Isihluzi se-fluorescence yinxalenye ebalulekileyo ye-fluorescence microscope. Isixokelelwano esiqhelekileyo sinezihluzo ezisisiseko ezintathu: isihluzo sokuvuselela, isihluzo sokukhupha kunye nesibuko se-dichroic. Ngokuqhelekileyo zipakishwa kwityhubhu ukuze iqela lifakwe kunye kwi-microscope.
Sisebenza njani isihluzo se-fluorescence?
Isihluzi Sokuvuselela
Izihluzi ze-excitation zisasaza ukukhanya kobude obuthile kwaye zithintele amanye amaza amaza. Zingasetyenziswa ukuvelisa imibala eyahlukileyo ngokucofa isihluzo ukuvumela umbala omnye kuphela. Izihluzo ezivuselelayo ziza kwiindidi eziphambili ezimbini - izihluzo zokudlula ezinde kunye nezihluzo zokupasa band. I-exciter idla ngokuba sisihluzo se-bandpass esidlula kuphela i-wavelengths efunxwe yi-fluorophore, ngaloo ndlela icutha uchulumanco lweminye imithombo ye-fluorescence kunye nokuthintela ukukhanya kwe-excitation kwi-fluorescence emission band. Njengoko kuboniswe ngumgca ohlaza okwesibhakabhaka kumfanekiso, i-BP yi-460-495, oku kuthetha ukuba inokudlula kuphela kwi-fluorescence ye-460-495nm.
Ibekwe ngaphakathi kwindlela yokukhanya ye-fluorescence microscope kwaye ihluze onke amaza okukhanya omthombo wokukhanya ngaphandle koluhlu lwe-fluorophore excitation. Isihluzi esincinci sosasazo siyalela ukuqaqamba kunye nokuqaqamba kwemifanekiso. Ubuncinane obungama-40% wosulelo kulo naliphi na isihluzo sovuselelo siyacetyiswa ngohlobo lokuba usasazo lube ngumlinganiselo>85%. I-bandwidth yesihluzo sovuselelo kufuneka sibe ngaphakathi ngokupheleleyo kuluhlu lovuselelo lwe-fluorophore ukuze ubude obuphakathi beziko (CWL) lwesihluzi lusondele kangangoko kwincopho yovuselelo lwamaza e-fluorophore. Isihluzo soxinzelelo lwe-optical density (OD) sichaza ubumnyama bomfanekiso ongasemva; I-OD ngumlinganiselo wendlela isihluzi esithintela ngayo i-wavelengths ngaphandle koluhlu lothumelo okanye i-bandwidth. Ubuncinci be-OD ye-3.0 iyanconywa kodwa i-OD ye-6.0 okanye enkulu ifanelekile.
Isihluzi Sokukhupha
Izihluzi ezikhutshwayo zisebenza ngenjongo yokuvumela i-fluorescence enqwenelekayo esuka kwisampulu ukuba ifikelele kwisixhobo. Zithintela ubude obufutshane kwaye zinosulelo oluphezulu lwamaza amaza amade. Uhlobo lokucoca lukwanxulunyaniswa nenani, umz. BA510IF kumzobo (isithintelo sothintelo lokucoca isihluzo), olo phawu lubhekiselele kumgama wamaza kuma-50% ogqithiso lwalo lukhulu.
Iingcebiso ezifanayo zokucoca izihluzi zibambe ngokwenyani kwizihluzi ezikhutshwayo: ukuhanjiswa okuncinci, i-bandwidth, i-OD, kunye ne-CWL. Isihluzi sokukhutshwa kunye ne-CWL efanelekileyo, ubuncinane bokudluliselwa, kunye ne-OD indibaniselwano ibonelela ngeyona mifanekiso iqaqambileyo, kunye neyona ndlela inzulu yokuthintela, kwaye iqinisekisa ukubhaqwa kweempawu zokukhupha ezibuthathaka.
Isibuko seDichroic
Isibuko se-dichroic sibekwe phakathi kwe-excitation filter kunye ne-emission filter kwi-engile ye-45 ° kwaye sibonisa umqondiso we-excitation ngokubhekiselele kwi-fluorophore ngelixa uhambisa umqondiso wokukhupha kwi-detector. Iifilitha ezifanelekileyo ze-dichroic kunye ne-beam splitters zineenguqu ezibukhali phakathi kokubonakaliswa okuphezulu kunye nokuhanjiswa okuphezulu, kunye ne-> 95% ibonakaliso ye-bandwidth ye-excitation filter kunye nokuhanjiswa kwe> 90% kwi-bandwidth ye-emission filter. Khetha isihluzo esinobude obunqamlezayo bendlela (λ) ye-fluorophore engqondweni, ukunciphisa isibane esilahlekileyo kunye nokwandisa umlinganiselo womfanekiso we-fluorescent wesignali ukuya kwingxolo.
Isibuko se-dichroic kulo mfanekiso yi-DM505, ebizwa ngokuba yi-505 nanometers ubude be-wavelength kwi-50% yokuhanjiswa okukhulu kwesi sibuko. Ijiko lothumelo lwesi sibuko libonisa ukuhanjiswa okuphezulu ngaphezu kwe-505 nm, ukuhla okunyukayo ekuhanjisweni ukuya ngasekhohlo kwee-nanometers ezingama-505, kunye nokubonakala okuphezulu ngasekhohlo kwee-nanometers ezingama-505 kodwa kusenokubakho ukosulelo olungaphantsi kwe-505 nm.
Yintoni umahluko phakathi kokudlula ixesha elide kunye nezihluzo zokupasa ngebhendi?
Izihlungi ze-Fluorescence zinokwahlulwa zibe ziindidi ezimbini: i-long pass (LP) kunye ne-band pass (BP).
Izihluzi zokudlula ezinde zisasaza ubude bamaza amade kwaye zivale ezimfutshane. Ukunqunyulwa kwe-wavelength lixabiso kwi-50% yokuhanjiswa kwe-peak, kwaye zonke ii-wavelengths ezingaphezulu kwe-cut-on zihanjiswa ngeefilitha zokudlula ezide. Zihlala zisetyenziswa kwizipili ze-dichroic kunye nezihluzo eziphumayo. Izihluzi ze-Longpass kufuneka zisetyenziswe xa isicelo sifuna ukuqokelelwa okuphezulu okukhuphayo kwaye xa ucalucalulo olubonakalayo lunganqweneleki okanye luyimfuneko, oluyimeko ngokubanzi yophando oluvelisa uhlobo olunye olukhuphayo kwisampulu ezinamaqondo asezantsi ngokwentelekiso emvelaphi ye-autofluorescence.
Izihluzi zokudlula ibhendi zigqithisa kuphela ibhendi yobude obuthile, kwaye ivalele abanye. Banciphisa i-crosstalk ngokuvumela kuphela inxalenye eyomeleleyo ye-fluorophore emission spectrum ukuba idluliselwe, ukunciphisa ingxolo ye-autofluorescence kwaye ngaloo ndlela kuphuculwe umlinganiselo we-signal-to-noise kwiisampuli eziphezulu ze-autofluorescence, apho izihlungi zokudlula ixesha elide azikwazi ukunika.
Zingaphi iintlobo zesihluzi se-fluorescence seti i-BestScope inokubonelela?
Ezinye iintlobo eziqhelekileyo zokucoca ziquka izihluzi eziluhlaza, eziluhlaza, kunye ne-ultraviolet. Njengoko kubonisiwe kwitheyibhile.
| Iseti yesihluzi | Isihluzi Sokuvuselela | Isibuko seDichroic | Isihluzi sesithintelo | Isibane se-LED Ubude be-Wave | Isicelo |
| B | BP460-495 | DM505 | BA510 | 485nm | ·FITC: Indlela ye-Fluorescent antibody ·Acidine orenji: DNA, RNA ·Auramine: I-tubercle bacillus ·EGFP, S657, RSGFP |
| G | BP510-550 | DM570 | BA575 | 535nm | ·Rhodamine, TRITC: Indlela ye-antibody eFluorescent ·Propidium iodide: DNA ·RFP |
| U | BP330-385 | DM410 | BA420 | 365nm | ·Ukuqwalaselwa kwe-auto-fluorescence ·I-DAPI: Ibala le-DNA ·Hoechest 332528, 33342: isetyenziselwa ukudyobheka kweChromosome |
| V | BP400-410 | DM455 | BA460 | 405nm | ·Catecholamines ·5-hydroxy tryptamine ·Tetracycline: Skeleton, Amazinyo |
| R | BP620-650 | DM660 | BA670-750 | 640nm | ·Cy5 ·Alexa Fluor 633, Alexa Fluor 647 |
Iiseti zesihluzi ezisetyenziswa ekufumaneni i-fluorescence ziyilwe malunga nobude bamaza amakhulu asetyenziswa kwi-fluorescence applications, esekelwe kwii-fluorophores ezisetyenziswa kakhulu. Ngenxa yesi sizathu, zikwathiywa ngegama le-fluorophore ezijoliswe ekufanekiseni, njenge-DAPI (blue), i-FITC (eluhlaza) okanye i-TRITC (ebomvu) iityhubhu zokucoca.
| Iseti yesihluzi | Isihluzi Sokuvuselela | Isibuko seDichroic | Isihluzi sesithintelo | Isibane se-LED Ubude be-Wave |
| FITC | BP460-495 | DM505 | BA510-550 | 485nm |
| DAPI | BP360-390 | DM415 | BA435-485 | 365nm |
| I-TRITC | BP528-553 | DM565 | BA578-633 | 535nm |
| I-FL-Auramine | BP470 | DM480 | BA485 | 450nm |
| eTexas Red | BP540-580 | DM595 | BA600-660 | 560nm |
| mCherry | BP542-582 | DM593 | BA605-675 | 560nm |
Unokukhetha njani isihluzi se-fluorescence?
1. Umgaqo wokukhetha isihluzi se-fluorescence kukuvumela ukukhanya kwe-fluorescence / ukukhutshwa kudlule ekupheleni kwe-imaging kangangoko kunokwenzeka, kwaye kuvimbele ngokupheleleyo ukukhanya kwe-excitation ngexesha elifanayo, ukuze ufumane umlinganiselo ophezulu we-signal-to-noise. Ngokukodwa kwisicelo se-multiphoton excitation kunye ne-microscope epheleleyo yangaphakathi ebonisa, ingxolo ebuthathaka iya kubangela ukuphazamiseka okukhulu kwimpembelelo ye-imaging, ngoko ke imfuno yomqondiso kwisandi sengxolo iphezulu.
2. Yazi imincili kunye ne-spectrum ephumayo ye-fluorophore. Ukwakha iseti yokucoca i-fluorescence eyenza umgangatho ophezulu, umgangatho ophezulu wokuchasana kunye nemvelaphi emnyama, izihlungi zokuvuselela kunye ne-emission kufuneka zifezekise ukuhanjiswa okuphezulu kunye ne-ripple encinci ye-passband kwiingingqi ezihambelana ne-fluorophore excitation peaks okanye ukukhutshwa.
3. Qwalasela ukuqina kwezihluzo ze-fluorescence. Ezi zihluzo kufuneka zingathinteki kwimithombo yokukhanya ebukhali eyenza ukukhanya kwe-ultraviolet (UV) okunokukhokelela "ekutshisweni", ngakumbi kwisihluzo se-exciter njengoko siphantsi koxinzelelo olupheleleyo lomthombo wokukhanya.
IMifanekiso yeSampuli yeFluorescent eyahlukileyo
Izibonelelo ziqokelelwa kwaye zilungelelaniswe kwi-Intanethi, kwaye zisetyenziselwa ukufunda nokunxibelelana kuphela. Ukuba kukho naluphi na ulwaphulo-mthetho, nceda uqhagamshelane nathi ukuze ucime.
Ixesha lokuposa: Dec-09-2022