Ihe nzacha fluorescence bụ ihe dị mkpa na microscope fluorescence. Usoro a na-ahụkarị nwere nzacha atọ bụ isi: ihe nzacha mkpali, ihe nzacha ikuku na enyo dichroic. A na-anakọtakarị ha n'otu kube ka e wee tinye otu ahụ ọnụ n'ime microscope.
Kedu ka nzacha fluorescence si arụ ọrụ?
Ihe nzacha mkpali
Ihe nzacha na-akpali akpali na-ebunye ìhè nke otu ogologo ebili mmiri ma gbochie ogologo ebili mmiri ndị ọzọ. Enwere ike iji ha mepụta agba dị iche iche site n'imezi ihe nzacha ka ọ hapụ naanị otu agba. Ihe nzacha mkpali na-abịa n'ụdị isi abụọ - ihe nzacha ngafe ogologo na ihe nzacha ngafe band. Ihe na-akpali akpali na-abụkarị ihe nzacha bandpas nke na-agafe naanị ogologo ebili mmiri nke fluorophore na-etinye, si otú a na-ebelata mkpali nke isi mmalite ndị ọzọ nke fluorescence na igbochi ọkụ mkpali na band fluorescence emission band. Dị ka e gosiri site na-acha anụnụ anụnụ akara na ọnụ ọgụgụ, BP bụ 460-495, nke pụtara na ọ nwere ike gafere naanị site fluorescence nke 460-495nm.
A na-edobe ya n'ime ụzọ ọkụ nke microscope fluorescence ma na-ehichapụ ogologo ebili mmiri niile nke isi iyi ọkụ ma e wezụga maka oke mkpali fluorophore. Nfefe kacha nta nke nzacha na-egosi nchapụta na nchapụta nke onyonyo. A na-atụ aro opekempe nke 40% maka nzacha mkpali ọ bụla ka nnyefe ahụ dị mma> 85%. The bandwit nke excitation nzacha kwesịrị kpamkpam n'ime fluorophore excitation nso dị ka center wavelength (CWL) nke nzacha dị nso dị ka o kwere omume kacha elu excitation wavelength nke fluorophore. The excitation filter optical density (OD) na-akọwa ọchịchịrị onyinyo ndabere; OD bụ ihe nleba nke ọma ka nzacha na-egbochi ogologo ebili mmiri na-abụghị oke nnyefe ma ọ bụ bandwit. Opekempe OD nke 3.0 ka akwadoro mana OD nke 6.0 ma ọ bụ karịa dị mma.
Ihe nzacha ikuku
Ihe nzacha ikuku na-arụ ebumnuche nke ikwe ka fluorescence a na-achọsi ike sitere na nlele ruo n'ihe nchọta. Ha na-egbochi ogologo ebili mmiri dị mkpụmkpụ ma nwee nnyefe dị elu maka ogologo ebili mmiri dị ogologo. A na-ejikọta ụdị nzacha ahụ na ọnụọgụ, dịka BA510IF na ọnụ ọgụgụ (nzacha ihe mgbochi nnyonye anya), aha ahụ na-ezo aka n'ogologo ogologo na 50% nke nnyefe kachasị.
Otu ndụmọdụ ndị ahụ maka nzacha mkpali bụ eziokwu maka nzacha ikuku: nnyefe kacha nta, bandwit, OD na CWL. Ihe nzacha ikuku nwere ezigbo CWL, nnyefe opekempe, yana nchikota OD na-enye onyogho kachasị mma enwere ike, yana mgbochi kacha dị omimi ma na-eme ka a hụ na achọpụtara nrịbama ikuku kacha ada.
Dichroic mirror
A na-edobe enyo dichroic n'etiti ihe nzacha mkpali na ihe nzacha na-ekpo ọkụ na akụkụ 45 Celsius ma na-egosipụta mgbama mkpali n'ebe fluorophore nọ mgbe ọ na-ebufe mgbama ikuku n'ebe onye na-achọpụta ihe. Ihe nzacha dichroic dị mma na ndị na-ekesa beam nwere ntụgharị dị nkọ n'etiti ntụgharị uche kachasị na nnyefe kachasị, yana> 95% ntụgharị maka bandwit nke nzacha mkpali na nnyefe nke> 90% maka bandwit nke nzacha ikuku. Họrọ nzacha n'obi nrutu ogologo nrutu (λ) nke fluorophore, ka ibelata ọkụ na-akpafu ma na-ebuli oke mgbama ihe onyonyo nke fluorescent.
Ngosipụta dichroic dị na ọnụ ọgụgụ a bụ DM505, nke a na-akpọ ya n'ihi na 505 nanometers bụ ogologo ogologo na 50% nke nnyefe kachasị maka enyo a. Usoro nnyefe maka enyo a na-egosi nnyefe dị elu n'elu 505 nm, mgbada dị elu na nnyefe gaa n'aka ekpe nke 505 nanometers, na ntụgharị uche kachasị n'aka ekpe nke 505 nanometers ma ka nwere ike ịnwe nnyefe n'okpuru 505 nm.
Kedu ihe dị iche na nzacha ogologo ngafe na band pass?
Enwere ike kewaa ihe nzacha fluorescence ụzọ abụọ: ogologo ngafe (LP) na band pass (BP).
Ihe nzacha ngafe ogologo na-ebufe ogologo wavelength ma gbochie ndị dị mkpụmkpụ. Ogologo ogologo nke ịkpụ bụ uru dị na 50% nke nnyefe kacha elu, na ogologo oge niile dị n'elu ịkpụ na-ebufe site na nzacha ogologo ngafe. A na-ejikarị ha eme ihe na enyo dichroic na ihe nzacha ikuku. Ekwesịrị iji ihe nzacha Longpass mgbe ngwa ahụ chọrọ nchịkọta ikuku kacha yana mgbe ịkpa oke na-adịghị mma ma ọ bụ dị mkpa, nke na-abụkarị maka nyocha nke na-emepụta otu ụdị na-ebupụta n'ụdị nwere obere ọkwa ndabere autofluorescence.
Ihe nzacha band pass na-ebufe naanị otu band ogologo, ma gbochie ndị ọzọ. Ha na-ebelata crosstalk site n'ikwe ka naanị akụkụ kachasị ike nke fluorophore emission spectrum na-ebufe, na-ebelata ụda autofluorescence ma si otú a na-emeziwanye akara mgbaàmà na ụda na elu ndabere autofluorescence samples, nke ogologo ngafe nzacha enweghị ike inye.
Kedu ụdị nzacha nzacha fluorescence BestScope nwere ike ịnye?
Ụfọdụ ụdị nzacha a na-ahụkarị gụnyere nzacha anụnụ anụnụ, akwụkwọ ndụ akwụkwọ ndụ na ultraviolet. Dị ka e gosiri na tebụl.
| Ntọala nzacha | Ihe nzacha mkpali | Dichroic mirror | Ihe mgbochi mgbochi | Ọkụ LED Ogologo Ogologo | Ngwa |
| B | BP460-495 | DM505 | BA510 | 485nm | · FITC: Usoro mgbochi fluorescent · oroma acid: DNA, RNA Auramine: bacillus tubercle · EGFP, S657, RSGFP |
| G | BP510-550 | DM570 | BA575 | 535nm | Rhodamine, TRITC: Fluorescent antibody usoro · Propidium iodide: DNA · RFP |
| U | BP330-385 | DM410 | BA420 | 365nm | · Nleba anya fluorescence na akpaaka · DAPI: DNA ntụpọ · Hoechest 332528, 33342: ejiri mee ntụpọ chromosome |
| V | BP400-410 | DM455 | BA460 | 405nm | Catecholamines 5-hydroxy tryptamine · Tetracycline: ọkpụkpụ, ezé |
| R | BP620-650 | DM660 | BA670-750 | 640nm | · 5 Alexa Fluor 633, Alexa Fluor 647 |
A na-emepụta ihe nzacha a na-eji na-enweta fluorescence gburugburu ogologo ogologo ogologo nke a na-eji na ngwa fluorescence, nke dabere na fluorophores ndị a na-ejikarị eme ihe. N'ihi nke a, a na-akpọkwa ha aha fluorophore ha ezubere maka onyonyo, dị ka DAPI (acha anụnụ anụnụ), FITC (green) ma ọ bụ TRITC (red) nzacha cubes.
| Ntọala nzacha | Ihe nzacha mkpali | Dichroic mirror | Ihe mgbochi mgbochi | Ọkụ LED Ogologo Ogologo |
| 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 |
Kedu otu esi ahọrọ nzacha fluorescence?
1. Ụkpụrụ nke ịhọrọ fluorescence filter bụ ikwe ka ọkụ ọkụ / ọkụ ọkụ na-agafe na njedebe ihe ngosi dị ka o kwere mee, ma gbochie ọkụ ọkụ ọkụ kpamkpam n'otu oge ahụ, iji nweta akara ngosi kachasị elu na ụda mkpọtụ. Karịsịa maka ntinye nke multiphoton excitation na mkpokọta ntụgharị uche dị n'ime microscope, ụda na-adịghị ike ga-emekwa ka nnyonye anya dị ukwuu na mmetụta onyonyo, ya mere ihe a chọrọ maka mgbaàmà na ụda mkpọtụ dị elu.
2. Mara mkpali na ihe dị iche iche nke fluorophore. Iji wuo ihe nzacha fluorescence nke na-ewepụta onyonyo dị elu, dị elu nke nwere okirikiri ojii, ihe nzacha mkpali na ihe nzacha ga-enweta nnyefe dị elu site na iji mpe mpe mpe mpe mpe mpe mpe akwa gafere mpaghara ndị dabara na oke mkpali nke fluorophore ma ọ bụ ikuku.
3. Tụlee ịdịte aka nke nzacha fluorescence. Ihe nzacha ndị a ga-abụ nke na-agaghị ekwe omume ịnweta isi iyi ọkụ siri ike nke na-emepụta ọkụ ultraviolet (UV) nke nwere ike ibute "ọkụ", karịsịa nke nzacha ihe na-akpali akpali ka ọ na-edobe ike nke isi iyi ọkụ.
Foto dị iche iche nke Fluorescent
A na-anakọta ma hazie akụrụngwa ndị ahụ na ịntanetị, a na-ejikwa ya naanị maka mmụta na nkwukọrịta. Ọ bụrụ na enwere mmebi iwu, biko kpọtụrụ anyị ka ihichapụ.
Oge nzipu: Dec-09-2022