Fyuluta ya fluorescence ndi gawo lofunikira mu microscope ya fluorescence. Dongosolo lodziwika bwino lili ndi zosefera zitatu: fyuluta yosangalatsa, fyuluta yotulutsa mpweya ndi galasi la dichroic. Nthawi zambiri amaikidwa mu cube kuti gululo lilowetsedwe pamodzi mu microscope.
Kodi fyuluta ya fluorescence imagwira ntchito bwanji?
Zosefera Zosangalatsa
Zosefera zachisangalalo zimatumiza kuwala kwa utali wosiyanasiyana ndikutchinga mafunde ena. Atha kugwiritsidwa ntchito kupanga mitundu yosiyanasiyana pokonza zosefera kuti zilole mtundu umodzi wokha. Zosefera zosangalatsa zimabwera m'mitundu iwiri ikuluikulu - zosefera zazitali zazitali ndi zosefera za band pass. Chisangalalocho nthawi zambiri chimakhala fyuluta ya bandpass yomwe imadutsa mafunde okha omwe amatengedwa ndi fluorophore, motero kuchepetsa kusangalatsa kwa magwero ena a fulorosisi ndi kutsekereza kuwala kosangalatsa mu bandi yotulutsa fulorosenti. Monga momwe zasonyezedwera ndi mzere wa buluu pachithunzichi, BP ndi 460-495, zomwe zikutanthauza kuti ikhoza kudutsa mu fluorescence ya 460-495nm.
Imayikidwa mkati mwa njira yowunikira ya microscope ya fluorescence ndipo imasefa mafunde onse a gwero la kuwala kupatulapo fulorophore excitation range. Zosefera zochepa zimatengera kuwala ndi kunyezimira kwa zithunzi. Kupatsirana kochepera 40% kwa fyuluta iliyonse yosangalatsa kumalimbikitsidwa kotero kuti kufalitsa ndikoyenera> 85%. Bandiwifi ya fyuluta yosangalatsa iyenera kukhala mkati mwa chiwongolero cha fulorophore kotero kuti pakati wavelength (CWL) ya fyulutayo ikhale yoyandikana kwambiri ndi kutalika kwa chitonthozo chapamwamba cha fulorophore. The excitation fyuluta kuwala kachulukidwe (OD) kulamula chakumbuyo chithunzi mdima; OD ndi muyeso wa momwe fyuluta imatsekera bwino kutalika kwa mafunde kunja kwa mayendedwe opatsirana kapena bandwidth. Ochepera OD a 3.0 akulimbikitsidwa koma OD ya 6.0 kapena kupitilira apo ndi yabwino.
Zosefera Emission
Zosefera zotulutsa utsi zimagwira ntchito yolola kuti fulorosenti yofunikira kuchokera pachitsanzo ifike pa chowunikira. Amaletsa mafunde afupikitsa ndipo amakhala ndi kufalikira kwakukulu kwa mafunde ataliatali. Mtundu wa fyuluta umalumikizidwanso ndi nambala, mwachitsanzo BA510IF pachithunzicho (zosokoneza zotchinga fyuluta), dzinalo limatanthawuza kutalika kwa kutalika kwa 50% ya kufalikira kwake kwakukulu.
Zomwezo zosefera zosangalatsa zimakhala zowona pazosefera zotulutsa: kufalitsa kochepa, bandwidth, OD, ndi CWL. Zosefera zotulutsa zokhala ndi CWL yabwino, kufalitsa pang'ono, ndi kuphatikiza kwa OD kumapereka zithunzi zowoneka bwino kwambiri, zotsekereza zozama kwambiri, ndikuwonetsetsa kuti zizindikiritso zotulutsa pang'ono kwambiri.
Dichroic Mirror
Kalilore wa dichroic amayikidwa pakati pa fyuluta yosangalatsa ndi fyuluta yotulutsa mpweya pa ngodya ya 45 ° ndikuwonetsa chizindikiro cha chisangalalo chopita ku fluorophore pamene akutumiza chizindikiro cha mpweya ku chowunikira. Zosefera zabwino za dichroic ndi zogawanitsa zamitengo zimakhala ndi kusintha kwakuthwa pakati pa kuwunikira kwakukulu ndi kufalikira kwakukulu, ndi> 95% chiwonetsero cha bandwidth ya fyuluta yosangalatsa komanso kufalitsa> 90% pa bandwidth ya fyuluta yotulutsa. Sankhani fyuluta yomwe ili ndi kutalika kwa kutalika kwa msewu (λ) wa fulorophore m'maganizo, kuti muchepetse kuwala kosokera komanso kukulitsa chiyerekezo cha chithunzi cha fulorosenti ndi phokoso.
Magalasi a dichroic pachithunzichi ndi DM505, omwe amatchedwa chifukwa 505 nanometers ndi kutalika kwa 50% ya kufalikira kwakukulu kwa galasi ili. Njira yopatsira pagalasiyi ikuwonetsa kufalikira kwakukulu pamwamba pa 505 nm, kutsika kotsetsereka kumanzere kwa 505 nanometers, komanso kuwunikira kwambiri kumanzere kwa 505 nanometers komabe kumatha kufalikira kumunsi kwa 505 nm.
Kodi pali kusiyana kotani pakati pa zosefera zazitali ndi band pass?
Zosefera za fluorescence zitha kugawidwa m'mitundu iwiri: kupita kutali (LP) ndi band pass (BP).
Zosefera zazitali zimatulutsa mafunde aatali ndikuletsa zazifupi. Kudulidwa-pa wavelength ndi mtengo wa 50% wa kufala kwapamwamba, ndipo mafunde onse pamwamba pa odulidwa amafalitsidwa ndi zosefera zazitali. Amagwiritsidwa ntchito pafupipafupi mu magalasi a dichroic ndi zosefera zotulutsa. Zosefera za Longpass ziyenera kugwiritsidwa ntchito pomwe pulogalamuyo ikufuna kusonkhanitsa zinthu zambiri komanso ngati tsankho lachiwonekere silingafune kapena kuli kofunikira, zomwe nthawi zambiri zimakhala zofufuza zomwe zimapanga mtundu umodzi wokha wotulutsa mu zitsanzo zokhala ndi milingo yotsika yakumbuyo ya autofluorescence.
Zosefera za band pass zimangotumiza gulu linalake la wavelength, ndikuletsa ena. Amachepetsa crosstalk polola kuti gawo lamphamvu kwambiri la fluorophore emission spectrum lifalitsidwe, kuchepetsa phokoso la autofluorescence ndipo motero kumapangitsa kuti chiŵerengero cha signal-to-noise mu zitsanzo zapamwamba za autofluorescence, zomwe zosefera zazitali sizingapereke.
Ndi mitundu ingati ya fyuluta ya fluorescence yomwe BestScope ingapereke?
Mitundu ina yodziwika bwino ya zosefera imaphatikizapo zosefera zabuluu, zobiriwira, ndi za ultraviolet. Monga momwe tawonetsera mu tebulo.
| Zosefera Seti | Zosefera Zosangalatsa | Dichroic Mirror | Zosefera Zotchinga | Utali wa Wave Wave wa LED | Kugwiritsa ntchito |
| B | Mtengo wa BP460-495 | DM505 | Chithunzi cha BA510 | 485nm pa | FITC: Njira ya antibody ya fluorescent +Acidine lalanje: DNA, RNA Auramine: Tubercle bacillus ·EGFP, S657, RSGFP |
| G | Mtengo wa BP510-550 | DM570 | BA575 | 535nm pa | ·Rhodamine, TRITC: Njira ya antibody ya fluorescent Propidium iodide: DNA · RFP |
| U | BP330-385 | DM410 | Chithunzi cha BA420 | 365nm pa | · Kuwunika kwa auto-fluorescence ·DAPI: Kudetsa kwa DNA Hoechest 332528, 33342: yogwiritsidwa ntchito pakuyezera ma Chromosome |
| V | BP400-410 | DM455 | Chithunzi cha BA460 | 405nm pa | · Katekisimu 5-hydroxy tryptamine Tetracycline: Mafupa, Mano |
| R | Mtengo wa BP620-650 | DM660 | BA670-750 | 640nm pa | · Chi5 Alexa Fluor 633, Alexa Fluor 647 |
Zosefera zomwe zimagwiritsidwa ntchito pogula fulorosenti zimapangidwa mozungulira mafunde akuluakulu omwe amagwiritsidwa ntchito popanga ma fluorescence, omwe amakhala mozungulira ma fluorophores omwe amagwiritsidwa ntchito kwambiri. Pazifukwa izi, amatchulidwanso dzina la fluorophore omwe amapangidwira kuti azijambula, monga DAPI (buluu), FITC (wobiriwira) kapena TRITC (red) fyuluta cubes.
| Zosefera Seti | Zosefera Zosangalatsa | Dichroic Mirror | Zosefera Zotchinga | Utali wa Wave Wave wa LED |
| Mtengo wa FITC | Mtengo wa BP460-495 | DM505 | BA510-550 | 485nm pa |
| DAPI | BP360-390 | DM415 | BA435-485 | 365nm pa |
| Mtengo wa TRITC | Mtengo wa BP528-553 | DM565 | BA578-633 | 535nm pa |
| FL-Auramine | Mtengo wa BP470 | DM480 | BA485 | 450nm pa |
| Texas Red | Mtengo wa BP540-580 | DM595 | BA600-660 | 560nm pa |
| mCherry | Mtengo wa BP542-582 | DM593 | BA605-675 | 560nm pa |
Kodi mungasankhe bwanji fyuluta ya fluorescence?
1. Mfundo yosankha fyuluta ya fluorescence ndiyo kulola kuwala kwa fluorescence / kutuluka kwa mpweya kudutsa kumapeto kwa kujambula momwe zingathere, ndikutsekereza kwathunthu kuwala kosangalatsa panthawi imodzimodziyo, kuti mupeze chiŵerengero chapamwamba kwambiri cha chizindikiro ndi phokoso. Makamaka pakugwiritsa ntchito kusangalatsa kwa ma multiphoton ndi maikulosikopu yowoneka bwino yamkati, phokoso lofooka lipangitsanso kusokoneza kwakukulu pamaganizidwe, kotero kuti chiwongolero cha ma sign ndi phokoso ndichokwera kwambiri.
2. Dziwani chisangalalo ndi kutulutsa kwa fluorophore. Kupanga seti ya fyuluta ya fluorescence yomwe imapanga chithunzi chapamwamba, chosiyana kwambiri ndi maziko akuda, zosefera zachisangalalo ndi zotulutsa zotulutsa zimayenera kupititsa patsogolo kufalikira kwapang'onopang'ono kwa passband kumadera omwe amagwirizana ndi nsonga zachisangalalo za fluorophore kapena kutulutsa mpweya.
3. Ganizirani kulimba kwa zosefera za fulorosenti. Zoseferazi ziyenera kukhala zosasunthika ndi kuwala kwamphamvu komwe kumatulutsa kuwala kwa ultraviolet (UV) komwe kungayambitse "kutopa", makamaka fyuluta ya exciter chifukwa imayang'aniridwa ndi mphamvu yonse ya gwero lounikira.
Mitundu Yosiyanasiyana ya Zitsanzo za Fluorescent
Zothandizira zimasonkhanitsidwa ndikukonzedwa pa intaneti, ndipo zimagwiritsidwa ntchito pophunzira ndi kulumikizana. Ngati pali kuphwanya kulikonse, chonde titumizireni kuti tifufute.
Nthawi yotumiza: Dec-09-2022