Cov Kev Xav Tau Kev Npaj Rau Fluorescence Microscopy
Yuav Tsum Tau Npaj Cov Qauv rau Fluorescence Microscopy
(1) iav swb
Lub thickness ntawm cov iav swb yuav tsum nyob nruab nrab ntawm 0 8-L2mm. Ib tug swb uas tuab heev yuav nqus tau ntau lub teeb ntawm ib sab tes thiab tsis tuaj yeem tsom lub teeb pom kev zoo ntawm cov qauv ntawm qhov tod tes. Cov zawv zawg yuav tsum du, sib xws hauv tuab, thiab tsis muaj qhov pom tseeb autofluorescence. Qee zaum quartz slides yog siv.
(2) Npog iav
Lub thickness ntawm lub hau iav yog li ntawm 0.17mm, du. Txhawm rau ntxiv dag zog rau lub teeb pom kev zoo, ib qho kev cuam tshuam npog iav tuaj yeem siv tau, uas yog lub npog tshwj xeeb iav coated nrog ob peb txheej ntawm cov tshuaj (xws li magnesium fluoride) uas muaj kev cuam tshuam sib txawv ntawm lub teeb ntawm qhov sib txawv wavelengths, uas tuaj yeem ua rau lub teeb pom kev zoo. fluorescence smooth. Lub teeb excitation yog dhau los ntawm, thiab lub teeb excitation yog reflected, thiab qhov no reflected excitation teeb yuav excite tus qauv.
(3) Cov qauv
Cov ntaub so ntswg los yog lwm cov qauv yuav tsum tsis txhob tuab heev. Yog hais tias lub thickness yog tuab heev, feem ntau ntawm cov excitation teeb yuav tsum tau noj nyob rau hauv qis ib feem ntawm cov qauv, thaum lub sab sauv ncaj qha soj ntsuam los ntawm lub hom phiaj lens yuav tsis tau zoo siab heev. Tsis tas li ntawd, fluorescence tshwm sim los ntawm overlapping hlwb los yog impurities, keeb kwm yav dhau uas tsis yog tshwj xeeb staining cuam tshuam rau kev txiav txim.
(4) iav roj
Feem ntau, thaum soj ntsuam cov qauv nrog qhov tsaus ntuj nti fluorescence microscopes thiab roj immersion microscopes, yuav tsum tau siv roj immersion. Nws yog qhov zoo tshaj plaws los siv cov roj tshwj xeeb uas tsis muaj fluorescent immersion. Glycerin kuj tseem siv tau los hloov, thiab cov kua paraffin kuj tseem siv tau, tab sis qhov ntsuas qhov ntsuas qis qis, uas muaj kev cuam tshuam me ntsis ntawm cov duab zoo.
Nkag siab lub teeb lub voos xwmfab ntawm Fluorescence Microscopy
Fluorescence yog lub teeb uas electrons nyob rau hauv ib yam khoom nqus lub teeb lub zog los ntawm lub xeev uas tsis muaj zog mus rau lub high-zog lub xeev, thiab ces tso lub teeb thaum nws rov qab mus rau lub low-zog lub xeev. Nws yog tsis kub-kub radiated lub teeb-luminescence. Ntawd yog: cov khoom absorbs lub teeb luv-yoj, nkag mus rau hauv lub xeev zoo siab, thiab emits ntev-yoj lub teeb.
Txawm hais tias nws yog autofluorescence ntawm cov khoom, cov fluorescent dye los yog cov fluorescent protein qhia los ntawm fusion, nws yuav tsum tau zoo siab los ntawm ib tug tshwj xeeb wavelength ntawm lub teeb (Excitation). Tom qab cov electrons tsiv thiab poob lub zog, lawv emit lub teeb ntawm ib tug ntev wavelength (Emision). , uas tuaj yeem sau los ntawm kev kuaj xyuas kom ua tiav cov haujlwm ntawm kev txheeb xyuas cov fluorescence tshwj xeeb.
Lub teeb fluorescent yog dab tsi?
Nyob rau hauv fluorescence microscope soj ntsuam thiab duab, lub excitation lub teeb ntawm tej wavelength thiab cov coj ntev-wavelength emission teeb yog muab los ntawm lub fluorescence lub teeb lub voos xwmfab, thiaj li hais tias cov teeb liab fluorescence tuaj yeem sau los ntawm qhov muag liab qab, tso saib lossis lub koob yees duab. Yog li ntawd, lub fluorescence lub teeb lub voos xwmfab txiav txim seb yuav kuaj tau dab tsi Cov cuab yeej tseem ceeb ntawm lub teeb liab fluorescence, nws cov yam ntxwv muaj xws li EX: excitation wavelength filter parameters, EM: emission wavelength filter parameters and DM: dichotomous mirror parameters. Siv lub DAPI lub teeb lub voos xwmfab ntawm Revolve integrated fluorescence microscope ua piv txwv, EX: 385/30, EM: 450/50, DM: 425.
The light emitted by the light source passes through DAPI EX to obtain excitation light in a specific wavelength range, that is, light of 385±15nm, which specifically excites fluorescent substances that can only be excited within this range; the DM dichroic mirror separates the excitation light from the fluorescence Optical elements, as special mirrors, reflect only specific wavelengths of light and allow all other wavelengths to pass through, so only >425nm lub teeb tuaj yeem xa mus rau EM; EM emission lim yog siv los cais cov fluorescence emission los ntawm fluorophore los ntawm lwm yam keeb kwm yav dhau Optical ntsiab rau cais lub teeb. Emission lim xa lub teeb ntawm fluorescence wavelength los ntawm daim iav dichroic thaum thaiv tag nrho lwm lub teeb uas xau los ntawm lub teeb pom kev zoo (xws li los ntawm cov qauv lossis optics). Lub emitted lub teeb wavelength yog ntau dua EM yuav tsum tau pom, uas yog, tsuas yog lub teeb nyob rau hauv lub cheeb tsam ntawm 450 ± 25nm nkag mus rau hauv lub system nrhiav kom tau. Kev xaiv kom raug ntawm EX, EM lim, thiab DM dichotomies tuaj yeem pab cov neeg tshawb nrhiav kom ua tiav cov teeb liab-rau-lub suab sib piv (S / N).
