Sesefa sa fluorescence ke karolo ea bohlokoa ho microscope ea fluorescence.Sistimi e tloaelehileng e na le li-filters tse tharo tsa mantlha: filthara e tsosang takatso, sesefa se tsoang le seipone sa dichroic.Hangata li kenngoa ka har'a cube e le hore sehlopha se kenngoe hammoho ka microscope.
Sesefa sa fluorescence se sebetsa joang?
Sesefa se Hlahisang
Lisefe tse hlasimollang li fetisa khanya ea bolelele bo itseng ba maqhubu 'me li thibela maqhubu a mang.Li ka sebelisoa ho hlahisa mebala e fapaneng ka ho lokisa sefe ho lumella 'mala o le mong feela.Li-filters tse hlasimollang li tla ka mefuta e 'meli ea mantlha - li-filters tse telele le li-band pass filters.Sehlahisoa hangata ke sesefa sa bandpass se fetisang feela bolelele ba maqhubu a kentsoeng ke fluorophore, kahoo se fokotsa ho tsosoa ha mehloli e meng ea fluorescence le ho thibela lebone la ho hlasimolla letlapeng la fluorescence emission.Joalokaha ho bontšoa ke mola o moputsoa setšoantšong, BP ke 460-495, e bolelang hore e ka feta feela ka fluorescence ea 460-495nm.
E behiloe ka har'a tsela ea khanya ea microscope ea fluorescence 'me e sefa makhubu 'ohle a mohloli oa leseli ntle le sebaka sa ho hlasimolla ha fluorophore.Phetiso e fokolang ea sefe e laela ho khanya le ho phatsima ha litšoantšo.Ho khothaletsoa hore phetisetso e tlase ea 40% bakeng sa sefe kapa sefe se hlasimollang e le hore phetiso e ka ba> 85%.Bophara ba sefefo se hlasimollang bo lokela ho ba ka botlalo ka har'a sebaka sa ho hlasimolla ha fluorophore hoo sebaka sa "centre wavelength" (CWL) sa sefe se leng haufi ka hohle kamoo ho ka khonehang ho bophahamo ba maqhubu a phihlello ea fluorophore.The excitation filter optical density (OD) e laela lefifi la setšoantšo sa bokamorao;OD ke tekanyo ea hore na sefe se thibela hantle bolelele ba maqhubu ka ntle ho sebaka sa phetisetso kapa bandwidth.Ho khothalletsoa bonyane OD ea 3.0 empa OD ea 6.0 kapa ho feta e nepahetse.
Sesefa sa Emission
Li-filters tsa emission li sebeletsa sepheo sa ho lumella fluorescence e lakatsehang e tsoang sampoleng hore e fihle ho detector.Li thibela maqhubu a makhutšoane 'me li na le phetiso e phahameng bakeng sa maqhubu a malelele.Mofuta oa sefe o boetse o amahanngoa le palo, mohlala, BA510IF setšoantšong (seferefere sa litšitiso), lebitso leo le bolela bolelele ba maqhubu ho 50% ea phetisetso ea eona e phahameng.
Litlhahiso tse ts'oanang tsa li-filters tse hlasimollang li ts'oara 'nete bakeng sa lits'oants'o tsa tlhahiso: phetisetso e nyane, bandwidth, OD, le CWL.Setlhopha sa emission se nang le CWL e loketseng, phetisetso e fokolang, le motsoako oa OD se fana ka litšoantšo tse khanyang ka ho fetesisa, tse nang le thibelo e tebileng ka ho fetesisa, le ho netefatsa ho bonoa ha mats'oao a fokolang haholo.
Seipone sa Dichroic
Seipone sa dichroic se behiloe lipakeng tsa filthara e tsosang takatso le filthara e tsoang ka lehlakoreng la 45 ° 'me se bonahatsa lets'oao la ho hlasimolla le lebileng ho fluorophore ha se ntse se fetisetsa lets'oao la ho tsoa ho detector.Li-filters tse loketseng tsa dichroic le li-splitters tsa beam li na le liphetoho tse hlabang pakeng tsa ho bonahatsa ho fetisisa le phetisetso e kholo, e nang le > 95% e bonts'ang bakeng sa bandwidth ea filter ea excitation le phetisetso ea> 90% bakeng sa bandwidth ea emission filter.Kgetha sefe se nang le wavelength ya mateano (λ) kelellong ya fluorophore, ho fokotsa kganya e lahlehileng le ho hodisa karolelano ya senepe sa fluorescent le lerata.
Seipone sa dichroic setšoantšong sena ke DM505, e rehiloe joalo hobane 505 nanometers ke wavelength ho 50% ea phetisetso e kholo ea seipone sena.Mokokotlo oa phetisetso ea seipone sena o bonts'a phetisetso e phahameng kaholimo ho 505 nm, ho theoha ho hoholo ha phetisetso ka letsohong le letšehali la li-nanometer tse 505, le ponahalo e phahameng ka ho le letšehali ea li-nanometer tse 505 empa ho ntse ho ka ba le phetiso e ka tlase ho 505 nm.
Phapano ke efe lipakeng tsa li-filter tsa "long pass" le "band pass"?
Li-filters tsa fluorescence li ka aroloa ka mefuta e 'meli: long pass (LP) le band pass (BP).
Li-filters tse telele li fetisa maqhubu a malelele ebe li thibela tse khuts'oane.The cut-on wavelength ke boleng ba 50% ea phetisetso ea tlhōrō, 'me maqhubu ohle a ka holimo ho sehiloeng a fetisoa ke li-filters tse telele tsa ho feta.Hangata li sebelisoa ka liipone tsa dichroic le li-filters tsa emission.Lisefe tsa Longpass li lokela ho sebelisoa ha ts'ebeliso e hloka pokello e kholo ea tlhahiso le ha khethollo e sa lakatseheng kapa e sa hlokahale, eo hangata e leng taba ea li-probes tse hlahisang mofuta o le mong o ntšang mofuta o le mong oa mehlala e nang le maemo a tlase haholo a autofluorescence.
Lisefe tsa band pass li fetisa feela sehlopha se itseng sa wavelength, ebe li thibela tse ling.Ba fokotsa crosstalk ka ho lumella feela hore karolo e matla ka ho fetisisa ea fluorophore emission spectrum e fetisoe, e fokotsa lerata la autofluorescence 'me kahoo e ntlafatse tekanyo ea signal-to-lerata ka lisampole tse phahameng tsa autofluorescence, tseo li-filters tsa nako e telele li ke keng tsa fana ka tsona.
Ke mefuta e mekae ea li-filters tsa fluorescence tse ka fanoang ke BestScope?
Mefuta e meng e tloaelehileng ea li-filters e kenyelletsa li-filters tse putsoa, tse tala le tsa ultraviolet.Joalokaha ho bontšitsoe tafoleng.
| Sefa Sesefa | Sesefa se Hlahisang | Seipone sa Dichroic | Sethibelo Sefe | Lebone la LED Bolelele ba Leqhubu | Kopo |
| B | BP460-495 | DM505 | BA510 | 485nm | ·FITC: Mokhoa oa ho itšireletsa mafung ·Acidine orange: DNA, RNA Auramine: Tubercle bacillus ·EGFP, S657, RSGFP |
| G | BP510-550 | DM570 | BA575 | 535nm | ·Rhodamine, TRITC: Mokgwa wa selwantsha-mogare sa Fluorescent Propidium iodide: DNA ·RFP |
| U | BP330-385 | DM410 | BA420 | 365nm | ·Ho shebella ha fluorescence ka koloi ·DAPI: DNA staining ·Hoechest 332528, 33342: e sebedisetswa mabala a Chromosome |
| V | BP400-410 | DM455 | BA460 | 405nm | ·Katekolamine ·5-hydroxy tryptamine Tetracycline: Skeleton, Meno |
| R | BP620-650 | DM660 | BA670-750 | 640nm | ·Cy5 · Alexa Fluor 633, Alexa Fluor 647 |
Li-filters tse sebelisoang ho fumana fluorescence li entsoe ho potoloha maqhubu a maholo a sebelisoang lits'ebetsong tsa fluorescence, tse thehiloeng ho pota-pota li-fluorophore tse sebelisoang haholo.Ka lebaka lena, li boetse li bitsoa ka fluorophore tseo li reretsoeng ho li etsa litšoantšo, tse kang DAPI (blue), FITC (tala) kapa TRITC (red) filter cubes.
| Sefa Sesefa | Sesefa se Hlahisang | Seipone sa Dichroic | Sethibelo Sefe | Lebone la LED Bolelele ba Leqhubu |
| 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 Red | BP540-580 | DM595 | BA600-660 | 560nm |
| mCherry | BP542-582 | DM593 | BA605-675 | 560nm |
U ka khetha sefe ea fluorescence joang?
1. Molao-motheo oa ho khetha filthara ea fluorescence ke ho lumella khanya ea fluorescence / emission ho feta qetellong ea litšoantšo ka hohle kamoo ho ka khonehang, le ho thibela ka ho feletseng khanya ea ho hlasimolla ka nako e le 'ngoe, e le ho fumana tekanyo e phahameng ka ho fetisisa ea pontšo-ho-lerata.Haholo-holo bakeng sa ts'ebeliso ea li-multiphoton excitation le kakaretso ea ka hare ea microscope ea ho bonahatsa ka hare, lerata le fokolang le tla baka tšitiso e kholo ho phello ea litšoantšo, kahoo tlhokahalo ea karolelano ea pontšo le lerata e phahame.
2. Tseba mahlahahlaha le ho tsoa ha fluorophore.Ho aha sete ea sefe ea fluorescence e hlahisang setšoantšo sa boleng bo holimo, se fapaneng haholo se nang le bokamorao bo botšo, li-filters tse tsosang takatso le tse tsoang li lokela ho fihlella phetiso e phahameng ka ho fetella ho fokolang ho feta libaka tse tsamaellanang le litlhoro kapa mesi e hlahisoang ke fluorophore.
3. Nahana ka ho tšoarella ha li-filters tsa fluorescence.Lisefe tsena li tlameha ho se khone ho kenella mehloling ea khanya e matla e hlahisang leseli la ultraviolet (UV) le ka lebisang ho "cheleng", haholo-holo sefefo sa exciter kaha se laoloa ke matla a felletseng a mohloli oa khanya.
Lits'oants'o tse fapaneng tsa Mehlala ea Fluorescent
Lisebelisoa li bokelloa le ho hlophisoa Marang-rang, 'me li sebelisetsoa ho ithuta le puisano feela.Haeba ho na le tlolo ea molao, ka kopo ikopanye le rona ho hlakola.
Nako ea poso: Dec-09-2022