Flow cytometry yog ib txoj kev tshawb fawb cytological siv rau kev tsom xam qhov tob ntawm cov hlwb. Nws qhov zoo dua yog tias nws tso cai rau koj los kawm txhua lub xov tooj ntawm tus kheej. Hom kev tshuaj ntsuam no yuav pab ntsuas ntau qhov ntsuas hauv ntau pua lub hlwb hauv ib lub vib nas this. Raws li qhov tshwm sim, cytofluorimetry yog suav tias yog ib txoj hauv kev ceev tshaj plaws thiab muaj tseeb tshaj plaws ntawm kev tshuaj ntsuam tam sim no muaj rau cov kws tshawb fawb thiab kws kho mob.
Principle
Lub hauv paus ntsiab lus ntawm ntws cytometry yog raws li kev ntsuas ntawm lub teeb tawg thiab luminescence (fluorescence) ntawm cov hlwb. Lub cell ncua kev kawm ntawv yog kis raws li ib tug kwj ntawm kev kub ceev los ntawm lub cytometer cell, qhov twg nws yog irradiated nrog ib tug laser. Lub thiaj li hu ua hydrodynamic focusing kuj tau ua nyob rau ntawd. Nws lub tswv yim yog tias cov dej ntws los ntawm cov xov tooj ntawm tes nrog cov kev tshawb fawb hais ntawm qhov hluav taws xob ntws mus rau hauv lub dav hlau sab nraud, uas muaj qhov nrawm dua. Raws li qhov tshwm sim, cov khoom sib xyaw ua ke hauv cov saw hlau xaj.
Cov qe ua ntej tau sau nrog cov xim tshwj xeeb fluorescent (fluorochromes). Ua tsaug rau lawv, laser beamexcites ib tug thib ob glow. Cov teeb liab tau txais tau sau npe los ntawm cov khoom ntes. Tom qab ntawd, cov ntaub ntawv tau ua tiav siv software algorithms uas tso cai rau koj los suav cov neeg ntawm tes uas txawv ntawm qee qhov qauv.
Kev tshawb fawb nrog cov pa microscopy feem ntau tsis paub qhov txawv ntawm cov hlwb vim lawv zoo ib yam. Cytofluorimetry tuaj yeem muab lwm cov ntaub ntawv (DNA qauv kev ncaj ncees), txheeb xyuas cov protein qhia, cell ciaj sia.
Txij li kev ua kom zoo ntawm fluorochromes yuav tsum muaj cov kab teeb pom kev sib txawv, nrog rau ntau hom kev ntsuas, kev teeb tsa niaj hnub tau nruab nrog ntau qhov kev tshawb nrhiav (los ntawm 4 txog 30). Tus naj npawb ntawm cov laser emitters tuaj yeem yog los ntawm 1 txog 7. Cov khoom siv ntau dua tso cai rau ntau qhov kev tshawb fawb ntawm ntau yam khoom ntawm cov khoom ib zaug.
Qhov zoo thiab qhov tsis zoo
Cov txiaj ntsig ntawm kev ntws cytometry suav nrog:
- kev ua haujlwm siab nrawm (sau npe txog li 30 txhiab qhov xwm txheej hauv 1 thib ob);
- muaj peev xwm kawm ntau lub hlwb (txog 100 lab hauv ib qho qauv);
- Quantitating qhov siv ntawm lub teeb fluorescent;
- tsom xam ntawm txhua lub cell;
- kev tshawb fawb ib txhij ntawm cov txheej txheem heterogeneous;
- tsis siv neeg sib cais ntawm cov ntaub ntawv los ntawm cov pej xeem ntawm tes;
- kev pom zoo ntawm cov txiaj ntsig.
Lwm yam ntawm cov thev naus laus zis no yog qhov ntawdCov tshuaj ntsuam xyuas tuaj yeem ua xim nrog ntau cov tshuaj fluorescent. Ua tsaug rau qhov no, kev tshawb fawb ntau yam tshwm sim.
Qhov tsis zoo suav nrog qhov nyuaj ntawm cov cuab yeej siv thiab xav tau kev npaj tshwj xeeb.
Cytometers
Thawj cov khoom siv ntawm hom no tau tshwm sim nyob rau xyoo 1968 hauv tebchaws Yelemes, tab sis lawv tau dhau los ua ntau yam tom qab. Tam sim no, txhua yam khoom siv uas ua haujlwm los ntawm txoj kev ntws cytometry tuaj yeem muab faib ua 2 hom:
- khoom siv ntsuas hluav taws xob fluorescent (ob lossis ntau lub wavelengths), 10 ° thiab 90 ° lub teeb tawg (tsawg lub kaum sab xis thiab sab scatter detector);
- cov khoom siv uas, ntxiv rau kev ntsuas ntau lub xov tooj ntawm tes, txiav ua pawg raws li cov qauv no.
Lub tshuab xa mus rau pem hauv ntej yog tsim los txiav txim siab qhov loj ntawm lub xov tooj ntawm tes, thiab cov khoom siv sab nraud tso cai rau koj kom tau txais cov ntaub ntawv hais txog qhov muaj cov granules intracellular, qhov ntim piv ntawm cytoplasm thiab cov nucleus.
Classic cytometers, tsis zoo li lub teeb microscopes, tsis tso cai kom tau txais cov duab ntawm lub xovtooj. Txawm li cas los xij, nyob rau xyoo tas los no, cov khoom siv sib xyaw ua ke tau tsim muaj peev xwm sib txuas cov peev txheej ntawm lub tshuab tsom thiab lub cytofluorimeter. Lawv yuav tham hauv qab no.
Imaging Cytometers
Rau cov cuab yeej siv hauv classical flow cytometry,ib qho tshwj xeeb yog tus yam ntxwv: yog tias tsis tshua muaj xwm txheej tau sau npe hauv cov pej xeem ntawm cov tshuaj ntsuam xyuas, ces tsis muaj txoj hauv kev los ntsuas seb lawv cov ntsiab lus yog dab tsi. Cov khoom no tuaj yeem yog cov seem ntawm cov hlwb tuag lossis ib pawg tsawg ntawm lawv. Hauv cov khoom siv ib txwm siv, cov ntaub ntawv no tsis suav nrog cov xwm txheej dav dav, tab sis nws yog lawv uas tuaj yeem muaj txiaj ntsig tshwj xeeb rau kev tshawb fawb thiab tshuaj ntsuam xyuas.
Lub cim tshiab ntawm cov duab ntws cytometers tso cai rau koj los ntes cov duab ntawm txhua lub xov tooj ntawm tes hla hauv qhov ntws los ntawm thaj chaw ntes. Nws yog ib qho yooj yim kom pom nws los ntawm txhaj rau ntawm qhov sib thooj ntawm daim duab kos duab, uas tso tawm rau hauv lub computer saib.
thaj chaw thov
Flow cytometry yog ib txoj hauv kev siv dav hauv ntau yam tshuaj thiab kev tshawb fawb:
- immunology;
- oncology;
- transplantology (hloov cov pob txha liab, qia hlwb);
- hematology;
- toxicology;
- biochemistry (kev ntsuas cov kua qaub hauv lub cell, kev kawm ntawm lwm yam tsis muaj);
- pharmacology (tsim tshuaj tshiab);
- microbiology;
- parasitology thiab virology;
- dej hiav txwv (kev kawm ntawm phytoplankton los ntsuas lub xeev ntawm lub cev dej thiab lwm yam dej num);
- nanotechnology thiab microparticle tsom xam.
Immunology
Tib neeg lub cev tiv thaiv kab mob muaj ntau lub hlwb. Flow cytometry hauv kev tiv thaiv kab mob ua rau nws muaj peev xwm los ntsuas lawv cov qauv thiab kev ua haujlwm, uas yog, ua tiav morphofunctional.tsom xam.
Kev tshawb fawb no pab kom nkag siab qhov nyuaj ntawm kev tiv thaiv kab mob. Cell phenotypes hloov raws li kev ua kom los ntawm antigens, kev loj hlob ntawm pathologies, thiab lwm yam. Cytofluorometry tuaj yeem cais cov subpopulations ntawm lub cev tiv thaiv kab mob hauv qhov sib xyaw ua ke thiab ntsuas tag nrho lawv cov kev hloov pauv raws sijhawm.
Oncology
Ib qho ntawm cov haujlwm tseem ceeb tshaj plaws hauv oncology yog kev sib txawv ntawm cov hlwb raws li lawv hom. Lub hauv paus ntsiab lus ntawm kev tsom xam los ntawm kev ntws cytometry hauv oncohematology yog ua raws li cov xwm txheej hauv qab no: thaum tus qauv raug kho nrog cov tshuaj fluorescent tshwj xeeb, nws khi rau cytoplasmic proteins. Tom qab kev faib tawm hauv cov hlwb nquag, nws cov ntsiab lus txo qis ib nrab. Raws li, qhov kev siv ntawm tes luminescence txo ob npaug.
Muaj lwm txoj hauv kev los kuaj xyuas cov cell proliferating:
- siv DNA-binding dyes (propidium iodide);
- siv daim ntawv lo uracil;
- sau npe ntawm qib nce ntawm kev qhia ntawm cyclin proteins, uas koom nrog kev tswj hwm ntawm lub voj voog ntawm tes.
