Filter-ka-Fluorescence waa qayb muhiim ah oo ku jirta mikroskoob-ka fluorescence. Nidaamka caadiga ah wuxuu leeyahay saddex filtarrada aasaasiga ah: filter filter, filter filter iyo muraayad dichroic. Waxay caadi ahaan ku baakadeeyaan cube si kooxda loo wada geliyo mikroskoob.
Sidee buu u shaqeeyaa shaandhada fluorescence?
Shaandhaynta xiisaha
Shaandhooyinka xamaasadda leh waxay gudbiyaan iftiinka mawjadaha dhererka gaarka ah waxayna xannibaan hirarka dhererka kale. Waxaa loo isticmaali karaa in lagu soo saaro midabyo kala duwan iyadoo la hagaajinayo shaandhada si loogu oggolaado hal midab oo keliya. filtarrada xamaasadu waxay u kala baxaan laba nooc oo waaweyn - filtarrada dhaafka dheer iyo filtarrada ka gudubka band. Dareen-qaaduhu inta badan waa filter-ka-bandheeyaha kaas oo dhaafa kaliya mawjadaha dhaadheer ee uu nuugo fluorophore-ku, sidaas awgeed waxa ay yaraynaysaa xamaasadda ilaha kale ee fluorescence iyo xannibaadda iftiinka kicinta ee qaybta qiiqa qiiqa. Sida ku cad xariiqda buluuga ah ee shaxanka, BP waa 460-495, taas oo macnaheedu yahay in ay ka gudbi karto oo kaliya fluorescence 460-495nm.
Waxa lagu meeleeyaa jidka iftiinka ee mikroskoob-ka florescence waxayna shaandhaysaa dhammaan hirarka dhaadheer ee isha iftiinka marka laga reebo qiyaasta kicinta fluorophore. Shaandhaynta ugu yar ee gudbinta ayaa tilmaamaysa dhalaalka iyo dhalaalka sawirada. Ugu yaraan 40% gudbinta shaandhada kicinta ayaa lagula talinayaa si gudbintu ay ugu habboon tahay> 85%. Baaxadda shaandhada kicinta waa in ay gebi ahaanba ku dhex jirtaa qiyaasta kicinta fluorophore sida in mawjadda dhererka dhexe (CWL) ee shaandhadu ay ugu dhowdahay inta suurtogalka ah mawjada ugu sarreysa ee fluorophore-ka. Cufnaanta indhaha ee shaandhada xiisaha leh (OD) waxay tilmaamaysaa mugdiga sawirka asalka; OD waa cabbirka sida fiican ee shaandhuhu u xannibo hirarka hirarka ee ka baxsan baaxadda gudbinta ama baaxadda baaxadda. Ugu yaraan OD ee 3.0 ayaa lagula talinayaa laakiin OD ah 6.0 ama ka weyn ayaa ku habboon.
Filterka sii daaya
Shaandhooyinka sii daaya waxay u adeegaan ujeeddada u oggolaanshaha fluorescence-ka la jecel yahay ee muunada si ay u gaaraan baaraha. Waxay xannibaan hirarka dhererka gaaban waxayna leeyihiin gudbinta sare ee hirarka dhaadheer. Nooca filtarku waxa kale oo uu la xidhiidha nambar, tusaale BA510IF ee shaxanka (shaandhaynta xannibaadda faragelinta), calaamadayntaas waxa ay tilmaamaysaa hirarka dhererka 50% ee gudbintiisa ugu badan.
Talooyinka la midka ah ee filtarrada kicinta ayaa run u ah filtarrada qiiqa: gudbinta ugu yar, bandwidth, OD, iyo CWL. Shaandheeyaha sii daaya ee leh CWL ugu habboon, gudbinta ugu yar, iyo isku darka OD wuxuu bixiyaa sawirada ugu iftiimaya ee suurtogalka ah, oo leh xannibaadda ugu qoto dheer ee suurtogalka ah, waxayna hubisaa in la ogaado calaamadaha qiiqa ugu hooseeya.
Muraayada Dichroic
Muraayad dichroic ah ayaa la dhex dhigayaa inta u dhaxaysa shaandhada kicinta iyo shaandhada qiiqa qiiqa xagal 45° waxayna ka tarjumaysaa calaamada kicinta ee ku wajahan fluorophore iyadoo u gudbinaysa calaamada qiiqa ee dhinaca baaraha. Shaandhooyinka dichroic ee ku habboon iyo kala qaybinta biimku waxay leeyihiin isbeddel fiiqan oo u dhexeeya milicsiga ugu sarreeya iyo gudbinta ugu badan, oo leh> 95% milicsiga xajmiga xajmiga shaandhada kicinta iyo gudbinta> 90% ee xawaaraha shaandhada qiiqa. Dooro shaandhada maskaxda ku haysa mowjadda isgoysyada (λ) ee fluorophore, si aad u yarayso iftiinka lumaya oo aad sare ugu qaaddo saamiga sawirka fluorescent-to-sanaq.
Muraayada dichroic ee jaantuskan waa DM505, sidaas darteed waxaa loo magacaabay sababtoo ah 505 nanometer waa hirarka dhererka 50% ee gudbinta ugu badan ee muraayadan. Qalooca gudbinta ee muraayaddan ayaa muujinaysa gudbinta sare ee ka sarreysa 505 nm, hoos u dhac ku yimid gudbinta bidix ee 505 nanometer, iyo milicsiga ugu badan ee bidix ee 505 nanometers laakiin wali waxaa laga yaabaa in qaar gudbin ah oo ka hooseeya 505 nm.
Waa maxay faraqa u dhexeeya baaska dheer iyo filtarrada baaska?
Shaandheeyayaasha Fluorescence waxa loo qaybin karaa laba nooc: Pass dheer (LP) iyo band pass (BP).
Shaandhooyinka baska dheer ayaa gudbiya mawjadaha dhaadheer waxayna xannibaan kuwa gaagaaban. Mawjada dhererka la jaray waa qiimaha 50% ee gudbinta ugu sarreysa, iyo dhammaan hirarka dhererka ka sarreeya goynta waxaa gudbiya filtarrada baaska dheer. Inta badan waxaa lagu isticmaalaa muraayadaha dichroic iyo filtarrada qiiqa. filtarrada Longpass waa in la isticmaalaa marka codsigu u baahan yahay ururinta ugu badnaan qiiqa iyo marka takoorid spectral aan la rabin ama lagama maarmaanka ah, taas oo guud ahaan kiiska baaritaanka soo saara hal nooc oo sii daaya ee muunado leh heerar hoose ee autofluorescence asalka.
Shaandheeyayaasha kaarka baska waxa ay gudbiyaan oo kaliya qayb mawjadaha dhererka dhererka ah, oo ay xannibaan kuwa kale. Waxay yareeyaan is-dhaafsiga iyaga oo u oggolaanaya kaliya qaybta ugu xoogga badan ee qiiqa qiiqa fluorophore in la gudbiyo, waxay yareeyaan dhawaaqa autofluorescence oo sidaas darteed waxay hagaajiyaan saamiga calaamad-ilaa-sanqa ee muunadaha autofluorescence asalka sare, kuwaas oo filtarrada gudbinta dheer aysan bixin karin.
Immisa nooc oo ah shaandhada shaandhada florescence-ka BestScope ayaa bixin karta?
Qaar ka mid ah noocyada caadiga ah ee filtarrada waxaa ka mid ah buluug, cagaar, iyo filtarrada ultraviolet. Sida shaxda ka muuqata.
| Qaabka shaandhaynta | Shaandhaynta xiisaha | Muraayada Dichroic | Filterka xannibaadda | Nalalka LED dhererka hirarka | Codsiga |
| B | BP460-495 | DM505 | BA510 | 485nm | FITC: Habka kahortaga jidhka ee fluorescent orange acid: DNA, RNA · Auramine: Bacillus tubercle · EGFP, S657, RSGFP |
| G | BP510-550 | DM570 | BA575 | 535nm | Rhodamine, TRITC: Habka kahortaga jidhka ee fluorescent · Propidium iodide: DNA · RFP |
| U | BP330-385 | DM410 | BA420 | 365nm | U fiirsashada tooska ah ee fluorescence DAPI: midabaynta DNA · Hoechest 332528, 33342: loo isticmaalo midabaynta Chromosome |
| V | BP400-410 | DM455 | BA460 | 405nm | · Catecholamines · 5-hydroxy tryptamine Tetracycline: Qalfoofka, Ilkaha |
| R | BP620-650 | DM660 | BA670-750 | 640nm | · Cy5 Alex Fluor 633, Alexa Fluor 647 |
Shaandhooyinka shaandhaynta ee loo isticmaalo soo iibsiga fluorescence waxa lagu nashqadeeyay hareeraha mawjadaha dhaadheer ee loo isticmaalo codsiyada fluorescence, kaas oo ku salaysan fluorophores-ka aadka loo isticmaalo. Sababtan awgeed, waxaa sidoo kale loogu magac daray fluorophore-ka loogu talagalay sawir-qaadista, sida DAPI (buluug), FITC (cagaar) ama TRITC (casaan) xabadaha shaandhada.
| Qaabka shaandhaynta | Shaandhaynta xiisaha | Muraayada Dichroic | Filterka xannibaadda | Nalalka LED dhererka hirarka |
| FITC | BP460-495 | DM505 | BA510-550 | 485nm |
| DAPI | BP360-390 | DM415 | BA435-485 | 365nm |
| TRITC | BP528-553 | DM565 | BA578-633 | 535nm |
| FL-Auramine | BP470 | DM480 | BA485 | 450nm |
| Casaan Texas | BP540-580 | DM595 | BA600-660 | 560nm |
| mCherry | BP542-582 | DM593 | BA605-675 | 560nm |
Sideed u doorataa shaandhada fluorescence?
1. Mabda'a xulashada shaandhada fluorescence waa in la oggolaado iftiinka fluorescence / iftiinka iftiinka inuu dhex maro dhammaadka sawir-qaadista inta ugu badan ee suurtogalka ah, oo gebi ahaanba xannibo iftiinka kicinta isla waqti isku mid ah, si loo helo saamiga ugu sarreeya ee signal-to-sanqa. Gaar ahaan codsiga kicinta badan ee sawir-qaadista iyo wadarta mikroskoob ee milicsiga gudaha, qaylada daciifka ah waxay sidoo kale keeni doontaa faragelin weyn oo saameyn ku yeelata sawirka, markaa shuruudaha calaamadda saamiga qaylada ayaa ka sarreeya.
2. Ogow xamaasadda iyo qiiqa qiiqa ee fluorophore-ka. Si loo dhiso shaandhada shaandhada oo soo saarta muuqaal tayo sare leh, muuqaal is barbar dhig ah oo leh asal madow, xamaasad iyo filtarrada sii daaya waa in ay gaaraan gudbin sare oo leh baasboorka ugu yar ee ka sarreeya gobollada u dhigma meelaha ugu sarreeya ee kacsanaanta fluorophore ama qiiqa.
3. Tixgeli adkeysiga filtarrada fluorescence. Shaandheeyayaashani waa inay ahaadaan kuwo aan ka ilaalin ilaha iftiinka aadka u daran ee dhaliya iftiinka ultraviolet (UV) ee u horseedi kara "gubasho", gaar ahaan shaandhada exciter maadaama ay hoos ugu dhacayso xoogga buuxa ee isha iftiinka.
Sawirada Tusaalaha Fluorescent ee kala duwan
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Waqtiga boostada: Dec-09-2022