ʻO kahi kānana fluorescence kahi mea nui i ka microscope fluorescence. ʻO kahi ʻōnaehana maʻamau he ʻekolu kānana kumu: kahi kānana excitation, kahi kānana emission a me kahi aniani dichroic. Hoʻokomo pinepine ʻia lākou i loko o kahi pahu i hoʻokomo pū ʻia ka hui i loko o ka microscope.
Pehea e hana ai kahi kānana fluorescence?
Kānana hoʻohauʻoli
Hoʻouna nā kānana hoʻohiwahiwa i ka mālamalama o kahi lōʻihi nalu kikoʻī a pale i nā lōʻihi nalu ʻē aʻe. Hiki ke hoʻohana ʻia e hana i nā kala like ʻole ma ke kani ʻana i ka kānana e ʻae i hoʻokahi kala wale nō. Loaʻa nā kānana hauʻoli i ʻelua ʻano nui - nā kānana hele lōʻihi a me nā kānana band pass. ʻO ka exciter ka mea maʻamau i kahi kānana bandpass e hele wale i nā lōʻihi hawewe i hoʻomoʻa ʻia e ka fluorophore, no laila e hōʻemi ana i ka hauʻoli o nā kumu ʻē aʻe o ka fluorescence a me ka pale ʻana i ke kukui excitation i loko o ka pūʻulu emission fluorescence. E like me ka mea i hōʻike ʻia e ka laina polū ma ke kiʻi, ʻo BP ka 460-495, ʻo ia ka mea hiki ke hele wale i ka fluorescence o 460-495nm.
Hoʻonoho ʻia ia i loko o ke ala hoʻomālamalama o kahi microscope fluorescence a kānana i nā lōʻihi hawewe āpau o ke kumu kukui koe wale nō ka laula hoʻohiwahiwa fluorophore. ʻO ka hoʻoili liʻiliʻi loa o ke kānana e kuhikuhi i ka ʻōlinolino a me ka nani o nā kiʻi. ʻO ka liʻiliʻi o 40% ka hoʻouna ʻana no kēlā me kēia kānana excitation i manaʻo ʻia e like me ke kūpono o ka hoʻouna ʻana ma> 85%. ʻO ka bandwidth o ka kānana hoʻohiwahiwa e pono i loko o ka laulā hoʻohiwahiwa fluorophore e like me ka lōʻihi o ka nalu waena (CWL) o ka kānana i kahi kokoke loa i ka lōʻihi o ka hawewe o ka fluorophore. ʻO ka kānana hoʻohiwahiwa optical density (OD) e kuhikuhi i ka pōʻeleʻele kiʻi hope; ʻO OD kahi ana o ka maikaʻi o ke kānana i ka pale ʻana i nā lōʻihi nalu ma waho o ka pae hoʻouna a i ʻole ka bandwidth. Manaʻo ʻia ka OD liʻiliʻi o 3.0 akā kūpono ka OD o 6.0 a ʻoi aku paha.
Kānana hoʻokuʻu
Hana nā kānana hoʻokuʻu i ke kumu o ka ʻae ʻana i ka fluorescence i makemake ʻia mai ka hāpana e hiki i ka mea ʻike. Kāohi lākou i nā hawewe pōkole a loaʻa ka hoʻouna kiʻekiʻe no nā lōʻihi hawewe lōʻihi. Hoʻopili pū ʻia ke ʻano kānana me kahi helu, e laʻa i ka BA510IF ma ke kiʻi (pena keakea hoʻopili), ʻo ia koho ʻana i ka lōʻihi hawewe ma 50% o kona hoʻouna ʻana.
ʻO nā ʻōlelo aʻoaʻo like no nā kānana hauʻoli e paʻa no nā kānana hoʻokuʻu: ka liʻiliʻi liʻiliʻi, ka bandwidth, OD, a me CWL. ʻO kahi kānana hoʻokuʻu me ka CWL kūpono, ka liʻiliʻi liʻiliʻi, a me ka hui OD e hāʻawi i nā kiʻi ʻālohilohi loa, me ka hoʻopaʻa ʻana hohonu loa, a e hōʻoia i ka ʻike ʻana i nā hōʻailona hoʻokuʻu ʻia.
ʻO ke aniani Dichroic
Hoʻonoho ʻia ke aniani dichroic ma waena o ka kānana hoʻohiwahiwa a me ka kānana hoʻokuʻu ʻana ma kahi kihi 45 ° a hōʻike i ka hōʻailona hoʻāla i ka fluorophore i ka wā e hoʻouna ana i ka hōʻailona hoʻokuʻu i ka mea ʻike. ʻO nā kānana dichroic maikaʻi a me nā beam splitters he mau hoʻololi koʻikoʻi ma waena o ka noʻonoʻo kiʻekiʻe a me ka hoʻoili kiʻekiʻe loa, me kahi> 95% noʻonoʻo no ka bandwidth o ka kānana excitation a me ka hoʻoili ʻana o> 90% no ka bandwidth o ka kānana emission. E koho i ka kānana me ka lōʻihi nalu kuʻekuʻe (λ) o ka fluorophore i ka noʻonoʻo, e hōʻemi i ke kukui hele a hoʻonui i ka ratio hōʻailona-a-noise kiʻi fluorescent.
ʻO ke aniani dichroic i kēia kiʻi ʻo ka DM505, i kapa ʻia no ka mea ʻo 505 nanometers ka lōʻihi o ka nalu ma 50% o ka hoʻoili kiʻekiʻe no kēia aniani. Hōʻike ka pihi hoʻouna no kēia aniani i ka neʻe kiʻekiʻe ma luna o 505 nm, kahi hāʻule kiʻekiʻe o ka hoʻouna ʻana ma ka hema o 505 nanometers, a me ka noʻonoʻo kiʻekiʻe loa i ka hema o 505 nanometers akā e loaʻa ana paha kahi hoʻouna ma lalo o 505 nm.
He aha ka ʻokoʻa ma waena o nā kānana pass long a me band pass?
Hiki ke hoʻokaʻawale ʻia nā kānana fluorescence i ʻelua ʻano: lolo lōʻihi (LP) a me band pass (BP).
Hoʻouna nā kānana hele lōʻihi i nā nalu lōʻihi a pale i nā mea pōkole. ʻO ka lōʻihi o ka nalu ʻoki ʻia ka waiwai ma 50% o ka hoʻouna ʻana i ka piko, a ʻo nā lōʻihi a pau ma luna o ka ʻoki ʻia e hoʻouna ʻia e nā kānana hele lōʻihi. Hoʻohana pinepine ʻia lākou i nā aniani dichroic a me nā kānana emission. Pono e hoʻohana ʻia nā kānana Longpass i ka wā e koi ai ka noi i ka hōʻiliʻili hoʻokuʻu kiʻekiʻe loa a inā ʻaʻole makemake ʻia ka hoʻokae spectral a pono ʻole, ʻo ia ka hihia no nā probes e hoʻoulu i hoʻokahi ʻano hoʻokuʻu i nā specimens me nā pae haʻahaʻa o ka autofluorescence hope.
Hoʻouna wale nā kānana band pass i kekahi pūna hawewe lōʻihi, a pale i nā mea ʻē aʻe. Hoʻemi lākou i ka crosstalk ma ka ʻae ʻana i ka ʻāpana ikaika loa o ka spectrum emission fluorophore e hoʻouna ʻia, e hoʻemi i ka walaʻau autofluorescence a pēlā e hoʻomaikaʻi ai i ka ratio hōʻailona-a-noise i nā laʻana autofluorescence kiʻekiʻe, ʻaʻole hiki ke hāʻawi i nā kānana lōʻihi.
ʻEhia nā ʻano kānana fluorescence i hiki iā BestScope ke hāʻawi?
ʻO kekahi mau ʻano kānana maʻamau me nā kānana uliuli, ʻōmaʻomaʻo, a me ultraviolet. E like me ka mea i hoikeia ma ka papa.
| Hoʻonohonoho kānana | Kānana hoʻohauʻoli | ʻO ke aniani Dichroic | Kānana Kānāwai | kukui LED Ka lōʻihi o ka nalu | Palapala noi |
| B | BP460-495 | DM505 | BA510 | 485nm | ·FITC: ʻO ke ʻano antibody fluorescent · ʻAlani Acidine: DNA, RNA ·Auramine: Tubercle bacillus ·EGFP, S657, RSGFP |
| G | BP510-550 | DM570 | BA575 | 535nm | · Rhodamine, TRITC: ʻO ke ʻano antibody fluorescent · Propidium iodide: DNA ·RFP |
| U | BP330-385 | DM410 | BA420 | 365nm | · Ka nānā 'ana i ka 'auto-fluorescence ·DAPI: ʻāhu DNA · Hoechest 332528, 33342: hoʻohana ʻia no ke kala ʻana o Chromosome |
| V | BP400-410 | DM455 | BA460 | 405nm | · Catecholamine · 5-hydroxy tryptamine ·Tetracycline: iwi, niho |
| R | BP620-650 | DM660 | BA670-750 | 640nm | · Cy5 ·Alexa Fluor 633, Alexa Fluor 647 |
Hoʻolālā ʻia nā ʻāpana kānana i hoʻohana ʻia i ka loaʻa ʻana o ka fluorescence a puni nā nalu nui i hoʻohana ʻia i nā noi fluorescence, i hoʻokumu ʻia a puni nā fluorophores i hoʻohana nui ʻia. No kēia kumu, kapa ʻia lākou ma muli o ka fluorophore i manaʻo ʻia no ke kiʻi ʻana, e like me DAPI (uliuli), FITC ('ōmaʻomaʻo) a i ʻole TRITC (ʻulaʻula) kānana cubes.
| Hoʻonohonoho kānana | Kānana hoʻohauʻoli | ʻO ke aniani Dichroic | Kānana Kānāwai | kukui LED Ka lōʻihi o ka nalu |
| 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 |
| ʻO Texas ʻulaʻula | BP540-580 | DM595 | BA600-660 | 560nm |
| mCherry | BP542-582 | DM593 | BA605-675 | 560nm |
Pehea ʻoe e koho ai i kahi kānana fluorescence?
1. ʻO ke kumumanaʻo o ke koho ʻana i ka kānana fluorescence e ʻae i ke kukui fluorescence / emission e hele i ka hopena kiʻi e like me ka hiki, a hoʻopaʻa loa i ke kukui excitation i ka manawa like, i loaʻa ai ka lākiō hōʻailona kiʻekiʻe loa. Maikaʻi no ka hoʻohana ʻana i ka multiphoton excitation a me ka nui o ka microscope noʻonoʻo i loko, ʻo ka leo nāwaliwali e hoʻopilikia nui i ka hopena kiʻi, no laila ʻoi aku ka kiʻekiʻe o ke koi no ka hōʻailona noise ratio.
2. E ʻike i ke ʻano o ka hoʻoulu a me ka hoʻokuʻu ʻana o ka fluorophore. No ke kūkulu ʻana i kahi hoʻonohonoho kānana fluorescence e hoʻopuka i kahi kiʻi kiʻekiʻe, kiʻi like ʻole me kahi ʻeleʻele ʻeleʻele, pono e hoʻokō i nā kānana kiʻekiʻe me ka liʻiliʻi passband ripple ma luna o nā ʻāpana e pili ana i nā kiʻekiʻe o ka fluorophore excitation peaks.
3. E noʻonoʻo i ka lōʻihi o nā kānana fluorescence. Pono e pale ʻole kēia mau kānana i nā kumu kukui ikaika e hoʻoulu i ke kukui ultraviolet (UV) hiki ke alakaʻi i ka "burnout", ʻo ia hoʻi o ka kānana exciter no ka mea e kau ʻia ana i ka ikaika piha o ke kumu hoʻomālamalama.
Nā Kiʻi Laʻana Fluorescent ʻokoʻa
ʻOhi ʻia nā kumuwaiwai a hoʻonohonoho ʻia ma ka Pūnaewele, a hoʻohana wale ʻia no ke aʻo ʻana a me ke kamaʻilio. Inā loaʻa kekahi hewa, e ʻoluʻolu e kelepona mai iā mākou e holoi.
Ka manawa hoʻouna: Dec-09-2022