Akayunguruzo ka fluorescence nikintu cyingenzi muri microscope ya fluorescence.Sisitemu isanzwe ifite ibice bitatu byibanze muyunguruzi: akayunguruzo gashimishije, akayunguruzo gasohoka hamwe nindorerwamo ya dicroic.Mubisanzwe bapakirwa muri cube kugirango itsinda ryinjizwe hamwe muri microscope.
Nigute filteri ya fluorescence ikora?
Akayunguruzo
Akayunguruzo gashimishije kohereza urumuri rwumurongo wihariye kandi ugahagarika ubundi burebure.Birashobora gukoreshwa kubyara amabara atandukanye muguhuza akayunguruzo kugirango yemere ibara rimwe gusa.Ibyishimo bishungura biza muburyo bubiri bwingenzi - birebire birengana byungurura na bande byungurura.Ubusanzwe ibyuka ni akayunguruzo kanyuze hejuru yuburebure bwa florofore gusa, bityo bikagabanya umunezero wandi masoko ya fluorescence no guhagarika urumuri rwo kwishima mumutwe wa florescence.Nkuko bigaragazwa numurongo wubururu ku ishusho, BP ni 460-495, bivuze ko ishobora kunyura muri fluorescence ya 460-495nm.
Ishyirwa munzira yo kumurika microscope ya fluorescence hanyuma ikayungurura uburebure bwumuraba wose wumucyo usibye urwego rwa fluorophore.Akayunguruzo byibuze kohereza byerekana ubwiza nubwiza bwamashusho.Nibura byibuze 40% byoherezwa kubintu byose byungururwa birasabwa kuburyo kwanduza ari byiza> 85%.Umuyoboro mugari wa filteri ishimishije ugomba kuba rwose murwego rwo kwinezeza kwa fluorophore kuburyo uburebure bwumurongo wo hagati (CWL) bwiyungurura bwegereye ibishoboka byose kugirango uburebure bwa tekinike ya fluorophore.Akayunguruzo gashimishije optique yuzuye (OD) itegeka umwijima w'ishusho umwijima;OD ni igipimo cyukuntu akayunguruzo gahagarika uburebure bwumurongo hanze yumurongo wa interineti cyangwa umurongo.Nibura OD ya 3.0 irasabwa ariko OD ya 6.0 cyangwa irenga nibyiza.
Akayunguruzo
Akayunguruzo kohereza imyuka itanga intego yo kwemerera fluorescence yifuzwa kuva icyitegererezo kugera kuri detector.Zibuza uburebure bwumurongo muto kandi zifite umuvuduko mwinshi kuburebure burebure.Ubwoko bwa Muyunguruzi nabwo bujyanye numubare, urugero BA510IF mu gishushanyo (intambamyi ya barrière filteri), iryo zina ryerekeza ku burebure bwa 50% byoherejwe cyane.
Ibyifuzo bimwe kubishungura byungurura bifata ukuri kubisohoka muyungurura: kohereza byibuze, umurongo mugari, OD, na CWL.Akayunguruzo koherezwa hamwe na CWL nziza, ihererekanyabubasha ntarengwa, hamwe na OD itanga amashusho meza cyane ashoboka, hamwe no guhagarika byimbitse, kandi ikanagaragaza ibimenyetso byerekana ibyuka bihumanya.
Indorerwamo ya Dichroic
Indorerwamo ya dicroic ishyirwa hagati ya filteri yo kwishima no kuyungurura ibyuka ku nguni ya 45 ° kandi ikagaragaza ikimenyetso cyibyishimo kuri fluorophore mugihe cyohereza ibimenyetso byangiza ikirere.Ibyiza bya dicroic muyunguruzi no gutandukanya ibice bifite inzibacyuho ityaye hagati yo kugaragarira cyane no kwanduza cyane, hamwe na> 95% byerekana umurongo mugari wa filteri ishimishije hamwe no kohereza> 90% kumurongo mugari wa filteri yangiza.Hitamo akayunguruzo hamwe nuburebure bwumurambararo (λ) wa fluorophore mubitekerezo, kugirango ugabanye urumuri rwayobye kandi ugabanye ishusho ya fluorescent yerekana ibimenyetso-by-urusaku.
Indorerwamo ya dichroic kuri iyi shusho ni DM505, yitwa rero kuko nanometero 505 nuburebure bwumurongo kuri 50% byokwirakwiza kwinshi kuriyi ndorerwamo.Gukwirakwiza umurongo kuri iyi ndorerwamo byerekana kohereza hejuru hejuru ya 505 nm, kugabanuka gukabije kwihererekanyabubasha ibumoso bwa nanometero 505, hamwe no kugaragariza cyane ibumoso bwa nanometero 505 ariko birashobora kuba bifite kohereza munsi ya 505 nm.
Ni irihe tandukaniro riri hagati yinzira ndende na bande yungurura?
Akayunguruzo ka Fluorescence gashobora kugabanywamo ubwoko bubiri: inzira ndende (LP) na bande ya bande (BP).
Inzira ndende zungurura zohereza uburebure burebure no guhagarika bigufi.Gukata-kumurongo wuburebure nigiciro kuri 50% yikwirakwizwa ryimpinga, kandi uburebure bwumurongo wose hejuru yo gukata byanduzwa ninzira ndende.Zikoreshwa cyane mumirorerwamo ya dicroic hamwe nayungurura imyuka.Akayunguruzo ka Longpass kagomba gukoreshwa mugihe porogaramu isaba kwegeranya imyuka ihumanya ikirere kandi mugihe ivangura ridasanzwe ritifuzwa cyangwa rikenewe, mubisanzwe mubisanzwe kubushakashatsi butanga ubwoko bumwe bwohereza imyuka mubigereranyo bifite urwego ruto ugereranije na autofluorescence.
Bande pass filtri yohereza gusa umurongo muremure wumurongo, hanyuma uhagarike izindi.Bagabanya inzira nyabagendwa bemerera gusa igice kinini cyimyuka ya fluorophore yoherejwe, kugabanya urusaku rwa autofluorescence bityo bikazamura igipimo cyerekana-urusaku mumasoko maremare ya autofluorescence ntangarugero, filtri ndende idashobora gutanga.
Ni ubuhe bwoko bwa filteri ya fluorescence ishiraho BestScope ishobora gutanga?
Bimwe mubisanzwe byungurura birimo ubururu, icyatsi, na ultraviolet muyunguruzi.Nkuko bigaragara mu mbonerahamwe.
| Gushungura | Akayunguruzo | Indorerwamo ya Dichroic | Akayunguruzo | Itara rya LED Uburebure | Gusaba |
| B | BP460-495 | DM505 | BA510 | 485nm | · FITC: Uburyo bwa antibody ya Fluorescent · Acideine orange: ADN, RNA · Auramine: Indwara y'igituntu · EGFP, S657, RSGFP |
| G | BP510-550 | DM570 | BA575 | 535nm | · Rhodamine, TRITC: Uburyo bwa antibody ya Fluorescent · Iyode ya Propidium: ADN · RFP |
| U | BP330-385 | DM410 | BA420 | 365nm | · Kwitegereza Auto-fluorescence · DAPI: ADN yanduye · Hoechest 332528, 33342: ikoreshwa kuri Chromosome |
| V | BP400-410 | DM455 | BA460 | 405nm | · Catecholamine · 5-hydroxy tryptamine · Tetracycline: Skeleton, Amenyo |
| R | BP620-650 | DM660 | BA670-750 | 640nm | · Cy5 · Ibicurane bya Alexa 633, Alexa Fluor 647 |
Akayunguruzo gakoreshwa mukugura kwa fluorescence kwakozwe hafi yuburebure bukomeye bwumurongo ukoreshwa muri porogaramu ya fluorescence, ishingiye kuri fluorofore ikoreshwa cyane.Kubera iyo mpamvu, nanone bitiriwe fluorophore bagenewe gushushanya, nka DAPI (ubururu), FITC (icyatsi) cyangwa TRITC (umutuku) filteri.
| Gushungura | Akayunguruzo | Indorerwamo ya Dichroic | Akayunguruzo | Itara rya LED Uburebure |
| 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 Umutuku | BP540-580 | DM595 | BA600-660 | 560nm |
| mCherry | BP542-582 | DM593 | BA605-675 | 560nm |
Nigute ushobora guhitamo akayunguruzo ka fluorescence?
1. Ihame ryo guhitamo akayunguruzo ka fluorescence ni ukureka urumuri rwa fluorescence / imyuka isohoka ikanyura mumashusho yerekana amashusho ashoboka, kandi igahagarika burundu itara ryibyishimo icyarimwe, kugirango tubone igipimo kinini cyerekana-urusaku.Cyane cyane mugukoresha moteri ya moteri ya mikorosikopi hamwe na microscope yimbere yimbere, urusaku rudakomeye narwo ruzatera intambamyi zikomeye kumashusho, bityo rero ibisabwa kugirango ibimenyetso byerekana urusaku ni byinshi.
2. Menya ibintu byishimisha hamwe nibisohoka bya fluorophore.Kugirango wubake akayunguruzo ka fluorescence gatanga ishusho nziza-nziza, itandukanye cyane nigishusho cyumukara, kwishima no kuyungurura ibyuka bigomba kugera kumurongo mwinshi hamwe na passband ntoya ihindagurika mukarere gahuye nimpinga ya fluorophore.
3. Reba kuramba kwa fluorescence muyunguruzi.Akayunguruzo kagomba kutabangamira urumuri rukomeye rutanga urumuri ultraviolet (UV) rushobora kuganisha kuri "gucana", cyane cyane muyungurura ibintu kuko bikorerwa ubukana bwuzuye bw'isoko.
Amashusho atandukanye ya Fluorescent
Ibikoresho byakusanyirijwe hamwe kandi bitunganijwe kuri enterineti, kandi bikoreshwa gusa mukwiga no gutumanaho.Niba hari ihohoterwa, nyamuneka twandikire kugirango dusibe.
Igihe cyo kohereza: Ukuboza-09-2022