{"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\/lt\/mikroskopija-kaip-mokslininkai-panaudoja-fluorescencija-kad-pamatytu-lasteles-vidu\/","title":{"rendered":"Mikroskopija: Kaip mokslininkai naudoja fluorescencij\u0105, kad pamatyt\u0173 l\u0105steli\u0173 vid\u0173"},"content":{"rendered":"

Didel\u0117 mokslininko darbo dalis - kurti ir atlikti eksperimentus.<\/p>\n\n\n\n

Laboratorini\u0173 metod\u0173 derinys pad\u0117s atsakyti \u012f daugum\u0105 mokslinink\u0173 si\u016blom\u0173 klausim\u0173, o darbo eiga, leid\u017eianti pasi\u016blyti naujus metodus, priklauso nuo mokslininko i\u0161silavinimo ir patirties.<\/p>\n\n\n\n

Biologams l\u0105steli\u0173 atvaizdai gali daug pasakyti apie tai, kas vyksta su j\u0173 tiriamais procesais ir mechanizmais.<\/p>\n\n\n\n

\u0160viesos mikroskopija<\/strong> yra labai paplit\u0119s biologijos moksl\u0173 metodas.<\/p>\n\n\n\n

Naudodami da\u017eus, antik\u016bnus ir fluorescencinius zondus, mokslininkai mikroskopo l\u0105stel\u0117se mato vaizdus to, kas kitu atveju b\u016bt\u0173 per ma\u017ea, kad b\u016bt\u0173 galima pamatyti ir net suvokti.<\/p>\n\n\n\n

Fluorescenciniai mikroskopai<\/strong> ir fluorochrom\u0173 naudojimas tapo \u012fmanoma 1930 m.1<\/sup><\/a>, o \u0161iandien galima naudoti daugyb\u0119 fluorochrom\u0173 derini\u0173 baltymams, organel\u0117ms ir l\u0105steli\u0173 bei audini\u0173 strukt\u016broms da\u017eyti.<\/p>\n\n\n\n

Fluorochromai (arba fluoroforai)<\/strong> tai molekul\u0117s, kurios, su\u017eadintos tam tikro bangos ilgio \u0161viesa, skleid\u017eia tam tikro bangos ilgio \u0161vies\u0105, kuri\u0105 u\u017efiksuoja mikroskopo l\u0119\u0161iai ir paver\u010dia tikru vaizdu.<\/p>\n\n\n\n

Fluorescencijos, l\u0119\u0161i\u0173 ir kamer\u0173 derinys leid\u017eia mums u\u017efiksuoti l\u0105stel\u0117se vykstan\u010di\u0173 proces\u0173 vaizdus \u012fvairiais aspektais.<\/p>\n\n\n\n

Pavyzd\u017eiui, naudodami mikroskop\u0105 2,5x arba 4x objektyvu galime matyti platesn\u012f pel\u0117s smegen\u0173 gabal\u0117lio vaizd\u0105, o 63x objektyvu - smulkias tiriamo aktino citoskeleto detales tame pa\u010diame m\u0117ginyje.<\/p>\n\n\n\n

\u0160iems tyrimams atlikti galime naudoti antik\u016bnus arba da\u017eus, nukreiptus prie\u0161 konkre\u010dius l\u0105stel\u0117je ar audinyje esan\u010dius baltymus, o antik\u016bnas paprastai turi fluorofor\u0105.<\/p>\n\n\n\n

\u0160tokso poslinkis paai\u0161kina \u0161\u012f rei\u0161kin\u012f: fluoroforai praranda vibracin\u0119 energij\u0105 spinduliuojamos \u0161viesos pavidalu, kai i\u0161 su\u017eadintos b\u016bsenos pereina atgal \u012f pagrindin\u0119 b\u016bsen\u0105. Fluorescencijos mikroskopai suteikia \u0161viesos fluoroforui su\u017eadinti ir priima jo i\u0161spinduliuot\u0105 \u0161vies\u0105. Spinduliuojam\u0105 \u0161vies\u0105 galima u\u017efiksuoti objektyvu, apdoroti CCD kamera ir paversti skaitmeniniu vaizdu.<\/p>\n\n\n\n

Ta\u010diau apie l\u0105steli\u0173 vaizdo gavim\u0105 pakalb\u0117sime v\u0117liau. Dabar tur\u0117tume jus supa\u017eindinti su pavyzd\u017eiais ir patarimais, kaip atlikti pagrindinius veiksmus prie\u0161 \u012fgyjant vaizd\u0105.<\/p>\n\n\n\n

Kaip parinkti ir derinti \u012fvairi\u0173 tip\u0173 da\u017eus ir antik\u016bnus, kad pamatytume ir suprastume l\u0105steli\u0173 ar audini\u0173 organoid\u0173 ir baltym\u0173 ry\u0161ius?<\/p>\n\n\n\n

Pirmiausia mokslininkai turi nustatyti, kokius antik\u016bnus ir da\u017eus naudoti, remdamiesi savo tyrimais.<\/p>\n\n\n\n

Pavyzd\u017eiui, \u0161iame straipsnyje<\/a>Mendon\u00e7a band\u0117 \u012fvertinti katijonini\u0173 kiet\u0173j\u0173 lipid\u0173 nanodaleli\u0173 (cSLN) poveik\u012f ir galim\u0105 rizik\u0105 \u017eiurk\u0117ms. Kasmet kuriama ir tiriama daugyb\u0117 nanodaleli\u0173, kuriomis siekiama pagerinti vaist\u0173 ar gen\u0173 perdavim\u0105 daugeliui lig\u0173 gydyti. Vienas i\u0161 \u012fdomi\u0173 \u0161io tyrimo klausim\u0173 buvo, ar nanodalel\u0117s gali pasiekti smegenis, \u012fveikdamos kraujo-smegen\u0173 barjer\u0105. \u0160is barjeras apsaugo m\u016bs\u0173 smegenis nuo cirkuliuojan\u010di\u0173 toksin\u0173 ar patogen\u0173, tod\u0117l paprastai nepageidautina, kad molekul\u0117s kirst\u0173 \u0161\u012f barjer\u0105. Ta\u010diau \u0161iuo konkre\u010diu atveju, Mendon\u00e7a's<\/a> buvo siekiama, kad nanodalel\u0117s \u012fveikt\u0173 barjer\u0105 ir pasiekt\u0173 smegenis, kad ateityje jomis b\u016bt\u0173 galima tiekti vaistus ar genus.<\/p>\n\n\n\n

Nor\u0117dami patikrinti, ar nanodaleli\u0173 yra smegen\u0173 parenchimoje, autoriai naudojo endotelio l\u0105steli\u0173 kraujagysli\u0173 \u017eymekl\u012f RECA-1 (pavaizduotas raudona spalva), o l\u0105steli\u0173 branduoliai buvo da\u017eomi m\u0117lynu da\u017eikliu DAPI (4\u2032,6-diamidino-2-fenilindolas). U\u017e kraujagysli\u0173 rib\u0173 taip pat galima pasteb\u0117ti ma\u017eus \u017ealius ta\u0161kelius, \u017eymin\u010dius nanodaleles, o tai rei\u0161kia, kad jos pasiek\u0117 smegen\u0173 parenchim\u0105.<\/p>\n\n\n\n

Per\u017ei\u016br\u0117kite toliau pateikt\u0105 infografik\u0105 su atvaizdu.<\/p>\n\n\n

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

Supraskime, k\u0105 daro RECA-1 antik\u016bnas (raudonas).<\/p>\n\n\n\n

\u0160ie antik\u016bnai sukurti kaip specifiniai zondai, nukreipti \u012f konkret\u0173 antigen\u0105 (m\u016bs\u0173 atveju - \u012f baltym\u0105 RECA-1).<\/p>\n\n\n\n

Juos galima \u017eym\u0117ti fluoroforu arba v\u0117liau atpa\u017einti antriniu antik\u016bnu, sujungtu su fluoroforu.<\/p>\n\n\n\n

Tod\u0117l, su\u017eadinus m\u0117gin\u012f \u0161viesos \u0161altiniu, konkretus baltymas, kurio ie\u0161kote, bus atpa\u017eintas j\u016bs\u0173 m\u0117ginyje pagal tam tikro bangos ilgio \u0161viesos spinduliavim\u0105.<\/a>.<\/p>\n\n\n\n

DAPI atveju \u0161is da\u017eiklis yra branduoli\u0173 ir nukleosom\u0173 kontrbarometras, kuris, prisijung\u0119s prie DNR AT sri\u010di\u0173, skleid\u017eia m\u0117lyn\u0105 fluorescencij\u0105.<\/p>\n\n\n\n

Kaip sukurti imunofluorescencin\u0119 grup\u0119? <\/strong><\/p>\n\n\n\n

Prad\u0117kite nuo \u0161i\u0173 \u017eingsni\u0173:<\/strong><\/p>\n\n\n\n

    \n
  1. Nusipirkite (arba pasiskolinkite! Mokslas tur\u0117t\u0173 b\u016bti labai naudingas!) tyrimams reikaling\u0173 antik\u016bn\u0173 ir da\u017eikli\u0173. Pirmenyb\u0119 teikite pirminiams antik\u016bnams (be zond\u0173), o antrinius antik\u016bnus, b\u016bdingus \u0161eimininko r\u016b\u0161iai, pirkite i\u0161 pirmini\u0173 antik\u016bn\u0173. Pavyzd\u017eiui, jei naudojate pirmin\u012f antik\u016bn\u0105, pagamint\u0105 triu\u0161iams, naudokite antrin\u012f antik\u016bn\u0105 prie\u0161 triu\u0161\u012f. Tai u\u017etikrins specifi\u0161kum\u0105. <\/li>\n\n\n\n
  2. Naudojant antrinius antik\u016bnus, \u017eym\u0117tus fluoroforais, signalas sustipr\u0117s, nes bus aptikta daugiau antigen\u0173 vienam pirminiam antik\u016bnui. Be to, tai yra dinami\u0161kesnis b\u016bdas \u012fvairiems tyrimams parengti, nes tyr\u0117jas gali keisti grup\u0117s spalvas pagal savo poreikius. <\/li>\n\n\n\n
  3. Kitas svarbus \u017eingsnis - patikrinti, kokius filtrus galima naudoti mikroskope. Tur\u0117tum\u0117te \u012fsitikinti, kad j\u016bs\u0173 fluorofor\u0173 su\u017eadinimo ir i\u0161metimo bangos ilgiai yra su\u017eadinimo ir i\u0161metimo filtruose; prie\u0161ingu atveju negal\u0117site u\u017efiksuoti savo zond\u0173 i\u0161metimo \u0161viesos. Galite naudoti Fluorescencijos spektr\u0173 per\u017ei\u016bra<\/a> patikrinti suderinamum\u0105.<\/li>\n\n\n\n
  4. \u012esitikinkite, kad vis\u0173 j\u016bs\u0173 fluorofor\u0173 ir da\u017eikli\u0173 su\u017eadinimo ir i\u0161metimo bangos ilgiai nesutampa toje pa\u010dioje analiz\u0117je, Fluorescencijos spektr\u0173 per\u017ei\u016bra<\/a> yra puikus pasirinkimas. Jie apima beveik visus turimus fluoroforus!<\/li>\n<\/ol>\n\n\n\n

    Galiausiai patikrinkite hipotetinio eksperimento pavyzd\u012f, kuriame nukleino r\u016bg\u0161tims \u017eym\u0117ti naudojamas Hoechst 33258, o pirminis antik\u016bnas prie\u0161 RECA-1 \u017eymimas antriniu antik\u016bnu Alexa Fluor 647.<\/p>\n\n\n\n

    Geriausia b\u016bt\u0173 naudoti mikroskop\u0105 su DAPI kubeliu (su\u017eadinimas 377\/50, emisija 447\/60) ir CY5 kubeliu (su\u017eadinimas 628\/40, emisija 685\/40). Vis\u0105 \u0161i\u0105 informacij\u0105 \u012fterp\u0117me \u012f Fluorescencijos spektr\u0173 per\u017ei\u016bra<\/a> ir gavome abiej\u0173 da\u017eikli\u0173 spektrus bei abiej\u0173 kub\u0173 da\u017eni\u0173 juostos plo\u010dius (\u017ei\u016br\u0117kite spektr\u0105 infografike auk\u0161\u010diau).<\/p>\n\n\n\n

    \u0160is hipotetinis ra\u0161inys yra geras pavyzdys, kai fluorofor\u0173 spektrai patenka \u012f su\u017eadinimo ir i\u0161metimo filtrus, tod\u0117l tyr\u0117jas gali kuo geriau u\u017efiksuoti savo m\u0117ginius.<\/p>\n\n\n\n

    Dabar metas eiti \u012f laboratorij\u0105 ir visk\u0105 i\u0161bandyti prakti\u0161kai!<\/p>\n\n\n\n

    Tikiuosi, kad \u0161ie patarimai pad\u0117s jums atliekant kit\u0105 laboratorin\u012f eksperiment\u0105. S\u0117km\u0117s!<\/p>\n\n\n\n

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

      \n
    1. Fluorescencin\u0117s mikroskopijos \u012fvadas. Nikon's MicroscopyU <\/em><\/a>https:\/\/www.microscopyu.com\/techniques\/fluorescence\/introduction-to-fluorescence-microscopy<\/a>. \u017di\u016br\u0117ta 2021-04-11 17:20:40.<\/a><\/li>\n<\/ol>","protected":false},"excerpt":{"rendered":"

      Didel\u0117 mokslininko darbo dalis - kurti ir atlikti eksperimentus. Laboratorini\u0173 metod\u0173 derinys pad\u0117s atsakyti \u012f daugum\u0105 mokslinink\u0173 pasi\u016blyt\u0173 klausim\u0173, o darbo eiga, leid\u017eianti pasi\u016blyti naujus metodus, priklauso nuo mokslininko i\u0161silavinimo ir patirties. Biologams l\u0105steli\u0173 vaizdai gali daug pasakyti apie tai, kas vyksta su [...]<\/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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