{"id":12979,"date":"2021-06-08T20:10:05","date_gmt":"2021-06-08T23:10:05","guid":{"rendered":"https:\/\/mindthegraph.com\/blog\/?p=12979"},"modified":"2023-01-05T14:22:32","modified_gmt":"2023-01-05T17:22:32","slug":"microscopy-how-scientists-use-fluorescence-to-see-inside-cells","status":"publish","type":"post","link":"https:\/\/mindthegraph.com\/blog\/sk\/mikroskopia-ako-vedci-pouzivaju-fluorescenciu-na-pozorovanie-vnutra-buniek\/","title":{"rendered":"Mikroskopia: Ako vedci vyu\u017e\u00edvaj\u00fa fluorescenciu, aby videli do vn\u00fatra buniek"},"content":{"rendered":"

Ve\u013ekou s\u00fa\u010das\u0165ou pr\u00e1ce vedca je navrhovanie a vykon\u00e1vanie experimentov.<\/p>\n\n\n\n

Kombin\u00e1cia laborat\u00f3rnych techn\u00edk odpovie na v\u00e4\u010d\u0161inu ot\u00e1zok, ktor\u00e9 vedci navrhuj\u00fa, a pracovn\u00fd postup pri navrhovan\u00ed nov\u00fdch met\u00f3d z\u00e1vis\u00ed od vzdelania a sk\u00fasenost\u00ed vedca.<\/p>\n\n\n\n

Obr\u00e1zky buniek m\u00f4\u017eu biol\u00f3gom poveda\u0165 ve\u013ea o tom, \u010do sa deje v procesoch a mechanizmoch, ktor\u00e9 sk\u00famaj\u00fa.<\/p>\n\n\n\n

Sveteln\u00e1 mikroskopia<\/strong> je ve\u013emi roz\u0161\u00edren\u00e1 technika v biologick\u00fdch ved\u00e1ch.<\/p>\n\n\n\n

Pou\u017eitie farb\u00edv, protil\u00e1tok a fluorescen\u010dn\u00fdch sond umo\u017e\u0148uje vedcom vidie\u0165 v bunk\u00e1ch mikroskopu obrazy toho, \u010do by inak bolo pr\u00edli\u0161 mal\u00e9 na to, aby sa dalo vidie\u0165 a dokonca pochopi\u0165.<\/p>\n\n\n\n

Fluorescen\u010dn\u00e9 mikroskopy<\/strong> a pou\u017eitie fluorochr\u00f3mov bolo mo\u017en\u00e9 v roku 19301<\/sup><\/a>a dnes je mo\u017en\u00e9 farbi\u0165 prote\u00edny, organely a \u0161trukt\u00fary v bunk\u00e1ch a tkaniv\u00e1ch mnoh\u00fdmi kombin\u00e1ciami fluorochr\u00f3mov.<\/p>\n\n\n\n

Fluorochr\u00f3my (alebo fluorofory)<\/strong> s\u00fa molekuly, ktor\u00e9 po excit\u00e1cii \u0161pecifickou vlnovou d\u013a\u017ekou svetla vy\u017earuj\u00fa svetlo definovanej vlnovej d\u013a\u017eky, ktor\u00e9 zachytia objekt\u00edvy mikroskopu a premenia ho na skuto\u010dn\u00fd obraz.<\/p>\n\n\n\n

Kombin\u00e1cia fluorescencie, \u0161o\u0161oviek a kamier n\u00e1m umo\u017e\u0148uje sn\u00edma\u0165 procesy vo vn\u00fatri buniek z mnoh\u00fdch r\u00f4znych poh\u013eadov a aspektov.<\/p>\n\n\n\n

Pomocou mikroskopu m\u00e1me napr\u00edklad \u0161ir\u0161\u00ed poh\u013ead na pl\u00e1tok my\u0161ieho mozgu pri objekt\u00edve 2,5x alebo 4x a mal\u00e9 detaily sk\u00faman\u00e9ho akt\u00ednov\u00e9ho cytoskeletu v tej istej vzorke pri objekt\u00edve 63x.<\/p>\n\n\n\n

Na tieto testy m\u00f4\u017eeme pou\u017ei\u0165 protil\u00e1tky alebo farbiv\u00e1 proti \u0161pecifick\u00fdm prote\u00ednom pr\u00edtomn\u00fdm v bunke alebo tkanive, pri\u010dom protil\u00e1tka je zvy\u010dajne vybaven\u00e1 fluoroforom.<\/p>\n\n\n\n

Stokesov posun vysvet\u013euje tento jav: fluorofory str\u00e1caj\u00fa vibra\u010dn\u00fa energiu vo forme vy\u017earovan\u00e9ho svetla, ke\u010f prech\u00e1dzaj\u00fa z excitovan\u00e9ho stavu sp\u00e4\u0165 do z\u00e1kladn\u00e9ho stavu. Fluorescen\u010dn\u00e9 mikroskopy poskytn\u00fa svetlo na excit\u00e1ciu fluoroforu a prij\u00edmaj\u00fa jeho emitovan\u00e9 svetlo. Emitovan\u00e9 svetlo sa m\u00f4\u017ee zachyti\u0165 objekt\u00edvom, spracova\u0165 v CCD kamere a transformova\u0165 na digit\u00e1lny obraz.<\/p>\n\n\n\n

O z\u00edskavan\u00ed obrazu buniek v\u0161ak budeme hovori\u0165 nesk\u00f4r. Teraz by sme v\u00e1m mali predstavi\u0165 pr\u00edklady a tipy na hlavn\u00e9 kroky pred z\u00edskan\u00edm sn\u00edmky.<\/p>\n\n\n\n

Ako vyber\u00e1me a kombinujeme r\u00f4zne typy farb\u00edv a protil\u00e1tok, aby sme videli a pochopili vz\u0165ahy medzi organelami a prote\u00ednmi vo vn\u00fatri buniek alebo tkan\u00edv?<\/p>\n\n\n\n

Najprv musia vedci na z\u00e1klade svojho v\u00fdskumu ur\u010di\u0165, ktor\u00e9 protil\u00e1tky a farbiv\u00e1 pou\u017ei\u0165.<\/p>\n\n\n\n

Napr\u00edklad, v tomto \u010dl\u00e1nku<\/a>Mendon\u00e7a sa sna\u017eil vyhodnoti\u0165 \u00fa\u010dinky a potenci\u00e1lne rizik\u00e1 kati\u00f3nov\u00fdch tuh\u00fdch lipidov\u00fdch nano\u010dast\u00edc (cSLN) u potkanov. Ka\u017edoro\u010dne sa vyv\u00edja a sk\u00fama mnoho nano\u010dast\u00edc, ktor\u00fdch cie\u013eom je zlep\u0161i\u0165 dod\u00e1vku liekov alebo g\u00e9nov na lie\u010dbu mnoh\u00fdch ochoren\u00ed. Jednou zo zauj\u00edmav\u00fdch ot\u00e1zok v tejto \u0161t\u00fadii bolo, \u010di sa nano\u010dastice dok\u00e1\u017eu dosta\u0165 do mozgu prekonan\u00edm hematoencefalickej bari\u00e9ry. T\u00e1to bari\u00e9ra chr\u00e1ni n\u00e1\u0161 mozog pred cirkuluj\u00facimi tox\u00ednmi alebo patog\u00e9nmi a zvy\u010dajne nie je \u017eiaduce, aby molekuly t\u00fato bari\u00e9ru prekra\u010dovali. Ale v tomto konkr\u00e9tnom pr\u00edpade, Mendon\u00e7a's<\/a> Cie\u013eom bolo, aby nano\u010dastice pre\u0161li cez bari\u00e9ru a dostali sa do mozgu, kde by sa v bud\u00facnosti mohli pou\u017ei\u0165 na dod\u00e1vanie liekov alebo g\u00e9nov.<\/p>\n\n\n\n

V snahe zisti\u0165, \u010di s\u00fa nano\u010dastice pr\u00edtomn\u00e9 v mozgovom parench\u00fdme, autori pou\u017eili marker endotelov\u00fdch buniek pre cievy s n\u00e1zvom RECA-1 (zn\u00e1zornen\u00fd \u010dervenou farbou), zatia\u013e \u010do bunkov\u00e9 jadr\u00e1 boli farben\u00e9 farbivom s n\u00e1zvom DAPI (4,6-diamidino-2-fenylindol), ktor\u00e9 je modr\u00e9. M\u00f4\u017eeme tie\u017e pozorova\u0165 mal\u00e9 zelen\u00e9 bodky pre nano\u010dastice mimo ciev, \u010do znamen\u00e1, \u017ee sa dostali do mozgov\u00e9ho parench\u00fdmu.<\/p>\n\n\n\n

Pozrite si infografiku s vyobrazen\u00edm ni\u017e\u0161ie.<\/p>\n\n\n

\n
\"Mind<\/a><\/figure><\/div>\n\n\n

Pochopme, \u010do rob\u00ed protil\u00e1tka proti RECA-1 (\u010derven\u00e1).<\/p>\n\n\n\n

Tieto protil\u00e1tky s\u00fa navrhnut\u00e9 tak, aby sl\u00fa\u017eili ako \u0161pecifick\u00e9 sondy a s\u00fa zameran\u00e9 na \u0161pecifick\u00fd antig\u00e9n (v na\u0161om pr\u00edpade na prote\u00edn RECA-1).<\/p>\n\n\n\n

M\u00f4\u017eu by\u0165 ozna\u010den\u00e9 fluoroforom alebo nesk\u00f4r rozpoznan\u00e9 sekund\u00e1rnou protil\u00e1tkou spojenou s fluoroforom.<\/p>\n\n\n\n

Preto po excit\u00e1cii vzorky sveteln\u00fdm zdrojom, \u0161pecifick\u00fd prote\u00edn, ktor\u00fd h\u013ead\u00e1te, sa vo va\u0161ej vzorke rozpozn\u00e1 na z\u00e1klade emisie svetla \u0161pecifickej vlnovej d\u013a\u017eky<\/a>.<\/p>\n\n\n\n

V pr\u00edpade DAPI je toto farbivo protifarbivom jadier a nukleoz\u00f3mov a po naviazan\u00ed na AT oblasti DNA vy\u017earuje modr\u00fa fluorescenciu.<\/p>\n\n\n\n

Ako navrhn\u00fa\u0165 panel pre imunofluorescenciu? <\/strong><\/p>\n\n\n\n

Za\u010dnite t\u00fdmito krokmi:<\/strong><\/p>\n\n\n\n

    \n
  1. K\u00fapte si (alebo si po\u017ei\u010dajte! Veda by mala ve\u013emi spolupracova\u0165!) protil\u00e1tky a farbiv\u00e1 potrebn\u00e9 pre v\u00e1\u0161 v\u00fdskum. Uprednost\u0148ujte prim\u00e1rne protil\u00e1tky (bez sond) a z prim\u00e1rnej protil\u00e1tky si k\u00fapte sekund\u00e1rne protil\u00e1tky \u0161pecifick\u00e9 pre hostite\u013esk\u00fd druh. Ak napr\u00edklad pou\u017e\u00edvate prim\u00e1rnu protil\u00e1tku vyroben\u00fa u kr\u00e1likov, pou\u017eite sekund\u00e1rnu protil\u00e1tku proti kr\u00e1likom. To zaru\u010d\u00ed \u0161pecifickos\u0165. <\/li>\n\n\n\n
  2. Pou\u017eitie sekund\u00e1rnych protil\u00e1tok ozna\u010den\u00fdch fluorof\u00f3rmi zv\u00fd\u0161i v\u00e1\u0161 sign\u00e1l t\u00fdm, \u017ee sa na jednu prim\u00e1rnu protil\u00e1tku deteguje viac antig\u00e9nov. Takisto je to dynamickej\u0161\u00ed sp\u00f4sob vypracovania r\u00f4znych testov, preto\u017ee umo\u017e\u0148uje v\u00fdskumn\u00edkovi modifikova\u0165 farby v paneli na z\u00e1klade jeho potrieb. <\/li>\n\n\n\n
  3. \u010eal\u0161\u00edm d\u00f4le\u017eit\u00fdm krokom je skontrolova\u0165, ak\u00e9 filtre m\u00f4\u017eete v mikroskope pou\u017ei\u0165. Mali by ste sa uisti\u0165, \u017ee excita\u010dn\u00e9 a emisn\u00e9 vlnov\u00e9 d\u013a\u017eky va\u0161ich fluoroforov le\u017eia vo vn\u00fatri excita\u010dn\u00fdch a emisn\u00fdch filtrov; inak nebudete m\u00f4c\u0165 zachyti\u0165 emisn\u00e9 svetlo z va\u0161ich sond. M\u00f4\u017eete pou\u017ei\u0165 Prehliada\u010d fluorescen\u010dn\u00fdch spektier<\/a> skontrolova\u0165 kompatibilitu.<\/li>\n\n\n\n
  4. Aby ste sa uistili, \u017ee sa excita\u010dn\u00e9 a emisn\u00e9 vlnov\u00e9 d\u013a\u017eky v\u0161etk\u00fdch va\u0161ich fluoroforov a farb\u00edv neprekr\u00fdvaj\u00fa v r\u00e1mci tej istej anal\u00fdzy, Prehliada\u010d fluorescen\u010dn\u00fdch spektier<\/a> je skvelou vo\u013ebou. Pokr\u00fdvaj\u00fa takmer v\u0161etky dostupn\u00e9 fluorofory!<\/li>\n<\/ol>\n\n\n\n

    Nakoniec skontrolujte pr\u00edklad hypotetick\u00e9ho experimentu, v ktorom m\u00e1me Hoechst 33258 pre nukleov\u00e9 kyseliny a prim\u00e1rnu protil\u00e1tku proti RECA-1 ozna\u010den\u00fa sekund\u00e1rnou protil\u00e1tkou Alexa Fluor 647.<\/p>\n\n\n\n

    Ide\u00e1lne by bolo pou\u017ei\u0165 mikroskop s kockou DAPI (excit\u00e1cia 377\/50 a emisia 447\/60) a kockou CY5 (excit\u00e1cia 628\/40 a emisia 685\/40). V\u0161etky tieto inform\u00e1cie sme vlo\u017eili na Prehliada\u010d fluorescen\u010dn\u00fdch spektier<\/a> a z\u00edskali sme spektr\u00e1 pre obe farbiv\u00e1 a \u0161\u00edrky p\u00e1sma pre obe kocky (pozrite si spektrum v infografike vy\u0161\u0161ie).<\/p>\n\n\n\n

    T\u00e1to hypotetick\u00e1 esej je dobr\u00fdm pr\u00edkladom, kde spektr\u00e1 fluoroforov patria do excita\u010dn\u00fdch a emisn\u00fdch filtrov, \u010do umo\u017e\u0148uje v\u00fdskumn\u00edkovi \u010do najlep\u0161ie zachyti\u0165 jeho vzorky.<\/p>\n\n\n\n

    Teraz je \u010das \u00eds\u0165 do laborat\u00f3ria a v\u0161etko uplatni\u0165 v praxi!<\/p>\n\n\n\n

    D\u00fafam, \u017ee v\u00e1m tieto tipy pom\u00f4\u017eu pri va\u0161om \u010fal\u0161om laborat\u00f3rnom experimente. Ve\u013ea \u0161\u0165astia!<\/p>\n\n\n\n

    Odkazy:<\/strong><\/p>\n\n\n\n

      \n
    1. \u00davod do fluorescen\u010dnej mikroskopie. MikroskopiaU spolo\u010dnosti Nikon <\/em><\/a>https:\/\/www.microscopyu.com\/techniques\/fluorescence\/introduction-to-fluorescence-microscopy<\/a>. Pr\u00edstupn\u00e9 2021-04-11 17:20:40.<\/a><\/li>\n<\/ol>","protected":false},"excerpt":{"rendered":"

      Ve\u013ekou s\u00fa\u010das\u0165ou pr\u00e1ce vedca je navrhovanie a vykon\u00e1vanie experimentov. Kombin\u00e1cia laborat\u00f3rnych techn\u00edk odpovie na v\u00e4\u010d\u0161inu ot\u00e1zok navrhnut\u00fdch vedcami a pracovn\u00fd postup pri navrhovan\u00ed nov\u00fdch met\u00f3d z\u00e1vis\u00ed od vzdelania a sk\u00fasenost\u00ed vedca. Biol\u00f3gom m\u00f4\u017eu obr\u00e1zky buniek poveda\u0165 ve\u013ea o tom, \u010do sa deje s [...]<\/p>","protected":false},"author":18,"featured_media":12982,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":[],"categories":[66,959,958,28,38],"tags":[51,554,962],"yoast_head":"\nMicroscopy: How Scientists Use Fluorescence To See Inside Cells -<\/title>\n<meta name=\"description\" content=\"Fluorescence microscopy can say a lot about a cell process through image. 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