Fitar mai kyalli wani abu ne mai mahimmanci a cikin microscope mai kyalli.Tsari na yau da kullun yana da matattara na asali guda uku: matattara mai ban sha'awa, tacewar iska da madubi dichroic.Yawanci ana tattara su a cikin kubu domin a saka ƙungiyar tare a cikin na'urar hangen nesa.
Ta yaya tace fluorescence ke aiki?
Tace zumudi
Tace masu ban sha'awa suna watsa haske na takamaiman tsayin raƙuman ruwa kuma suna toshe sauran tsayin raƙuman ruwa.Ana iya amfani da su don samar da launuka daban-daban ta hanyar daidaita tacewa don ba da damar launi ɗaya kawai.Fitar da zazzagewa ta zo cikin manyan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'in tsiro) da na numfashi” da na numfashi na numfashi na numfashi na numfashi na huhu da kuma matattara mai tsayi mai tsayi mai tsawo da kuma matattarar wucewa ta band.Mai jan hankali yawanci matatar bandpass ce wacce ke wuce tsawon zangon da fluorophore ke sha, don haka yana rage zumudi na sauran hanyoyin haske da kuma toshe haske mai jan hankali a cikin rukunin hayaki mai kyalli.Kamar yadda aka nuna ta layin shuɗi a cikin adadi, BP shine 460-495, wanda ke nufin cewa kawai zai iya wucewa ta hanyar haske na 460-495nm.
Ana sanya shi a cikin hanyar haskakawa na na'urar hangen nesa mai haske kuma tana tace duk tsawon raƙuman haske na tushen hasken ban da kewayon tashin hankali na fluorophore.Mafi ƙarancin watsawa tace yana nuna haske da haske na hotuna.Ana ba da shawarar mafi ƙarancin watsawa 40% don kowane tacewa mai jan hankali kamar yadda watsawar ya dace> 85%.Ya kamata bandwidth na matatar tashin hankali ya kasance gaba ɗaya a cikin kewayon tashin hankali na fluorophore kamar yadda tsakiyar zango (CWL) na tace yana da kusanci kamar yadda zai yiwu zuwa tsayin tsayin tashin hankali na fluorophore.Ƙwaƙwalwar haɓakawar gani (OD) tana nuna duhun hoton bango;OD shine ma'auni na yadda tacewa ke toshe tsawon raƙuman ruwa a wajen kewayon watsawa ko bandwidth.Ana ba da shawarar mafi ƙarancin OD na 3.0 amma OD na 6.0 ko mafi girma shine manufa.
Tace Fitar
Fitar da iska tana aiki da manufar barin kyakyawan haske daga samfurin don isa ga mai ganowa.Suna toshe gajeriyar raƙuman ruwa kuma suna da babban watsawa don tsayin raƙuman ruwa.Nau'in tacewa kuma yana da alaƙa da lamba, misali BA510IF a cikin adadi (tatar da shingen tsangwama), wannan nadi yana nufin tsayin daka a 50% na iyakar watsawa.
Shawarwari iri ɗaya don masu tace tashin hankali suna riƙe gaskiya ga masu tacewa: ƙaramar watsawa, bandwidth, OD, da CWL.Fitar da iska tare da madaidaicin CWL, mafi ƙarancin watsawa, da haɗin OD yana ba da mafi kyawun hotuna masu yuwuwa, tare da mafi zurfin yuwuwar toshewa, kuma yana tabbatar da gano sigina mafi ƙarancin fitarwa.
Dichroic Mirror
Ana sanya madubi na dichroic tsakanin matatar tashin hankali da tace fitarwa a kusurwar 45 ° kuma yana nuna siginar motsa jiki zuwa fluorophore yayin watsa siginar fitarwa zuwa ga mai ganowa.Madaidaicin matattarar dichroic da masu rarraba katako suna da sauye-sauye masu kaifi tsakanin matsakaicin tunani da matsakaicin watsawa, tare da> 95% tunani don bandwidth na matattarar tashin hankali da watsawa> 90% don bandwidth na fitarwar fitarwa.Zaɓi tacewa tare da tsaka-tsakin raƙuman ruwa (λ) na fluorophore a hankali, don rage ɓoyayyen haske da ƙara girman siginar hoto-zuwa amo.
Dichroic madubi a cikin wannan adadi shi ne DM505, don haka mai suna saboda 505 nanometers shine tsayin daka a 50% na matsakaicin watsawa na wannan madubi.Wurin watsawa na wannan madubi yana nuna babban watsawa sama da 505 nm, raguwa mai zurfi a watsa zuwa hagu na 505 nanometers, da matsakaicin nuni zuwa hagu na 505 nanometers amma har yanzu yana iya samun wasu watsawa ƙasa da 505 nm.
Menene bambanci tsakanin dogon wucewa da matattarar wucewar band?
Za a iya raba filtata masu haske zuwa nau'i biyu: dogon wucewa (LP) da band pass (BP).
Dogayen matattarar wucewa suna watsa dogon zango kuma suna toshe gajerun.Matsakaicin tsayin da aka yanke shine ƙimar a 50% na watsawa kololuwa, kuma duk tsayin raƙuman da ke sama da yanke-kan ana watsa su ta hanyar matattarar wucewa mai tsayi.Ana amfani da su akai-akai a cikin madubin dichroic da masu tacewa.Ya kamata a yi amfani da matatun Longpass lokacin da aikace-aikacen ke buƙatar mafi girman tarin hayaki kuma lokacin da ba a son nuna wariya ko kuma ya zama dole, wanda galibi shine yanayin binciken da ke haifar da nau'in fitarwa guda ɗaya a cikin samfurori tare da ƙananan matakan baya na autofluorescence.
Matsakaicin madaidaicin bandeji kawai yana watsa wani yanki mai tsayi, kuma yana toshe wasu.Suna rage yawan magana ta hanyar ba da damar kawai mafi ƙarfi na nau'in watsawar fluorophore don watsawa, rage amo ta atomatik kuma don haka inganta siginar-zuwa amo a cikin manyan samfuran autofluorescence na baya, wanda masu tacewa mai tsawo ba zai iya bayarwa ba.
Nawa nau'ikan saitin tacewa mai kyalli BestScope zai iya bayarwa?
Wasu nau'ikan filtata gama gari sun haɗa da shuɗi, kore, da matatun ultraviolet.Kamar yadda aka nuna a tebur.
| Saitin Tace | Tace zumudi | Dichroic Mirror | Tace mai shinge | LED fitila Tsawon Wave | Aikace-aikace |
| B | Saukewa: BP460-495 | DM505 | BA510 | 485nm ku | · FITC: Hanyar antibody fluorescent · Acid orange: DNA, RNA · Auramine: Tubercle bacillus EGFP, S657, RSGFP |
| G | Saukewa: BP510-550 | DM570 | BA575 | 535nm ku | Rhodamine, TRITC: Hanyar antibody fluorescent · Propidium iodide: DNA · RFP |
| U | Saukewa: BP330-385 | DM410 | BA420 | 365nm ku | · Lura ta atomatik DAPI: Tabon DNA · Hoechest 332528, 33342: An yi amfani da shi don tabon Chromosome |
| V | Saukewa: BP400-410 | DM455 | BA460 | 405nm ku | · Catecholamines · 5-hydroxy tryptamine · Tetracycline: kwarangwal, hakora |
| R | Saukewa: BP620-650 | DM660 | BA670-750 | 640nm ku | ·Cy5 Alexa Fluor 633, Alexa Fluor 647 |
Saitunan tacewa waɗanda ake amfani da su wajen siyan kyalli an ƙera su a kusa da manyan tsayin daka da ake amfani da su a aikace-aikacen hasken wuta, wanda ya dogara ne akan mafi yawan amfani da fluorophores.Saboda wannan dalili, ana kuma sanya musu suna bayan fluorophore da aka yi nufin su don yin hoto, kamar DAPI (blue), FITC (kore) ko TRITC (ja) cubes filter.
| Saitin Tace | Tace zumudi | Dichroic Mirror | Tace mai shinge | LED fitila Tsawon Wave |
| FITC | Saukewa: BP460-495 | DM505 | BA510-550 | 485nm ku |
| DAPI | Saukewa: BP360-390 | DM415 | BA435-485 | 365nm ku |
| TRITC | Saukewa: BP528-553 | DM565 | BA578-633 | 535nm ku |
| FL-Auramine | Farashin BP470 | DM480 | BA485 | 450nm ku |
| Texas Red | Saukewa: BP540-580 | DM595 | BA600-660 | 560nm ku |
| mCherry | Saukewa: BP542-582 | DM593 | BA605-675 | 560nm ku |
Ta yaya ake zabar tace mai kyalli?
1. Ka'idar zaɓin tacewa mai walƙiya shine barin ƙyalli / haske mai haske ya wuce ta ƙarshen hoton kamar yadda zai yiwu, kuma gaba ɗaya ya toshe hasken tashin hankali a lokaci guda, don samun mafi girman sigina-zuwa amo rabo.Musamman don aikace-aikacen motsa jiki na multiphoton da kuma jimlar hangen nesa na ciki, ƙararrawar ƙarar za ta haifar da tsangwama ga tasirin hoto, don haka buƙatun sigina zuwa rabon amo ya fi girma.
2. Sanin tashin hankali da fitar da bakan fluorophore.Don gina saitin tacewa mai kyalli wanda ke haifar da babban inganci, hoto mai bambanci tare da baƙar fata, tashin hankali da tacewa ya kamata a cimma babban watsawa tare da ƙaramin fasfo ɗin wucewa akan yankuna waɗanda suka dace da kololuwar tashin hankali ko hayaƙin fluorophore.
3. Yi la'akari da dorewar tacewa mai kyalli.Dole ne waɗannan masu tacewa su kasance marasa ƙarfi ga tushen haske masu ƙarfi waɗanda ke haifar da hasken ultraviolet (UV) wanda zai iya haifar da “ƙonawa”, musamman ma matattarar exciter yayin da aka juyar da ita ga cikakken ƙarfin hasken.
Hotunan Samfuran Fluorescent Daban-daban
Ana tattara albarkatun kuma ana tsara su akan Intanet, kuma ana amfani dasu kawai don koyo da sadarwa.Idan akwai wani cin zarafi, da fatan za a tuntuɓe mu don sharewa.
Lokacin aikawa: Dec-09-2022