{"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\/cs\/mikroskopie-jak-vedci-pouzivaji-fluorescenci-k-videni-vnitrku-bunek\/","title":{"rendered":"Mikroskopie: Jak v\u011bdci pomoc\u00ed fluorescence nahl\u00ed\u017eej\u00ed do nitra bun\u011bk?"},"content":{"rendered":"

Velkou \u010d\u00e1st pr\u00e1ce v\u011bdce tvo\u0159\u00ed navrhov\u00e1n\u00ed a prov\u00e1d\u011bn\u00ed experiment\u016f.<\/p>\n\n\n\n

Kombinace laboratorn\u00edch technik odpov\u00ed na v\u011bt\u0161inu ot\u00e1zek navr\u017een\u00fdch v\u011bdci a pracovn\u00ed postup p\u0159i navrhov\u00e1n\u00ed nov\u00fdch metod z\u00e1vis\u00ed na vzd\u011bl\u00e1n\u00ed a zku\u0161enostech v\u011bdce.<\/p>\n\n\n\n

Obr\u00e1zky bun\u011bk mohou biolog\u016fm hodn\u011b napov\u011bd\u011bt o tom, co se d\u011bje s procesy a mechanismy, kter\u00e9 studuj\u00ed.<\/p>\n\n\n\n

Sv\u011bteln\u00e1 mikroskopie<\/strong> je v biologick\u00fdch v\u011bd\u00e1ch velmi roz\u0161\u00ed\u0159enou technikou.<\/p>\n\n\n\n

Pou\u017eit\u00ed barviv, protil\u00e1tek a fluorescen\u010dn\u00edch sond umo\u017e\u0148uje v\u011bdc\u016fm vid\u011bt v bu\u0148k\u00e1ch mikroskopu obrazy toho, co by jinak bylo p\u0159\u00edli\u0161 mal\u00e9 na to, aby to bylo mo\u017en\u00e9 vid\u011bt a v\u016fbec pochopit.<\/p>\n\n\n\n

Fluorescen\u010dn\u00ed mikroskopy<\/strong> a pou\u017eit\u00ed fluorochrom\u016f bylo mo\u017en\u00e9 v roce 19301<\/sup><\/a>, a dnes je mo\u017en\u00e9 barvit proteiny, organely a struktury v bu\u0148k\u00e1ch a tk\u00e1n\u00edch mnoha kombinacemi fluorochrom\u016f.<\/p>\n\n\n\n

Fluorochromy (nebo fluorofory)<\/strong> jsou molekuly, kter\u00e9 po excitaci specifickou vlnovou d\u00e9lkou sv\u011btla vyza\u0159uj\u00ed sv\u011btlo o definovan\u00e9 vlnov\u00e9 d\u00e9lce, kter\u00e9 zachyt\u00ed \u010do\u010dky mikroskopu a p\u0159em\u011bn\u00ed je na skute\u010dn\u00fd obraz.<\/p>\n\n\n\n

Kombinace fluorescence, \u010do\u010dek a kamer n\u00e1m umo\u017e\u0148uje po\u0159\u00eddit obraz proces\u016f uvnit\u0159 bun\u011bk v mnoha r\u016fzn\u00fdch pohledech a aspektech.<\/p>\n\n\n\n

Pomoc\u00ed mikroskopu m\u00e1me nap\u0159\u00edklad \u0161ir\u0161\u00ed pohled na pl\u00e1tek my\u0161\u00edho mozku v objektivu 2,5x nebo 4x a drobn\u00e9 detaily zkouman\u00e9ho aktinov\u00e9ho cytoskeletu ve stejn\u00e9m vzorku v objektivu 63x.<\/p>\n\n\n\n

Pro tyto testy m\u016f\u017eeme pou\u017e\u00edt protil\u00e1tky nebo barviva proti specifick\u00fdm protein\u016fm p\u0159\u00edtomn\u00fdm v bu\u0148ce nebo tk\u00e1ni, p\u0159i\u010dem\u017e protil\u00e1tka je obvykle opat\u0159ena fluoroforem.<\/p>\n\n\n\n

Stokes\u016fv posun vysv\u011btluje tento jev: fluorofory ztr\u00e1cej\u00ed vibra\u010dn\u00ed energii ve form\u011b vyza\u0159ovan\u00e9ho sv\u011btla, kdy\u017e se z excitovan\u00e9ho stavu vracej\u00ed zp\u011bt do z\u00e1kladn\u00edho stavu. Fluorescen\u010dn\u00ed mikroskopy poskytnou sv\u011btlo k excitaci fluoroforu a p\u0159ijmou jeho emitovan\u00e9 sv\u011btlo. Emitovan\u00e9 sv\u011btlo lze zachytit objektivem, zpracovat v CCD kame\u0159e a p\u0159ev\u00e9st na digit\u00e1ln\u00ed obraz.<\/p>\n\n\n\n

Ale o z\u00edsk\u00e1v\u00e1n\u00ed sn\u00edmk\u016f bun\u011bk si pov\u00edme pozd\u011bji. Nyn\u00ed bychom v\u00e1s m\u011bli sezn\u00e1mit s p\u0159\u00edklady a tipy na hlavn\u00ed kroky p\u0159ed po\u0159\u00edzen\u00edm sn\u00edmku.<\/p>\n\n\n\n

Jak vyb\u00edr\u00e1me a kombinujeme r\u016fzn\u00e9 typy barviv a protil\u00e1tek, abychom vid\u011bli a pochopili vztahy mezi organelami a proteiny uvnit\u0159 bun\u011bk nebo tk\u00e1n\u00ed?<\/p>\n\n\n\n

Nejprve mus\u00ed v\u011bdci na z\u00e1klad\u011b sv\u00e9ho v\u00fdzkumu ur\u010dit, kter\u00e9 protil\u00e1tky a barviva pou\u017eij\u00ed.<\/p>\n\n\n\n

Nap\u0159\u00edklad, v tomto \u010dl\u00e1nku<\/a>Mendon\u00e7a se sna\u017eil vyhodnotit \u00fa\u010dinky a potenci\u00e1ln\u00ed rizika kationick\u00fdch pevn\u00fdch lipidov\u00fdch nano\u010d\u00e1stic (cSLN) u potkan\u016f. Ka\u017edoro\u010dn\u011b je vyv\u00edjeno a studov\u00e1no mnoho nano\u010d\u00e1stic, jejich\u017e c\u00edlem je zlep\u0161it dod\u00e1vku l\u00e9\u010div nebo gen\u016f pro l\u00e9\u010dbu mnoha nemoc\u00ed. Jednou ze zaj\u00edmav\u00fdch ot\u00e1zek v t\u00e9to studii bylo, zda se nano\u010d\u00e1stice dok\u00e1\u017eou dostat do mozku p\u0159ekon\u00e1n\u00edm hematoencefalick\u00e9 bari\u00e9ry. Tato bari\u00e9ra chr\u00e1n\u00ed n\u00e1\u0161 mozek p\u0159ed cirkuluj\u00edc\u00edmi toxiny nebo patogeny a obvykle nen\u00ed \u017e\u00e1douc\u00ed, aby molekuly tuto bari\u00e9ru p\u0159ekra\u010dovaly. Ale v tomto konkr\u00e9tn\u00edm p\u0159\u00edpad\u011b, Mendon\u00e7a's<\/a> C\u00edlem bylo, aby nano\u010d\u00e1stice pronikly p\u0159es bari\u00e9ru a dostaly se do mozku, kde by v budoucnu mohly b\u00fdt pou\u017eity k doprav\u011b l\u00e9k\u016f nebo gen\u016f.<\/p>\n\n\n\n

Auto\u0159i cht\u011bli zjistit, zda jsou nano\u010d\u00e1stice p\u0159\u00edtomny v mozkov\u00e9m parenchymu, a proto pou\u017eili endoteli\u00e1ln\u00ed bun\u011b\u010dn\u00fd marker pro c\u00e9vy s n\u00e1zvem RECA-1 (zn\u00e1zorn\u011bn\u00fd \u010derven\u011b), zat\u00edmco bun\u011b\u010dn\u00e1 j\u00e1dra byla obarvena barvivem DAPI (4\u2032,6-diamidino-2-fenylindol), kter\u00e9 je modr\u00e9. M\u016f\u017eeme tak\u00e9 pozorovat mal\u00e9 zelen\u00e9 te\u010dky pro nano\u010d\u00e1stice mimo c\u00e9vy, co\u017e znamen\u00e1, \u017ee se dostaly do mozkov\u00e9ho parenchymu.<\/p>\n\n\n\n

Pod\u00edvejte se na n\u00ed\u017ee uvedenou infografiku s reprezentativn\u00edm obr\u00e1zkem.<\/p>\n\n\n

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

Pochop\u00edme, co d\u011bl\u00e1 protil\u00e1tka proti RECA-1 (\u010derven\u00e1).<\/p>\n\n\n\n

Tyto protil\u00e1tky jsou navr\u017eeny tak, aby slou\u017eily jako specifick\u00e9 sondy a byly zam\u011b\u0159eny na specifick\u00fd antigen (v na\u0161em p\u0159\u00edpad\u011b na protein RECA-1).<\/p>\n\n\n\n

Mohou b\u00fdt ozna\u010deny fluoroforem nebo pozd\u011bji rozpozn\u00e1ny sekund\u00e1rn\u00ed protil\u00e1tkou nav\u00e1zanou na fluorofor.<\/p>\n\n\n\n

Proto po excitaci vzorku sv\u011bteln\u00fdm zdrojem, specifick\u00fd protein, kter\u00fd hled\u00e1te, bude ve vzorku rozpozn\u00e1n na z\u00e1klad\u011b emise sv\u011btla specifick\u00e9 vlnov\u00e9 d\u00e9lky.<\/a>.<\/p>\n\n\n\n

V p\u0159\u00edpad\u011b DAPI je toto barvivo protibarvivem jader a nukleozom\u016f a p\u0159i vazb\u011b na AT oblasti DNA emituje modrou fluorescenci.<\/p>\n\n\n\n

Jak navrhnout panel pro imunofluorescenci? <\/strong><\/p>\n\n\n\n

Za\u010dn\u011bte t\u011bmito kroky:<\/strong><\/p>\n\n\n\n

    \n
  1. Nakupte (nebo si p\u016fj\u010dte! V\u011bda by m\u011bla b\u00fdt velmi kooperativn\u00ed!) protil\u00e1tky a barviva nezbytn\u00e1 pro v\u00e1\u0161 v\u00fdzkum. D\u00e1vejte p\u0159ednost prim\u00e1rn\u00edm protil\u00e1tk\u00e1m (bez sond) a z prim\u00e1rn\u00edch protil\u00e1tek kupujte sekund\u00e1rn\u00ed protil\u00e1tky specifick\u00e9 pro hostitelsk\u00fd druh. Nap\u0159\u00edklad pokud pou\u017e\u00edv\u00e1te prim\u00e1rn\u00ed protil\u00e1tku vyrobenou u kr\u00e1l\u00edk\u016f, pou\u017eijte sekund\u00e1rn\u00ed protil\u00e1tku proti kr\u00e1l\u00edk\u016fm. T\u00edm zaru\u010d\u00edte specifi\u010dnost. <\/li>\n\n\n\n
  2. Pou\u017eit\u00ed sekund\u00e1rn\u00edch protil\u00e1tek zna\u010den\u00fdch fluorofory zv\u00fd\u0161\u00ed sign\u00e1l t\u00edm, \u017ee detekuje v\u00edce antigen\u016f na jednu prim\u00e1rn\u00ed protil\u00e1tku. Rovn\u011b\u017e se jedn\u00e1 o dynami\u010dt\u011bj\u0161\u00ed zp\u016fsob vypracov\u00e1n\u00ed r\u016fzn\u00fdch test\u016f, proto\u017ee umo\u017e\u0148uje v\u00fdzkumn\u00edkovi m\u011bnit barvy v panelu na z\u00e1klad\u011b jeho pot\u0159eb. <\/li>\n\n\n\n
  3. Dal\u0161\u00edm d\u016fle\u017eit\u00fdm krokem je zkontrolovat, jak\u00e9 filtry lze v mikroskopu pou\u017e\u00edt. M\u011bli byste se ujistit, \u017ee excita\u010dn\u00ed a emisn\u00ed vlnov\u00e1 d\u00e9lka va\u0161eho fluoroforu le\u017e\u00ed uvnit\u0159 excita\u010dn\u00edch a emisn\u00edch filtr\u016f; jinak nebudete moci zachytit emisn\u00ed sv\u011btlo va\u0161ich sond. M\u016f\u017eete pou\u017e\u00edt Prohl\u00ed\u017ee\u010d fluorescen\u010dn\u00edch spekter<\/a> zkontrolovat kompatibilitu.<\/li>\n\n\n\n
  4. Ujist\u011bte se, \u017ee se excita\u010dn\u00ed a emisn\u00ed vlnov\u00e9 d\u00e9lky v\u0161ech va\u0161ich fluorofor\u016f a barviv nep\u0159ekr\u00fdvaj\u00ed v r\u00e1mci jednoho testu, Prohl\u00ed\u017ee\u010d fluorescen\u010dn\u00edch spekter<\/a> je skv\u011blou volbou. Pokr\u00fdvaj\u00ed t\u00e9m\u011b\u0159 v\u0161echny dostupn\u00e9 fluorofory!<\/li>\n<\/ol>\n\n\n\n

    Nakonec se pod\u00edvejte na p\u0159\u00edklad hypotetick\u00e9ho experimentu, kde m\u00e1me pro nukleov\u00e9 kyseliny Hoechst 33258 a prim\u00e1rn\u00ed protil\u00e1tku proti RECA-1 zna\u010denou sekund\u00e1rn\u00ed protil\u00e1tkou Alexa Fluor 647.<\/p>\n\n\n\n

    V ide\u00e1ln\u00edm p\u0159\u00edpad\u011b bychom pou\u017eili mikroskop s kostkou DAPI (excitace 377\/50 a emise 447\/60) a kostkou CY5 (excitace 628\/40 a emise 685\/40). V\u0161echny tyto informace jsme vlo\u017eili na Prohl\u00ed\u017ee\u010d fluorescen\u010dn\u00edch spekter<\/a> a z\u00edskal spektra obou barviv a \u0161\u00ed\u0159ky p\u00e1sma pro ob\u011b kostky (pod\u00edvejte se na spektra v infografice v\u00fd\u0161e).<\/p>\n\n\n\n

    Tato hypotetick\u00e1 esej je dobr\u00fdm p\u0159\u00edkladem toho, \u017ee spektra fluorofor\u016f pat\u0159\u00ed do excita\u010dn\u00edho a emisn\u00edho filtru, co\u017e umo\u017e\u0148uje v\u00fdzkumn\u00edkovi co nejl\u00e9pe zachytit jeho vzorky.<\/p>\n\n\n\n

    Nyn\u00ed je \u010das j\u00edt do laborato\u0159e a v\u0161e vyzkou\u0161et v praxi!<\/p>\n\n\n\n

    Douf\u00e1m, \u017ee v\u00e1m tyto tipy pomohou p\u0159i va\u0161em dal\u0161\u00edm laboratorn\u00edm experimentu. Hodn\u011b \u0161t\u011bst\u00ed!<\/p>\n\n\n\n

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

      \n
    1. \u00davod do fluorescen\u010dn\u00ed mikroskopie. MikroskopieU spole\u010dnosti Nikon <\/em><\/a>https:\/\/www.microscopyu.com\/techniques\/fluorescence\/introduction-to-fluorescence-microscopy<\/a>. P\u0159\u00edstup 2021-04-11 17:20:40.<\/a><\/li>\n<\/ol>","protected":false},"excerpt":{"rendered":"

      Velkou \u010d\u00e1st pr\u00e1ce v\u011bdce tvo\u0159\u00ed navrhov\u00e1n\u00ed a prov\u00e1d\u011bn\u00ed experiment\u016f. Kombinace laboratorn\u00edch technik odpov\u00ed na v\u011bt\u0161inu ot\u00e1zek navr\u017een\u00fdch v\u011bdci a pracovn\u00ed postup p\u0159i navrhov\u00e1n\u00ed nov\u00fdch metod z\u00e1vis\u00ed na vzd\u011bl\u00e1n\u00ed a zku\u0161enostech v\u011bdce. Biolog\u016fm mohou sn\u00edmky bun\u011bk hodn\u011b napov\u011bd\u011bt o tom, co se d\u011bje v organismu, a to i v p\u0159\u00edpad\u011b, \u017ee [...]<\/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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