela yesikrosiksi iyadingeka.