Isihlungi se-fluorescence siyingxenye ebalulekile kusibonakhulu se-fluorescence. Isistimu evamile inezihlungi ezintathu eziyisisekelo: isihlungi esivusa amadlingozi, isihlungi esikhipha umoya nesibuko se-dichroic. Avamise ukupakishwa ku-cube ukuze iqembu lifakwe ndawonye kusibonakhulu.
Sisebenza kanjani isihlungi se-fluorescence?
Isihlungi Sokuthokozisa
Izihlungi ezivusa amadlingozi zidlulisa ukukhanya kobude obuthile begagasi futhi zivimbe amanye amaza amaza. Angasetshenziselwa ukukhiqiza imibala ehlukene ngokulungisa isihlungi ukuvumela umbala owodwa kuphela. Izihlungi ezivusa amadlingozi ziza ngezinhlobo ezimbili eziyinhloko — izihlungi zephasi ende nezihlungi ze-band pass. I-exciter ivamise ukuba isihlungi se-bandpass esidlula kuphela ubude begagasi obumuncwe i-fluorophore, ngaleyo ndlela sinciphise ukuvukwa kweminye imithombo ye-fluorescence futhi sivimbe ukukhanya okuvusa amadlingozi kubhendi yokukhipha i-fluorescence. Njengoba kuboniswe umugqa ohlaza okwesibhakabhaka emfanekisweni, i-BP ingu-460-495, okusho ukuthi ingadlula kuphela ku-fluorescence ye-460-495nm.
Ifakwa ngaphakathi kwendlela yokukhanya yesibonakhulu se-fluorescence futhi ihlunge wonke ubude beza omthombo wokukhanya ngaphandle kwebanga le-fluorophore excitation. Ubuncane bokudluliswa kwesihlungi busho ukukhanya nokucwebezela kwezithombe. Kunconywe ukudluliswa okungama-40% kwanoma yisiphi isihlungi esivusa amadlingozi ukuze ukudlulisa kube ngokufanelekile >85%. Umkhawulokudonsa wesihlungi esivusa amadlingozi kufanele ube ngaphakathi ngokuphelele kwebanga lokuvusa i-fluorophore ukuze i-wavelength emaphakathi (CWL) yesihlungi ibe seduze ngangokunokwenzeka kubude begagasi be-excitation obuphezulu be-fluorophore. I-excitation filter optical density (OD) isho ubumnyama besithombe sangemuva; I-OD isilinganiso sokuthi isihlungi siwavimba kahle kangakanani ubude bamaza ngaphandle kwebanga lokudlulisela noma umkhawulokudonsa. I-OD encane engu-3.0 iyanconywa kodwa i-OD engu-6.0 noma ngaphezulu ilungile.
Isihlungi Sokuphuma
Izihlungi ezikhishwayo zisebenzisa inhloso yokuvumela i-fluorescence efiselekayo evela kusampula ukuthi ifinyelele kumtshina. Avimba amaza amaza amafushane futhi anokudluliselwa okuphezulu kwamaza amade amade. Uhlobo lwesihlungi luphinde luhlotshaniswe nenombolo, isb. BA510IF emfanekisweni (isihlungi sesithiyo sokuphazamiseka), leso sihloko sibhekisela kubude begagasi obungu-50% bokudluliswa kwaso okuphezulu.
Izincomo ezifanayo zezihlungi ezivusa amadlingozi zibambelela ngokuqinile kuzihlungi ezikhishwayo: ubuncane bokudlulisa, umkhawulokudonsa, i-OD, ne-CWL. Isihlungi se-emission esine-CWL ekahle, ukudluliselwa okuncane, kanye nenhlanganisela ye-OD sinikeza izithombe ezigqama kakhulu, ezinokuvimba okujulile okungase kube khona, futhi siqinisekisa ukutholwa kwamasignali akhipha umoya omncane kakhulu.
I-Dichroic Mirror
Isibuko se-dichroic sibekwe phakathi kwesihlungi esivusa amadlingozi nesihlungi esikhishwayo nge-engeli engu-45° futhi sibonisa isignali yokuvusa ibheke ku-fluorophore kuyilapho sidlulisela isignali yokukhipha umtshina kumtshina. Izihlungi ezifanelekile ze-dichroic nezihlukanisi zemishayo zinokushintsha okubukhali phakathi kokubonisa okuphezulu nokudluliswa okuphezulu, okunokuboniswa >95% komkhawulokudonsa wesihlungi esivusa amadlingozi kanye nokudluliswa okungu->90% komkhawulokudonsa wesihlungi se-emission. Khetha isihlungi esinobude be-wavelength bokuhlangana (λ) be-fluorophore engqondweni, ukuze unciphise ukukhanya okunhlanhlathayo futhi wandise isilinganiso sesignali somsindo wesithombe se-fluorescent.
Isibuko se-dichroic kulesi sibalo yi-DM505, esiqanjwe kanjalo ngoba ama-nanometer angu-505 ubude begagasi obungu-50% wokudluliselwa okukhulu kwalesi sibuko. Ijika lalesi sibuko libonisa ukudluliswa okuphezulu okungaphezulu kuka-505 nm, ukwehla okukhulu kokudluliselwa kwesokunxele sama-nanometers angu-505, kanye nokubonakala okuphezulu kwesokunxele sama-nanometers angu-505 kodwa kusengenzeka kube nokudluliswa okuthile okungaphansi kuka-505 nm.
Uyini umehluko phakathi kwezihlungi zephasi ende nezihlungi ze-band pass?
Izihlungi ze-fluorescence zingahlukaniswa zibe izinhlobo ezimbili: i-long pass (LP) ne-band pass (BP).
Izihlungi zamaphasi amade zidlulisa ubude bezansi amade futhi zivimbe amafushane. I-wavelength enqunyiwe iyinani elingu-50% lokudluliswa okuphezulu, futhi wonke amaza amaza angaphezu kwe-cut-on adluliswa izihlungi zokudlula amade. Zivame ukusetshenziswa ezibukweni ze-dichroic kanye nezihlungi ze-emission. Izihlungi ze-Longpass kufanele zisetshenziswe lapho uhlelo lokusebenza ludinga ukuqoqwa kokukhishwayo okuphezulu nalapho ukucwaswa okubonwayo kungafiseleki noma kungadingeki, okuvamise ukuba yindaba yophenyo olukhiqiza uhlobo olulodwa olukhiphayo kumasampula anamazinga aphansi ngokuqhathaniswa we-autofluorescence yangemuva.
Izihlungi ze-band pass zidlulisa kuphela ibhendi ethile ye-wavelength, futhi ivimbe abanye. Banciphisa i-crosstalk ngokuvumela kuphela ingxenye enamandla kakhulu ye-fluorophore emission emission spectrum ukuthi idluliselwe, banciphise umsindo we-autofluorescence futhi ngaleyo ndlela bathuthukise isilinganiso sesignali-kuya-nomsindo kumasampuli aphezulu angemuva we-autofluorescence, izihlungi zokudlula amade ezingakwazi ukunikeza.
Zingaki izinhlobo zezihlungi ze-fluorescence ezinikezwa yi-BestScope?
Ezinye izinhlobo ezijwayelekile zezihlungi zifaka izihlungi eziluhlaza okwesibhakabhaka, eziluhlaza, kanye ne-ultraviolet. Njengoba kukhonjisiwe etafuleni.
| Isethi Yesihlungi | Isihlungi Sokuthokozisa | I-Dichroic Mirror | Isihlungi Sokuvimbela | Isibani se-LED Ubude begagasi | Isicelo |
| B | BP460-495 | DM505 | I-BA510 | 485nm | ·FITC: Indlela ye-Fluorescent antibody I-Acidine orange: DNA, RNA I-Auramine: I-Tubercle bacillus ·EGFP, S657, RSGFP |
| G | BP510-550 | DM570 | BA575 | 535nm | ·Rhodamine, TRITC: Indlela ye-Fluorescent antibody ·Propidium iodide: DNA ·I-RFP |
| U | BP330-385 | DM410 | I-BA420 | 365nm | ·Ukubonwa kwe-auto-fluorescence ·I-DAPI: Ukungcoliswa kwe-DNA ·Hoechest 332528, 33342: isetshenziselwa ukungcolisa i-Chromosome |
| V | BP400-410 | DM455 | BA460 | 405nm | ·Ama-catecholamines ·5-hydroxy tryptamine ·Tetracycline: Skeleton, Amazinyo |
| R | BP620-650 | DM660 | I-BA670-750 | 640nm | ·Cy5 ·I-Alexa Fluor 633, i-Alexa Fluor 647 |
Amasethi okuhlunga asetshenziswa ekutholweni kwe-fluorescence aklanywe azungeze amaza amaza amakhulu asetshenziswa ezinhlelweni ze-fluorescence, asekelwe kuma-fluorophore asetshenziswa kakhulu. Ngenxa yalesi sizathu, ziphinde ziqanjwe nge-fluorophore ezihloselwe ukuthwebula izithombe, njenge-DAPI (eluhlaza okwesibhakabhaka), i-FITC (eluhlaza) noma i-TRITC (ebomvu) yamakhyubhu okuhlunga.
| Isethi Yesihlungi | Isihlungi Sokuthokozisa | I-Dichroic Mirror | Isihlungi Sokuvimbela | Isibani se-LED Ubude begagasi |
| I-FITC | BP460-495 | DM505 | I-BA510-550 | 485nm |
| I-DAPI | BP360-390 | DM415 | I-BA435-485 | 365nm |
| I-TRITC | BP528-553 | DM565 | I-BA578-633 | 535nm |
| I-FL-Auramine | BP470 | DM480 | BA485 | 450nm |
| I-Texas Red | BP540-580 | DM595 | I-BA600-660 | 560nm |
| mCherry | BP542-582 | DM593 | I-BA605-675 | 560nm |
Ungasikhetha kanjani isihlungi se-fluorescence?
1. Umgomo wokukhetha isihlungi se-fluorescence ukuvumela ukukhanya kwe-fluorescence/okuphumayo kudlule ekugcineni kwesithombe ngangokunokwenzeka, futhi kuvinjwe ngokuphelele ukukhanya okuvusa amadlingozi ngesikhathi esifanayo, ukuze kutholwe isilinganiso esiphezulu sesignali-kuya-nomsindo. Ikakhulukazi ekusetshenzisweni kwesasasa le-multiphoton kanye nengqikithi ye-microscope yokubonisa yangaphakathi, umsindo obuthakathaka uzophinda ubangele ukuphazamiseka okukhulu kumthelela wesithombe, ngakho-ke isidingo sesilinganiso sesignali kumsindo siphezulu.
2. Yazi injabulo kanye ne-spectrum yokukhishwa kwe-fluorophore. Ukuze kwakhiwe isethi yesihlungi se-fluorescence esikhiqiza isithombe esiphezulu, esihluke kakhulu esinengemuva elimnyama, izihlungi ezivusa amadlingozi nezokukhipha umoya kufanele zifinyelele ukuhanjiswa okuphezulu nge-ripple encane yokudlula ibhande ezifundeni ezihambisana neziqongo zenjabulo ye-fluorophore noma ukukhishwa kwekhabhoni.
3. Cabangela ukuqina kwezihlungi ze-fluorescence. Lezi zihlungi akumelwe zikwazi ukumelana nemithombo yokukhanya eqinile ekhiqiza ukukhanya kwe-ultraviolet (UV) okungase kuholele “ekushiseni”, ikakhulukazi kwesihlungi se-exciter njengoba singaphansi kobukhulu obugcwele bomthombo wokukhanyisa.
Izithombe Ezihlukile Zesampula Le-Fluorescent
Izinsiza ziqoqwa futhi zihlelwe ku-inthanethi, futhi zisetshenziselwa ukufunda nokuxhumana kuphela. Uma kukhona ukwephulwa komthetho, sicela usithinte ukuze ukususe.
Isikhathi sokuthumela: Dec-09-2022