{"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\/lv\/mikroskopija-ka-zinatnieki-izmanto-fluorescenci-lai-redzetu-sunu-iekspusi\/","title":{"rendered":"Mikroskopija: K\u0101 zin\u0101tnieki izmanto fluorescenci, lai ieskat\u012btos \u0161\u016bnu iek\u0161ien\u0113?"},"content":{"rendered":"

Liela da\u013ca no zin\u0101tnieka darba pien\u0101kumiem ir eksperimentu pl\u0101no\u0161ana un veik\u0161ana.<\/p>\n\n\n\n

Laboratorijas meto\u017eu kombin\u0101cija atbild\u0113s uz liel\u0101ko da\u013cu zin\u0101tnieku ierosin\u0101to jaut\u0101jumu, un darba process, lai ierosin\u0101tu jaunas metodes, ir atkar\u012bgs no zin\u0101tnieka pieredzes un pieredzes.<\/p>\n\n\n\n

Biologiem \u0161\u016bnu att\u0113li var daudz ko past\u0101st\u012bt par to, kas notiek ar p\u0113t\u0101majiem procesiem un meh\u0101nismiem.<\/p>\n\n\n\n

Gaismas mikroskopija<\/strong> ir \u013coti izplat\u012bta metode biolo\u0123ijas zin\u0101tn\u0113s.<\/p>\n\n\n\n

Kr\u0101svielu, antivielu un fluoresc\u0113jo\u0161u zondes izmanto\u0161ana \u013cauj zin\u0101tniekiem mikroskopa \u0161\u016bn\u0101s redz\u0113t att\u0113lus, kas cit\u0101di b\u016btu p\u0101r\u0101k mazi, lai tos saskat\u012btu un pat saprastu.<\/p>\n\n\n\n

Fluorescences mikroskopi<\/strong> un fluorohromu izmanto\u0161ana k\u013cuva iesp\u0113jams 1930. gad\u0101.1<\/sup><\/a>, un m\u016bsdien\u0101s ir iesp\u0113jamas daudzas fluorohromu kombin\u0101cijas, lai iekr\u0101sotu olbaltumvielas, organellas un strukt\u016bras \u0161\u016bn\u0101s un audos.<\/p>\n\n\n\n

Fluorohromi (vai fluorofori)<\/strong> ir molekulas, kas, uzbudin\u0101tas ar noteikta vi\u013c\u0146a garuma gaismu, izstaro noteikta vi\u013c\u0146a garuma gaismu, kuru uztver mikroskopa l\u0113cas un p\u0101rveido faktisk\u0101 att\u0113l\u0101.<\/p>\n\n\n\n

Fluorescences, l\u0113cu un kameru kombin\u0101cija \u013cauj mums ieg\u016bt \u0161\u016bnu iek\u0161ien\u0113 notieko\u0161o procesu att\u0113lus da\u017e\u0101dos skatos un aspektos.<\/p>\n\n\n\n

Piem\u0113ram, izmantojot mikroskopu, ar 2,5x vai 4x objekt\u012bvu m\u0113s varam ieg\u016bt pla\u0161\u0101ku peles smadze\u0146u griezuma att\u0113lu, bet ar 63x objekt\u012bvu - s\u012bkas p\u0113t\u0101m\u0101 akt\u012bna citoskeleta deta\u013cas taj\u0101 pa\u0161\u0101 paraug\u0101.<\/p>\n\n\n\n

Lai veiktu \u0161os testus, m\u0113s varam izmantot antivielas vai kr\u0101svielas, kas v\u0113rstas pret konkr\u0113tiem olbaltumviel\u0101m, kuras atrodas \u0161\u016bn\u0101 vai audos, un antivielai parasti ir pievienots fluorofors.<\/p>\n\n\n\n

\u0160o par\u0101d\u012bbu izskaidro Stoksa nob\u012bde: fluorofori zaud\u0113 vibr\u0101cijas ener\u0123iju izstarot\u0101s gaismas veid\u0101, kad tie no uzbudin\u0101t\u0101 st\u0101vok\u013ca p\u0101riet atpaka\u013c pamatst\u0101vokl\u012b. Fluorescences mikroskopi nodro\u0161ina gaismu fluorofora uzbudin\u0101\u0161anai un sa\u0146em t\u0101 izstaroto gaismu. Izstaroto gaismu var uztvert ar objekt\u012bvu, apstr\u0101d\u0101t CCD kamer\u0101 un p\u0101rveidot digit\u0101l\u0101 att\u0113l\u0101.<\/p>\n\n\n\n

Bet par \u0161\u016bnu att\u0113lu ieg\u016b\u0161anu run\u0101sim v\u0113l\u0101k. Tagad iepaz\u012bstin\u0101sim j\u016bs ar piem\u0113riem un padomiem par galvenajiem so\u013ciem pirms att\u0113la ieg\u016b\u0161anas.<\/p>\n\n\n\n

K\u0101 m\u0113s izv\u0113lamies un kombin\u0113jam da\u017e\u0101du veidu kr\u0101svielas un antivielas, lai redz\u0113tu un izprastu attiec\u012bbas starp organel\u0113m un olbaltumviel\u0101m \u0161\u016bn\u0101s vai audos?<\/p>\n\n\n\n

Vispirms zin\u0101tniekiem ir j\u0101nosaka, kuras antivielas un kr\u0101svielas izmantot, pamatojoties uz veiktajiem p\u0113t\u012bjumiem.<\/p>\n\n\n\n

Piem\u0113ram, \u0161aj\u0101 rakst\u0101<\/a>Mendonca cent\u0101s nov\u0113rt\u0113t katjonu cieto lip\u012bdu nanoda\u013ci\u0146u (cSLN) ietekmi un iesp\u0113jamo risku \u017eurk\u0101m. Katru gadu tiek izstr\u0101d\u0101tas un p\u0113t\u012btas daudzas nanoda\u013ci\u0146as, kuru m\u0113r\u0137is ir uzlabot z\u0101\u013cu vai g\u0113nu pieg\u0101di daudzu slim\u012bbu \u0101rst\u0113\u0161anai. Viens no interesantajiem jaut\u0101jumiem \u0161aj\u0101 p\u0113t\u012bjum\u0101 bija, vai nanoda\u013ci\u0146as sp\u0113j sasniegt smadzenes, \u0161\u0137\u0113rsojot hematoencefalisko barjeru. \u0160\u012b barjera aizsarg\u0101 m\u016bsu smadzenes no cirkul\u0113jo\u0161iem toks\u012bniem vai patog\u0113niem, un parasti nav v\u0113lams, lai molekulas \u0161\u0137\u0113rsotu \u0161o barjeru. Bet \u0161aj\u0101 konkr\u0113taj\u0101 gad\u012bjum\u0101, Mendon\u00e7a's<\/a> m\u0113r\u0137is bija pan\u0101kt, lai nanoda\u013ci\u0146as \u0161\u0137\u0113rsotu barjeru un non\u0101ktu smadzen\u0113s, lai n\u0101kotn\u0113 var\u0113tu pieg\u0101d\u0101t z\u0101les vai g\u0113nus.<\/p>\n\n\n\n

V\u0113loties p\u0101rliecin\u0101ties, vai nanoda\u013ci\u0146as atrodas smadze\u0146u parenh\u012bm\u0101, autori izmantoja endot\u0113lija \u0161\u016bnu mar\u0137ieri, ko sauc par RECA-1 (att\u0113lots sarkan\u0101 kr\u0101s\u0101), bet \u0161\u016bnu kodolus iekr\u0101soja ar kr\u0101svielu DAPI (4\u2032,6-diamidino-2-fenilindols), kas ir zila. Var nov\u0113rot ar\u012b nelielus za\u013cus punkti\u0146us, kas nor\u0101da uz nanoda\u013ci\u0146\u0101m \u0101rpus asinsvadiem, kas noz\u012bm\u0113, ka t\u0101s ir sasniegu\u0161as smadze\u0146u parenh\u012bmu.<\/p>\n\n\n\n

Apl\u016bkojiet zem\u0101k redzamo infografiku ar att\u0113lojumu.<\/p>\n\n\n

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

Saprat\u012bsim, ko dara RECA-1 antiviela (sarkan\u0101 kr\u0101s\u0101).<\/p>\n\n\n\n

\u0160\u012bs antivielas ir izstr\u0101d\u0101tas k\u0101 specifiskas zondes, un t\u0101s ir v\u0113rstas pret konkr\u0113tu antig\u0113nu (m\u016bsu gad\u012bjum\u0101 - prote\u012bnu RECA-1).<\/p>\n\n\n\n

Tos var mar\u0137\u0113t ar fluoroforu vai v\u0113l\u0101k atpaz\u012bt ar sekund\u0101ro antivielu, kas saist\u012bta ar fluoroforu.<\/p>\n\n\n\n

T\u0101p\u0113c p\u0113c parauga uzbudin\u0101\u0161anas ar gaismas avotu, konkr\u0113tais prote\u012bns, ko mekl\u0113jat, paraug\u0101 tiks atpaz\u012bts p\u0113c gaismas emisijas konkr\u0113t\u0101 vi\u013c\u0146a garum\u0101.<\/a>.<\/p>\n\n\n\n

DAPI gad\u012bjum\u0101 \u0161\u012b kr\u0101sviela ir kodolu un nukleosomu pretkr\u0101sa, un, saistoties ar DNS AT apgabaliem, t\u0101 izstaro zilu fluorescenci.<\/p>\n\n\n\n

K\u0101 izveidot imunofluorescences paneli? <\/strong><\/p>\n\n\n\n

S\u0101ciet ar \u0161iem so\u013ciem:<\/strong><\/p>\n\n\n\n

    \n
  1. Ieg\u0101d\u0101jieties (vai aiz\u0146emieties! Zin\u0101tnei ir \u013coti labi j\u0101sadarbojas!) antivielas un kr\u0101svielas, kas nepiecie\u0161amas j\u016bsu p\u0113t\u012bjumam. Priek\u0161roku dodiet prim\u0101raj\u0101m antiviel\u0101m (bez zond\u0113m) un ieg\u0101d\u0101jieties sekund\u0101r\u0101s antivielas, kas ir specifiskas saimnieka sugai no prim\u0101r\u0101s antivielas. Piem\u0113ram, ja izmantojat prim\u0101ro antivielu, kas ra\u017eota tru\u0161iem, izmantojiet sekund\u0101ro antivielu pret tru\u0161iem. Tas garant\u0113s specifiskumu. <\/li>\n\n\n\n
  2. Izmantojot sekund\u0101r\u0101s antivielas, kas mar\u0137\u0113tas ar fluoroforiem, pastiprin\u0101siet sign\u0101lu, jo uz vienu prim\u0101ro antivielu tiks konstat\u0113ts vair\u0101k antig\u0113nu. Tas ir ar\u012b dinamisk\u0101ks veids, k\u0101 izstr\u0101d\u0101t da\u017e\u0101dus testus, jo \u013cauj p\u0113tniekam main\u012bt pane\u013ca kr\u0101sas, pamatojoties uz sav\u0101m vajadz\u012bb\u0101m. <\/li>\n\n\n\n
  3. V\u0113l viens svar\u012bgs solis ir p\u0101rbaud\u012bt, k\u0101di filtri ir pieejami mikroskop\u0101. P\u0101rliecinieties, ka j\u016bsu fluoroforu uzbudin\u0101juma un emisijas vi\u013c\u0146u garumi atrodas uzbudin\u0101juma un emisijas filtru iek\u0161pus\u0113; pret\u0113j\u0101 gad\u012bjum\u0101 j\u016bs nevar\u0113siet uztvert savu zondes emisijas gaismu. J\u016bs varat izmantot Fluorescences spektru p\u0101rl\u016bks<\/a> lai p\u0101rbaud\u012btu sader\u012bbu.<\/li>\n\n\n\n
  4. Lai p\u0101rliecin\u0101tos, ka visu j\u016bsu fluoroforu un kr\u0101svielu uzbudin\u0101juma un emisijas vi\u013c\u0146u garumi nep\u0101rkl\u0101jas vien\u0101 test\u0101, Fluorescences spektru p\u0101rl\u016bks<\/a> ir lieliska izv\u0113le. Tie aptver gandr\u012bz visus pieejamos fluoroforus!<\/li>\n<\/ol>\n\n\n\n

    Visbeidzot, p\u0101rbaudiet hipot\u0113tiska eksperimenta piem\u0113ru, kur\u0101 nukle\u012bnsk\u0101b\u0113m ir Hoechst 33258 un prim\u0101r\u0101 antiviela pret RECA-1, kas mar\u0137\u0113ta ar sekund\u0101ro antivielu Alexa Fluor 647.<\/p>\n\n\n\n

    Vislab\u0101k b\u016btu izmantot mikroskopu, kas apr\u012bkots ar DAPI kubu (uzbudin\u0101jums 377\/50 un emisija 447\/60) un CY5 kubu (uzbudin\u0101jums 628\/40 un emisija 685\/40). Visu \u0161o inform\u0101ciju m\u0113s ievietoj\u0101m Fluorescences spektru p\u0101rl\u016bks<\/a> un ieguva abu kr\u0101svielu spektrus un joslas platumu abiem kubiem (apskatiet spektru infografik\u0101 iepriek\u0161).<\/p>\n\n\n\n

    \u0160\u012b hipot\u0113tisk\u0101 eseja ir labs piem\u0113rs, kur fluoroforu spektri ietilpst uzbudin\u0101juma un emisijas filtru iek\u0161pus\u0113, \u013caujot p\u0113tniekam ieg\u016bt paraugus vislab\u0101kaj\u0101 iesp\u0113jamaj\u0101 veid\u0101.<\/p>\n\n\n\n

    Tagad ir pien\u0101cis laiks doties uz laboratoriju un visu \u012bstenot praks\u0113!<\/p>\n\n\n\n

    Es ceru, ka \u0161ie padomi pal\u012bdz\u0113s jums n\u0101kamaj\u0101 laboratorijas eksperiment\u0101. Veiksmi!<\/p>\n\n\n\n

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

      \n
    1. Ievads fluorescences mikroskopij\u0101. Nikon's MicroscopyU <\/em><\/a>https:\/\/www.microscopyu.com\/techniques\/fluorescence\/introduction-to-fluorescence-microscopy<\/a>. Piek\u013cuve 2021-04-11 17:20:40.<\/a><\/li>\n<\/ol>","protected":false},"excerpt":{"rendered":"

      Liela da\u013ca no zin\u0101tnieka darba pien\u0101kumiem ir eksperimentu pl\u0101no\u0161ana un veik\u0161ana. Laboratorijas meto\u017eu kombin\u0101cija atbild\u0113s uz liel\u0101ko da\u013cu zin\u0101tnieku ierosin\u0101to jaut\u0101jumu, un darba process, lai ierosin\u0101tu jaunas metodes, ir atkar\u012bgs no zin\u0101tnieka sagatavot\u012bbas un pieredzes. Biologiem \u0161\u016bnu att\u0113li var daudz ko past\u0101st\u012bt par to, kas notiek ar organismu.<\/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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