Fluorescence sefa chinhu chakakosha mu fluorescence microscope.Iyo yakajairwa sisitimu ine matatu ekutanga mafirita: yekunakisa sefa, emission sefa uye dichroic girazi.Anowanzo kuputirwa mucube kuitira kuti boka ripinzwe pamwechete mumaikorosikopu.
Sefa yefluorescence inoshanda sei?
Sefa Inofadza
Mafirita ekunakisa anoendesa mwenje weimwe wavelength uye anovhara mamwe mafungu.Anogona kushandiswa kugadzira mavara akasiyana nekugadzirisa sefa kuti ingobvumira ruvara rumwe chete kuburikidza.Mafirita ekusimudzira anouya mumhando mbiri huru - refu pass mafirita uye bhendi pass mafirita.Iyo exciter inowanzova bandpass sefa iyo inopfuura chete wavelengths yakabatwa nefluorophore, nokudaro kuderedza kunakidzwa kwemamwe masosi efluorescence uye kuvharira excitation chiedza mu fluorescence emission band.Sezvinoratidzwa nemutsara webhuruu mumufananidzo, BP i460-495, zvinoreva kuti inogona kungopfuura nepakati pefluorescence ye460-495nm.
Inoiswa mukati menzira yekuvhenekera yefluorescence microscope uye inosefa kunze kwese kwewavelengths yechiedza chechiedza kunze kweiyo fluorophore excitation range.Iyo sefa shoma yekufambisa inoraira kupenya uye kupenya kwemifananidzo.Iyo shoma ye40% kutapurirana kune chero excitation sefa inokurudzirwa zvekuti kutapurirana kuri kuita> 85%.Iyo bandwidth yefirita yekunakisa inofanirwa kunge iri mukati meiyo fluorophore excitation range zvekuti iyo center wavelength (CWL) yesefa iri padyo sezvinobvira kune peak excitation wavelength yefluorophore.Iyo excitation filter optical density (OD) inoraira iyo yekumashure mufananidzo rima;OD chiyero chekuti sefa inovharira sei mawavelength kunze kweyekufambisa renji kana bandwidth.Iyo shoma OD ye3.0 inokurudzirwa asi OD ye6.0 kana kupfuura ndiyo yakanaka.
Emission Sefa
Emission mafirita anoshanda chinangwa chekubvumira fluorescence inodiwa kubva kumuenzaniso kuti isvike pa detector.Vanovhara mapfupi ewavelengths uye vane hutachiwana hwepamusoro kwehurefu hwewavelengths.Rudzi rwesefa inobatanidzwawo nenhamba, semuenzaniso BA510IF mumufananidzo (interference barrier filter), iro zita rinoreva wavelength pa50% yekunyanya kutapurirana.
Iwowo kurudziro yemafirita ekusimudzira anobata echokwadi kune emission mafirita: mashoma kutapurirana, bandwidth, OD, uye CWL.Sefa yekuburitsa ine yakanakira CWL, kushoma kutapurirana, uye OD musanganiswa inopa inopenya inogoneka mifananidzo, ine yakadzama inovharika inovharika, uye inova nechokwadi chekuonekwa kwezvisina kusimba zvekuburitsa.
Dichroic Mirror
Dichroic girazi rinoiswa pakati pesefa yekukurudzira uye sefa yekuburitsa pakona ye45 ° uye inoratidza chiratidzo chekunakidzwa chakananga kufluorophore ichiendesa chiratidzo chekuburitsa kune detector.Ideal dichroic mafirita uye matanda anotsemuka ane shanduko dzakapinza pakati pekutarisa kwakanyanya uye yakanyanya kutapurirana, ine > 95% inoratidzira kune bandwidth yeiyo excitation sefa uye kutapurirana kwe> 90% yebandwidth yeemission filter.Sarudza iyo sefa ine mharadzano wavelength (λ) yefluorophore mundangariro, kuderedza kurasika-mwenje uye kuwedzera fluorescent mufananidzo chiratidzo-ku-ruzha reshiyo.
Dichroic mirror mumufananidzo uyu ndiyo DM505, yakadanwa kudaro nekuti 505 nanometers ndiyo yavelength pa50% yehukuru hwekutapurirana kwegirazi iri.Iyo yekutapurirana curve yeichi girazi inoratidza kutapurirana kwakakwira pamusoro pe505 nm, kudonha kwakadzika kwekutapurirana kuruboshwe kwe505 nanometers, uye kupenya kwakanyanya kuruboshwe kwe505 nanometers asi zvakadaro inogona kuve nekumwe kutapurirana pazasi 505 nm.
Ndeupi mutsauko pakati penguva refu uye bhendi pass filters?
Mafirita eFluorescence anogona kukamurwa kuita marudzi maviri: refu pass (LP) uye band pass (BP).
Masefa marefu ekupfuura anofambisa marefu mawavelengths uye anovhara mapfupi.Iyo yakachekwa-pawavelength ndiyo kukosha pa50% yekutapurirana kwepamusoro, uye ese mawavelength ari pamusoro peakachekwa anofambiswa neakareba mafirita.Iwo anowanzo shandiswa mune dichroic magirazi uye emission mafirita.Mafirita eLongpass anofanirwa kushandiswa kana chikumbiro chichida kuunganidzwa kwakanyanya uye kana rusaruro rusingafadzi kana kudikanwa, inova ndiyo nyaya yemaprobes anoburitsa mhando imwe chete inoburitsa mumienzaniso ine yakadzikira mwero wekumashure autofluorescence.
Band pass mafirita anongoendesa imwe wavelength bhendi, uye anovharira vamwe.Ivo vanoderedza crosstalk nekubvumira chete chikamu chakasimba che fluorophore emission spectrum kuti chifambiswe, kuderedza autofluorescence ruzha uye nekudaro vanovandudza chiratidzo-ku-ruzha reshiyo mune yakakwira kumashure autofluorescence samples, iyo yakareba kupfuura mafirita haigone kupa.
Mangani marudzi efluorescence seti seti BestScope inogona kupa?
Mamwe marudzi akajairika emafirita anosanganisira bhuruu, girini, uye ultraviolet mafirita.Sezvinoratidzwa mutafura.
| Sefa Seti | Sefa Inofadza | Dichroic Mirror | Barrier Sefa | Mwenje we LED Wave Length | Application |
| B | BP460-495 | DM505 | BA510 | 485nm | FITC: Fluorescent antibody nzira Acdine orenji: DNA, RNA ·Auramine: Tubercle bacillus ·EGFP, S657, RSGFP |
| G | BP510-550 | DM570 | BA575 | 535nm | ·Rhodamine, TRITC: Nzira yeFluorescent antibody Propidium iodide: DNA ·RFP |
| U | BP330-385 | DM410 | BA420 | 365nm | ·Auto-fluorescence observation ·DAPI: DNA staining Hoechest 332528, 33342: inoshandiswa kusvibisa Chromosome |
| V | BP400-410 | DM455 | BA460 | 405nm | ·Katecholamines ·5-hydroxy tryptamine Tetracycline: Skeleton, Mazino |
| R | BP620-650 | DM660 | BA670-750 | 640nm | ·Cy5 Alexa Fluor 633, Alexa Fluor 647 |
Masefa seti anoshandiswa mukutora fluorescence akagadzirwa akatenderedza mafungu makuru anoshandiswa mu fluorescence application, iyo yakavakirwa kutenderedza inonyanya kushandiswa fluorophores.Nechikonzero ichi, ivo zvakare vakatumidzwa zita re fluorophore yavanoitirwa kufungidzira, senge DAPI (bhuruu), FITC (girinhi) kana TRITC (tsvuku) sefa cubes.
| Sefa Seti | Sefa Inofadza | Dichroic Mirror | Barrier Sefa | Mwenje we LED Wave Length |
| 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 |
| Texas Red | BP540-580 | DM595 | BA600-660 | 560nm |
| mCherry | BP542-582 | DM593 | BA605-675 | 560nm |
Ungasarudza sei fluorescence sefa?
1. Nheyo yekusarudza fluorescence filter ndeyokurega chiedza chefluorescence / emission chichipfuura nepamagumo ekufungidzira sezvinobvira, uye kuvhara zvachose chiedza chekusimudzira panguva imwe chete, kuitira kuti uwane chiyero chepamusoro chechiratidzo-ku-ruzha.Kunyanya pakushandiswa kwemultiphoton excitation uye yakazara mukati inoratidzira maikorosikopu, iyo isina simba ruzha inokonzeresa kupindira kukuru kune yekufungidzira maitiro, saka chinodiwa chechiratidzo kune ruzha chiyero chakakwira.
2. Ziva mafambiro uye emission spectrum yefluorophore.Kugadzira seti yefluorescence seti inoburitsa yemhando yepamusoro, yepamusoro-inosiyana mufananidzo ine dema kumashure, mafaro uye emission mafirita anofanirwa kuwana kutapurirana kwepamusoro neashoma passband ripple pamusoro pematunhu anoenderana neiyo fluorophore excitation peaks kana emissions.
3. Funga kusimba kwemafirita efluorescence.Aya mafirita haafanire kuvharika kune akanyanya mwenje masosi anoburitsa ultraviolet (UV) mwenje unogona kutungamirira ku "kutsva", kunyanya yeexciter sefa sezvo ichiiswa pasi pekusimba kuzere kweruvheneko.
Iyo Yakasiyana Fluorescent Sample Images
Zviwanikwa zvinounganidzwa uye zvakarongeka paInternet, uye zvinongoshandiswa pakudzidza nekutaurirana.Kana paine chero kukanganisa, ndapota taura nesu kuti tibvise.
Nguva yekutumira: Zvita-09-2022