Lub fluorescence lim yog ib qho tseem ceeb ntawm fluorescence microscope.Ib qho kev raug kaw muaj peb lub ntsiab lus lim: ib qho kev lim dej excitation, lim emission thiab daim iav dichroic.Lawv feem ntau ntim rau hauv ib lub voos xwmfab kom cov pab pawg tau muab tso ua ke rau hauv lub microscope.
Lub fluorescence lim ua haujlwm li cas?
Excitation lim
Excitation filters kis lub teeb ntawm ib lub wavelength tshwj xeeb thiab thaiv lwm cov wavelengths.Lawv tuaj yeem siv los tsim cov xim sib txawv los ntawm kev kho lub lim kom tso cai rau ib qho xim los ntawm.Cov lim dej ua kom muaj zog tuaj nyob rau hauv ob hom tseem ceeb - cov ntxaij lim dej ntev dhau thiab cov lim lim.Cov exciter feem ntau yog cov lim bandpass uas hla tsuas yog cov wavelengths absorbed los ntawm fluorophore, yog li txo qis excitation ntawm lwm qhov chaw ntawm fluorescence thiab thaiv excitation teeb nyob rau hauv lub fluorescence emission band.Raws li pom los ntawm kab xiav hauv daim duab, BP yog 460-495, uas txhais tau hais tias nws tsuas tuaj yeem dhau los ntawm fluorescence ntawm 460-495nm.
Nws tau muab tso rau hauv txoj kev teeb pom kev ntawm lub tshuab hluav taws xob fluorescence thiab lim tawm tag nrho cov wavelengths ntawm lub teeb ci tshwj tsis yog rau fluorophore excitation ntau.Cov lim yam tsawg kawg nkaus xa tawm dictates lub brightness thiab brilliance ntawm cov duab.Yam tsawg kawg ntawm 40% kis tau tus mob rau ib qho excitation lim tau pom zoo xws li tias kev sib kis yog qhov zoo tshaj> 85%.Lub bandwidth ntawm lub lim excitation yuav tsum tag nrho nyob rau hauv lub fluorophore excitation ntau yam xws li qhov nruab nrab wavelength (CWL) ntawm lub lim yog ze li sai tau mus rau lub ncov excitation wavelength ntawm fluorophore.Lub excitation lim optical ntom (OD) dictates cov duab tom qab tsaus ntuj;OD yog ib qho kev ntsuas ntawm qhov zoo npaum li cas lub lim thaiv cov wavelengths sab nraud ntawm kev sib kis los yog bandwidth.Qhov tsawg kawg nkaus OD ntawm 3.0 raug pom zoo tab sis OD ntawm 6.0 lossis siab dua yog qhov zoo tagnrho.
Emission lim
Cov ntxaij lim dej emission ua haujlwm rau lub hom phiaj ntawm kev tso cai fluorescence uas xav tau los ntawm cov qauv kom ncav cuag lub ntes.Lawv thaiv qhov luv wavelengths thiab muaj kev sib kis tau ntev rau cov wavelengths ntev.Hom lim kuj tseem cuam tshuam nrog tus lej, piv txwv li BA510IF hauv daim duab (kev cuam tshuam cuam tshuam cov lim dej), qhov kev xaiv ntawd hais txog lub wavelength ntawm 50% ntawm nws qhov siab tshaj plaws kis tau tus mob.
Cov lus pom zoo tib yam rau cov lim lim tawm muaj tseeb rau cov lim dej tawm: qhov tsawg kawg nkaus kis tau tus mob, bandwidth, OD, thiab CWL.Lub emission lim nrog qhov zoo tshaj plaws CWL, qhov tsawg kawg nkaus kis tau tus mob, thiab OD ua ke muab cov duab ci ntsa iab tshaj plaws, nrog rau qhov tob tshaj plaws tau thaiv, thiab xyuas kom pom cov teeb liab faintest emission.
Daim iav Dichroic
Daim iav dichroic yog muab tso rau nruab nrab ntawm lub excitation lim thiab emission lim ntawm ib tug 45 ° lub kaum sab xis thiab qhia txog lub excitation teeb liab ntawm lub fluorophore thaum kis lub emission teeb liab mus rau lub detector.Qhov zoo tshaj plaws dichroic filters thiab beam splitters muaj kev hloov pauv ntawm qhov siab tshaj plaws ntawm kev xav thiab kev xa tawm siab tshaj plaws, nrog rau> 95% kev xav rau lub bandwidth ntawm lub lim lim dej thiab kev xa tawm ntawm> 90% rau bandwidth ntawm lub lim lim.Xaiv lub lim nrog qhov kev sib tshuam wavelength (λ) ntawm fluorophore hauv siab, kom txo qis qis-teeb pom kev zoo thiab ua kom pom qhov zoo tshaj plaws fluorescent duab teeb liab-rau- suab nrov piv.
Daim iav dichroic hauv daim duab no yog DM505, yog li lub npe vim 505 nanometers yog lub wavelength ntawm 50% ntawm qhov siab tshaj plaws kis tau tus mob rau daim iav no.Qhov kis tau tus mob nkhaus rau daim iav no qhia tau hais tias muaj kev sib kis siab tshaj 505 nm, qhov kev poob qis hauv kev xa mus rau sab laug ntawm 505 nanometers, thiab qhov kev xav tau siab tshaj plaws mus rau sab laug ntawm 505 nanometers tab sis tseem yuav muaj qee qhov kev sib kis hauv qab 505 nm.
Dab tsi yog qhov txawv ntawm qhov ntev pass thiab band pass lim?
Fluorescence lim tuaj yeem muab faib ua ob hom: ntev dhau (LP) thiab band dhau (BP).
Ntev dhau cov ntxaij lim dej xa cov wavelengths ntev thiab thaiv cov luv luv.Qhov kev txiav-on wavelength yog tus nqi ntawm 50% ntawm qhov kawg ntawm kev sib kis, thiab tag nrho cov wavelengths saum toj kawg nkaus yog kis tau los ntawm cov ntxaij lim dej ntev dhau.Lawv nquag siv hauv cov iav dichroic thiab emission lim.Longpass lim yuav tsum tau siv thaum daim ntawv thov yuav tsum tau ntau tshaj emission collection thiab thaum spectral kev ntxub ntxaug yog tsis xav tau los yog tsim nyog, uas yog feem ntau yog cov ntaub ntawv rau probes uas tsim ib tug emitting hom nyob rau hauv cov qauv uas muaj kuj tsawg theem ntawm autofluorescence tom qab.
Band dhau cov lim dej tsuas yog kis tau qee qhov wavelength band, thiab thaiv lwm tus.Lawv txo qis crosstalk los ntawm kev tso cai tsuas yog qhov muaj zog tshaj plaws ntawm fluorophore emission spectrum kis tau tus mob, txo cov suab nrov ntawm autofluorescence thiab yog li txhim kho cov teeb liab-rau- suab nrov piv hauv cov qauv autofluorescence siab tom qab, uas ntev dhau cov lim dej tsis tuaj yeem muab.
Muaj pes tsawg hom fluorescence lim teeb BestScope tuaj yeem muab tau?
Qee hom lim dej muaj xws li xiav, ntsuab, thiab ultraviolet lim.Raws li qhia nyob rau hauv lub rooj.
| Lim Teeb | Excitation lim | Daim iav Dichroic | Barrier Lim | LED teeb Wave Length | Daim ntawv thov |
| B | PEB 460-495 | TIAB SA DM505 | SIB 510 | 485nm ua | · FITC: Fluorescent antibody method · Acidine txiv kab ntxwv: DNA, RNA · Auramine: Tubercle bacillus · EGFP, S657, RSGFP |
| G | PEB 510-550 | SIB 570 | SIB 575 | 535nm ua | · Rhodamine, TRITC: Fluorescent antibody txoj kev Propidium iodide: DNA · RFP |
| U | PEB 330-385 | TIAB SA DM410 | SIB 420 | 365nm ua | · Auto-fluorescence soj ntsuam · DAPI: DNA staining · Hoechest 332528, 33342: siv rau Chromosome staining |
| V | PEB 400-410 | SIB 455 | SIB 460 | 405nm ua | · Catecholamines · 5-hydroxy tryptamine · Tetracycline: Skeleton, Cov hniav |
| R | PEB 620-650 | TIAB SA DM660 | IB 670-750 | 640nm ua | · Cy5 · Alexa Fluor 633, Alexa Fluor 647 |
Cov lim lim uas siv rau hauv cov khoom siv fluorescence yog tsim nyob ib ncig ntawm cov wavelengths loj siv hauv cov ntawv siv fluorescence, uas yog nyob ib ncig ntawm cov fluorophores siv tshaj plaws.Vim li no, lawv kuj muaj npe tom qab fluorophore lawv tau npaj rau kev yees duab, xws li DAPI (xiav), FITC (ntsuab) lossis TRITC (liab) lim cubes.
| Lim Teeb | Excitation lim | Daim iav Dichroic | Barrier Lim | LED teeb Wave Length |
| FITC | PEB 460-495 | TIAB SA DM505 | IB 510-550 | 485nm ua |
| DAPI | PEB 360-390 | SIB 415 | IB 435-485 | 365nm ua |
| TRITC | PEB 528-553 | TIAB SA 565 | IB 578-633 | 535nm ua |
| FL-Auramine | PEB 470 | SIB 480 | SIB 485 | 450nm ua |
| Texas Liab | PEB 540-580 | TIAB SA 595 | IB 600-660 | 560nm ua |
| mCherry | PEB 542-582 | SIB 593 | IB 605-675 | 560nm ua |
Koj xaiv lub lim fluorescence li cas?
1. Lub hauv paus ntsiab lus ntawm kev xaiv cov fluorescence lim yog cia lub teeb fluorescence / emission kis mus rau qhov kawg ntawm cov duab kom deb li deb tau, thiab tag nrho thaiv lub teeb excitation tib lub sijhawm, kom tau txais qhov siab tshaj plaws ntawm cov teeb liab-rau-suab nrov piv.Tshwj xeeb tshaj yog rau daim ntawv thov ntawm multiphoton excitation thiab tag nrho cov sab hauv lub tsom iav tsom iav, lub suab nrov tsis muaj zog yuav ua rau muaj kev cuam tshuam loj rau cov duab kos duab, yog li qhov yuav tsum tau ua rau lub teeb liab rau lub suab nrov piv yog siab dua.
2. Paub qhov excitation thiab emission spectrum ntawm fluorophore.Txhawm rau tsim cov fluorescence lim teeb uas tsim kom muaj cov duab zoo, zoo sib xws nrog cov keeb kwm yav dhau los dub, excitation thiab emission lim yuav tsum ua kom muaj kev sib kis tau zoo nrog tsawg kawg nkaus passband ripple hla cov cheeb tsam uas sib haum rau fluorophore excitation peaks lossis emissions.
3. Xav txog qhov ua tau ntev ntawm cov lim dej fluorescence.Cov lim dej no yuav tsum tsis txhob ua rau lub teeb pom kev hnyav uas tsim cov teeb pom kev ultraviolet (UV) uas tuaj yeem ua rau "burnout", tshwj xeeb tshaj yog cov lim dej exciter vim nws raug rau tag nrho cov kev siv ntawm lub teeb pom kev zoo.
Cov duab fluorescent txawv
Cov peev txheej tau sau thiab teeb tsa hauv Is Taws Nem, thiab tsuas yog siv rau kev kawm thiab kev sib txuas lus.Yog tias muaj kev ua txhaum cai, thov hu rau peb kom tshem tawm.
Post lub sij hawm: Dec-09-2022