Qhov txawv ntawm Fluorescence Microscope thiab Normal Microscope
1. Saib txoj kev teeb pom kev zoo
Txoj kev teeb pom kev ntawm fluorescence microscope feem ntau yog hom epi-hom, uas yog hais tias, lub teeb ci tau muab tso rau ntawm cov qauv ntsuas los ntawm lub hom phiaj lens.
2. Saib qhov kev daws teeb meem
Fluorescence microscopes siv lub teeb ultraviolet ua lub teeb ci, nrog lub wavelength luv luv, tab sis qhov kev daws teeb meem siab dua li ntawm cov khoom siv kho qhov muag zoo tib yam.
3, qhov sib txawv ntawm cov lim
Fluorescence microscope siv ob lub lim dej tshwj xeeb, siv rau pem hauv ntej ntawm lub teeb ci los lim tawm lub teeb pom kev, thiab siv nruab nrab ntawm lub hom phiaj lens thiab eyepiece los lim tawm ultraviolet lub teeb, uas tuaj yeem tiv thaiv tib neeg lub qhov muag.
Fluorescence microscope kuj yog ib hom ntawm optical microscope, feem ntau vim hais tias lub wavelength zoo siab los ntawm fluorescence microscope yog luv, yog li qhov no ua rau qhov sib txawv ntawm cov qauv thiab siv cov fluorescence microscope thiab lub microscope zoo tib yam. Feem ntau ntawm cov fluorescence microscopes muaj kev ua haujlwm zoo ntawm kev ntes lub teeb tsis muaj zog. , yog li nyob rau hauv tsis tshua muaj fluorescence tsis muaj zog, nws cov duab muaj peev xwm kuj zoo. Ua ke nrog kev txhim kho txuas ntxiv ntawm fluorescence microscopy nyob rau xyoo tas los no, suab nrov kuj tau txo qis heev. Yog li ntawd, ntau thiab ntau fluorescence microscopes tau siv.
Kev paub txog Ob-Photon Fluorescence Microscopy
Lub hauv paus ntsiab lus ntawm ob-photon excitation yog: nyob rau hauv cov ntaub ntawv ntawm high photon ceev, fluorescent molecules tuaj yeem nqus tau ob lub wavelength photons nyob rau tib lub sijhawm, thiab tom qab luv luv heev thiaj li hu ua lub xeev zoo siab lub neej, emit ib tug luv luv wavelength photon. ; cov nyhuv yog tib yam li siv photon nrog lub wavelength ib nrab ntawm qhov ntev wavelength kom excite ib fluorescent molecule. Ob-photon excitation yuav tsum muaj photon ceev, thiab thiaj li tsis ua kom puas hlwb, ob-photon microscopy siv high-zog hom-locked pulsed lasers. Lub laser no emits laser lub teeb nrog lub siab ncov lub zog thiab nruab nrab lub zog qis, nrog lub siab dav ntawm 100 femtoseconds thiab zaus ntawm 80 txog 100 MHz. Thaum lub siab tus lej aperture lub hom phiaj lens yog siv los tsom cov photons ntawm lub laser pulsed, photon ceev ntawm lub focal point ntawm lub hom phiaj lens yog siab tshaj, thiab ob-photon excitation tshwm sim tsuas yog nyob rau hauv lub focal point ntawm lub hom phiaj lens, yog li ob-photon microscope tsis xav tau confocal pinhole, uas txhim kho Fluorescence nrhiav kom tau efficiency.
Nyob rau hauv cov fluorescence tshwm sim, vim qhov tsawg photon ceev ntawm lub excitation teeb, ib tug fluorescent molecule tsuas nqus tau ib tug photon tib lub sij hawm, thiab ces emit ib tug fluorescent photon los ntawm kev hloov hluav taws xob, uas yog hu ua ib leeg-photon fluorescence. Rau cov txheej txheem fluorescence excitation nrog laser raws li lub teeb qhov chaw, ob-photon los yog ntau-photon fluorescence phenomenon yuav tshwm sim. Nyob rau lub sijhawm no, qhov kev siv ntawm lub teeb pom kev zoo siv yog siab, thiab qhov ceev ntawm photon ua tau raws li qhov yuav tsum tau ua kom cov fluorescent molecule absorbs ob photons tib lub sijhawm. Nyob rau hauv tus txheej txheem ntawm kev siv ib tug dav laser raws li lub excitation lub teeb qhov chaw, lub photon ceev tseem tsis txaus los tsim ob-photon absorption. Feem ntau, femtosecond pulsed laser siv, thiab nws lub zog tam sim no tuaj yeem ncav cuag qhov kev txiav txim ntawm megawatts. Yog li ntawd, lub wavelength ntawm ob-photon fluorescence yog luv dua lub wavelength ntawm lub excitation teeb, uas yog sib npaug rau cov nyhuv tsim los ntawm ib nrab-excitation wavelength excitation.
Ob-photon fluorescence microscopy muaj ntau yam zoo:
1) Lub teeb ntev-ntev tsis tshua muaj kev cuam tshuam los ntawm kev tawg ntawm lub teeb luv luv thiab tuaj yeem nkag mus rau cov qauv yooj yim;
2) Cov fluorescent molecules sab nraum lub dav hlau focal tsis zoo siab, yog li ntawd lub teeb excitation ntau tuaj yeem ncav cuag lub dav hlau focal, kom lub teeb excitation tuaj yeem nkag mus rau hauv cov qauv sib sib zog nqus;
3) Lub teeb ntev-ntev ze-infrared tsis tshua muaj tshuaj lom rau cov hlwb dua li lub teeb luv luv;
4) Thaum saib cov qauv nrog ob-photon microscopy, photobleaching thiab phototoxicity tsuas yog nyob ntawm lub dav hlau focal. Yog li ntawd, ob-photon microscopy yog qhov tsim nyog rau kev saib cov qauv tuab dua li ib leeg-photon microscopy, rau saib cov cell nyob, lossis rau kev ua cov kev sim photobleaching tas li.
Kev paub txog confocal fluorescence microscopy
Lub hauv paus ntsiab lus ntawm confocal fluorescence microscopy: siv lub teeb pom kev zoo los ua kom pom cov qauv, lub teeb me me nrog lub teeb pom kev zoo yog tsim rau ntawm lub dav hlau focal. tsim los ntawm cov nqaj splitter. Lub beam splitter xa cov fluorescence ncaj qha mus rau lub detector. Muaj ib tug pinhole nyob rau hauv pem hauv ntej ntawm lub teeb qhov chaw thiab lub detector, feem hu ua lub illumination pinhole thiab nrhiav kom pom pinhole. Qhov ntev geometric ntawm ob yog tib yam, txog 100-200 nm; txheeb ze rau lub teeb pom kev ntawm lub dav hlau focal, ob qho tib si yog conjugate, uas yog, lub teeb pom kev dhau los ntawm ntau lub lo ntsiab muag, thiab thaum kawg tuaj yeem tsom mus rau lub teeb pom kev zoo thiab qhov pom lub pinhole tib lub sijhawm. Nyob rau hauv txoj kev no, lub teeb los ntawm lub dav hlau focal tuaj yeem ua kom pom tseeb hauv qhov ntau ntawm qhov kev tshawb pom, thaum lub teeb tawg los ntawm sab saud lossis hauv qab ntawm lub dav hlau focal raug thaiv tawm ntawm lub qhov nrhiav pom thiab tsis tuaj yeem pom. Cov qauv yog scanned taw tes los ntawm taw tes nrog lub laser, thiab lub photomultiplier raj tom qab kuaj pom tus pinhole kuj tau txais cov duab confocal ntawm cov teeb pom kev sib txuas los ntawm qhov taw tes, uas tau hloov mus rau hauv cov teeb liab digital thiab xa mus rau lub computer, thiab thaum kawg sib sau ua ke. qhov screen rau hauv daim duab meej confocal ntawm tag nrho cov dav hlau focal. .
Txhua daim duab focal dav hlau yog qhov ua tau zoo ntawm qhov chaw hla ntawm cov qauv, thiab qhov kev sib tw kho qhov muag no ib txwm muaj qee qhov tuab, tseem hu ua optical hlais. Txij li thaum lub teeb siv ntawm lub focal point yog ntau ntau dua li ntawm qhov tsis yog-focal point, thiab lub dav hlau tsis focal yog lim tawm los ntawm lub pinhole, qhov tob ntawm thaj tsam ntawm lub confocal system yog kwv yees li xoom, thiab luam theej duab raws qhov Z-axis tuaj yeem paub kho qhov muag tomography, ua rau Kev Saib Xyuas ob-dimensional optical seem ntawm qhov chaw tsom ntawm cov qauv. Kev sib txuas ntawm XY dav hlau (focal dav hlau) scanning nrog Z-axis (optical axis) scanning, los ntawm accumulating ob-dimensional dluab ntawm successive khaubncaws sab nraud povtseg thiab ua tiav los ntawm tshwj xeeb computer software, ib tug peb-dimensional duab ntawm cov qauv yuav muab tau.
Ntawd yog, qhov kev tshawb pom tus pinhole thiab lub teeb pom kev pinhole yog ib txwm tsom mus rau tib lub ntsiab lus, kom cov fluorescence zoo siab sab nraum lub dav hlau tsom tsis tuaj yeem nkag mus rau qhov kev tshawb pom pinhole.
Lub ntsiab lus yooj yim ntawm kev ua haujlwm ntawm confocal laser yog tias nws siv lub laser ua lub teeb ci, thiab ntawm lub hauv paus ntawm cov tshuaj fluorescence microscope imaging, lub laser scanning ntaus ntawv thiab ib tug conjugate focusing ntaus ntawv txuas, thiab cov duab tau txais thiab ua. system yog tswj los ntawm lub computer.
