sted microscopy

suuppar resolution microscopy nirmmikkunna saankethikavidyakalilonna stimulated emission depletion (sted) microscopy. fluoroforukale thiranjeduttha nirjjeevamaakkunnathiloode ithu suuppar resolution chithrangal srishtikkukayum phokkal pooyintile prakaashatthinte vistheernnam kuraykkukayum angane oru nischitha systethinu nedaan kazhiyunna resolution vardhippikkukayum cheiyunnu. stephaan dablyu aanu ithu vikasippichath. 1994ll hell aandu jaann vichmaan, 1999ll hell aandu thomas clar ennivar aadyamaayi pareekshanaathmakamaayi pradharshippichu. hellinu athinte vikasanathinu 2014ll rasathanthrathinulla nobal sammanam labhichu. 1986ll vii.eiii. okhonin (instittyoott oof biophysics, yuu. ezz. ezz. aarr akkadami oof sciences, cyberian branch, crasnoyarsk) sted aashayathin pettant nediyirunnu. 1994ll eee pettant narakatthinum vichmaanum anjjaathamaayirunnu.
resolution vardhippikkunnathinaayi laitt microscopy defraction paridhi marikadakkaan aduthide vikasippichedutha niravadhi tharam suuppar resolution microscopy saankethikavidyakalil onnaanu sted microscopy. resolutionil mechappeduthal nedunnathinaayi biological saambilukal label cheyyaan saadhaaranayaayi upayogikkunna fluoroforukalude noonn-leanier prathikaranam chooshanam cheiyunna oru nirnnayaka pravartthana saankethikathayaanu sted, athaayath defraction paridhiyil thaazheyulla resolutionukalil chithrangal edukkaan sted anuvadikkunnu. foto activated localisation microscopy (paam), stokkaastik opttikkal reconstruction microscopy (stoom) thudangiya stokkaastik functional teknikkukalil ninnu ithu vyathyasthamaan, kaaranam eee reethikal ganithashaasthra maathrkakal upayogichu niravadhi settu defraction limittad emejukalil ninnu oru sub defraction paridhi punarnirmmikkunnu.
pashchaathalam.
parambaraagatha microscopy, prakaashatthinte vyathichalanathiloode nedaan kazhiyunna resolution parimithappedutthunnu. eee paridhi vivarikkan ernst abbe oru samavaakyam vikasippichu. samavaakyam ithaanu
ivide d ennathu vivaravyathyasa paridhi, λ ennathu prakaashatthinte tharamgadairkhyavum, Na ennathu samkhyaa appercher, allengil maadhyamathinte refractive soochikaye aapatana konile saine kondu gunikkunnu. n maathrkayude refractive soochikaye vivarikkunnu, α oru vasthuvinal prakasham shekharikkunna khara ardhakonam alakkunnu, λ ennathu maathrkaye uthejippikkaan upayogikkunna prakaashatthinte tharamgadairkhyamaanu, Na ennathu samkhyaa apercharaanu. uyarnna resolution nedunnathinu (i.iii. cheria dii moolyangal), hraswa tharamgadairkhyangal, uyarnna naa moolyangal (naa = enn cna) enniva opttimal aanu. ellah suuppar resolution reethikalum alakkunna maanadandamaanu eee vivarthana paridhi. sted fluorescencine thiranjedutthu nirjjeevamakkunnathinaal, parambaraagatha konfokkal microscopic pakaram mikacha resolution nedaan ithinu kazhiyum. oru electronine bhoosthiraavasthayil ninnu mattoru adisthaana oorjja nilayude aaveshabharithamaaya electronic avasthayilekku uthejippikkunnathiloodu saadhaarana fluorescence sambhavikkunnath (s0 ennathu s1 lake pookunnu), ithu bhoosthiraavasthayilekku (s1-nte) madangiyethiya sesham, s1-ll ninnu s0-ll vibrational oorjja nilayilekku veezhunnathiloode oru phottoon purappeduvikkunnu. phottoon purathuvarunnathinu munbu sted eee prakriya thadassappeduthunnu. aaveshabharithamaaya electron fluorescence parivarthanatthilekku praveshikkunnathinekkaal uyarnna vibration avasthayilekku vishramathilekku nirbandhitharaakunnu, ithu phottoon purathuvidunnathinu valathuvashathulla chithratthil kaanichirikkunnathupole chuvapp-shift cheyyaan kaaranamaakunnu. electron uyarnna vibrational avasthayilekku pookunnathinaal, rund avasthakaludeyum oorjja vyathyasam saadhaarana fluorescence vyathyaasathekkaal kuravaanu. oorjjam kurayunnathu tharamgadairkhyam uyarthukayum photonine spektrathinte chuvanna attathekku kooduthal neekkaan kaaranamaakukayum cheiyunnu. eee maattam rund tharam photonukale verthirikkukayum uthejippicha photonine avaganikkaan anuvadikkukayum cheiyunnu.
eee badal udvamanam sambhavikkaan nirbandhikkunnathinu, oru insidant phottoon fluorophoril adikkendathundu. ithu oru insident photoninaal adikkendathundu, stedinu rund prathyaaghaathangalundu. onnamathaayi, insidant photonukalude ennam eee udvamanathinte kaaryakshamathaye nerittu badhikkunnu, randaamathaayi, mathiyaya valia ennam photonukalude fluorescence upayogichu poornnamaayum adichamarthaan kazhiyum. fluorescence adichamarthaan aavashyamaaya valia ennam insident photonukal nedunnathinu, photonukal srishtikkaan upayogikkunna lazer uyarnna theevrathayullathaayirikkanam. nirbhagyavashaal, eee uyarnna theevrathayulla lazer fluorophore photobleaching prashnathilekku nayichekkaam. uyarnna theevrathayulla prakasham vazhi fluoroforukale nasippikkunnathinte peraanu photobleaching.
prakriya.
fluorescence purappeduvikkaan oru sentar phokkal spott sajeevamaayi avasheshippikkumbol saambilinte nirddishta pradeshangalil fluorescence kurayunnathiloode sted pravartthanangal nadathunnu. objective lensinte puple playininte savisheshathakalil maattam varuthunnathiloode eee phokkal eriya roopakalppana cheyyaan kazhiyum. eee defractive opttikkal moolakangalude ettavum saadhaaranamaaya aadyakaala udaaharanam, allengil, chuvade kaanichirikkunna dvimaana laattaral thadankalil upayogikkunna torres aakruthiyaanu. redu soonn illaathaakumbol green spott sajeevamaayi avasheshikkunnu. depletion laserinte vrithaakrithiyilulla dhruveekaranam, opttikkal vortexumaayi samyojippichaan eee doow srishtikkappedunnathu. eee doyude laattaral resolution saadhaaranayaayi 30 muthal 80 enm vareyaanu. ennirunnaalum, moolyam 2.4 enm aayi kuranjathaayi report cheythittundu. vyathyastha doses upayogichu, 100 enm kramatthil aksheeya resolution pradarshippichittundu. oru parishkaricha abeyude samavaakyam eee upavibhagam vivarana prameyathe ingane vivarikkunnu
soothravaakyam _ 2
soothravaakyam _ 3 maadhyamathinte refractive soochikayum soothravaakyam _ 4 intra kaavity theevrathayum soothravaakyam _ 5 saachureshan theevrathayumaanu. ivide formula _ 6 ennathu prayogicha (paramavadhi) sted theevrathayude anupaathavum saachureshan theevrathayum prakadippikkunna saachureshan gadakamaanu, formula _ 7.
stedinte falaprapti opttimais cheiyunnathinu, phokkal spottinte madhyabhaagathe vinashakaramaaya edapedal kazhiyunnathra thikanjathaayirikkanam. athu upayogikkaan kazhiyunna opttiksil chila niyanthranangal earppeduthunnu.
chayangal.
stedinte vikasanathinte thudakkathil, eee prakriyayil upayogikkaan kazhiyunna chaayangalude ennam valare parimithamaayirunnu. stedinte aadya saidhaanthika vivaranathilaanu rodamine bii enna peru nalkiyirikkunnath. thalphalamaayi, chuvanna spectrathil puranthallunna lazer aayirunnu aadyathe chayangal. jaiva samvidaanangalude sted vishakalanam anuvadikkunnathinu, chaayangalum lazer srothassukalum systethinu anusrithamaayi krameekarikkanam. eee samvidaanangalekkurichu mikacha vishakalanam nadathaanulla eee aagraham leaving sell sted, multikalar sted ennivayilekku nayichu, ennaal vardhicha pravarthanakshamathaye ulkkollunnathin kooduthal noothana chaayangalum eksaitteshan sisttangalum ithu aavasyappettittundu.
atharam oru purogathi rogaprathirodha label cheytha koshangalude vikasamaayirunnu. amide bondukaliloode aantibodikalumaayi bandhippichirikkunna sted fluorescent chaayangalaanu eee koshangal. eee saankethikavidyayude aadya upayogam oru chuvanna chayamaaya chayamaaya, oru dvitheeya aanti-mouse antibodiyumaayi chertthu. aaa aadya prayogam muthal, eee saankethikavidya pacha emitting, attoo 532, manja emitting, attoo 590, koodaathe adhika chuvapp emitting chayangal ennivayulppede valare vishalamaaya chayangalil prayogichu. koodaathe, rund niramulla sted nirmmikkaan eee reethiyilaan attoo attoo aadyamaayi upayogichathu.
aaplikkeshanukal.
kazhinja kure varshangalayi, sted sankeernavum valare nirddishtavumaaya oru saankethikavidyayil ninnu oru pothu fluorescence reethiyilekku vikasichu. thalphalamaayi, stedinte upayogakshamatha vipuleekarikkunnathinum kooduthal vivarangal nalkunnathinum niravadhi reethikal vikasippicheduthittundu.
gadanaaparamaaya vishakalanam.
prakriyayude thudakkam muthal, electron microscopy upayogichu maathram saadhyamaaya jolikal cheyyaan fluorescence microscopic sted anumathi nalkiyittundu. udaaharanathinu, upa-oorganail thalatthil protein ghadana vishakalanam vyakthamakkaan sted upayogichu. cytoskelital philamentukalude nireekshanamaanu eee thalatthilulla padanatthinte pothuvaaya thelivu. koodaathe, neurofilamentukal, aaktin, tubulin enniva palappozhum sted, konfokkal microscopukalude parihara shakti thaarathamyam cheyyaan upayogikkunnu.
sted upayogichu, membran fusione niyanthrikkunna manushya proteinaaya snap 25 parishodhikkumbol 70-90 enm laattaral resolution nediyittundu. snap 25 keniyude roopathinte pravarthanakshamathayil ninnu swathanthramaayi klasttarukal roopappeduthukayum klasttar cheytha vaakyaghadanayumaayi bandhippikkukayum cheiyunnuvennu eee nireekshanam theliyichittundu. mitocondria polulla sankeernamaaya avayavangalekkurichulla padanangal gadanaaparamaaya vishakalanathinayi sted microscopy ninnu prayojanam nedunnu. 50 enn. emm. ll thaazhe laattaral resolutionulla ishtaanusrithamaayi nirmmicha sted microscopukal upayogichu mitocondrial proteinukalaaya proteinukalaaya, toom20, vdac1 enniva nanoscale klasttarukalaayi vitharanam cheiyunnathaayi kandethi. mattoru patanam veettil nirmmicha sted microscopy, dna binding fluorescent dye enniva upayogichu, dna shakalangalude neelam konfokkal microscopy upayogichu parambaraagatha alavinekkaal valare krithyamaayi alakkunnu.
parasparabandhitha reethikal.
athinte pravartthanam kaaranam, sted microscopy palappozhum mattu uyarnna resolution reethikalodoppam upayogikkaam. electron, aattomik fozhsu microscopy ennivayude resolution sted resolutionekkaal mikachathaanu, pakshe aattomik fozhsu sted, shima muthalayavayumaayi samyojippikkunnathiloode. koshangalude kaadinyatthile mattangal nireekshikkumbol manushya andashaya cancer koshangalude aaktin cytoskelittan drishyavalkkarikkaan avarkk kazhinju.
multicolor.
proteinukalile ghadanayum pravartthanavum thammilulla aashrithathvam padikkaan sted upayogikkunnathile vardhichuvarunna prashnathinu marupadiyaayaanu multikalar sted vikasippichath. itharathilulla sankeernamaaya samvidaanathe kurichu padikkaan kuranjathu rund vyathyastha fluoroforukalenkilum upayogikkanam. rund fluorescent chaayangalum beam jodikalum upayogichu, synaptic, mitocondrial protein klasttarukalude collocalised imaging 5 enm vare resolution upayogichu saadhyamaan. synaptic vesicle proteinukalude vyathyastha poppuleshanukal escape synaptic bottanukalude misrithamallennu kaanikkan multikalar sted upayogichittundu. multy-life time imajingullah rund kalar sted upayogikkunnathiloode, three chaanal sted saadhyamaan.
live sell.
thudakkathil, jeevanulla koshangalumayi pravarthikkunnathinu upayogapradamaaya oru saankethikavidyayaayi sted kanakkaakkappettirunnu. nirbhagyavashaal, koshangale padikkaanulla eaka maargam plasma membranine jaiva chayangal upayogichu label cheyyuka ennathaayirunnu. sted fluorescence korileshan spectroscopy samyojippichu collustroll-mediated molecular complexukal sphingolippidukale keniyil peduthunnuvennu kaanichu, pakshe kshanikamaayi maathram. ennirunnaalum, fluorescent proteinukal maathrame oru jeevakoshathile eathenkilum oorgaanellukalo proteino drishyavalkkarikkaanulla kazhivu nalkunnulloo. sasthanikalude koshangal prakadippikkunna citrin-tubulininullil 50 enm laattaral resolutionil eee reethi pravarthikkunnuvennu kaanichu. sasthanikalude koshangalile gadanakal kandethunnathinupurame, sasyakoshangalude plasma membranile clustering vaii. eph. pii tagg cheytha pinn proteinukalude drishyavalkkaranathinum sted anuvadichittundu.
aduthide, pulsed phaar-redu lazer, clip eph-tagg, snap eph-tagg expression enniva upayogichu multikalar live-sell sted avatharippichu.
kedupaadukal illaatha mrugangalude thalachoril.
mouse courtexinte upariplavamaaya paalikal oru thalayottiyiloode aavarthichu chithreekarikkaan kazhiyum. ithu aazhchakalolam vyakthigatha dendritik nattellukalude vidhiyum aakruthiyum pinthudaraan anuvadikkunnu. rund niramulla sted upayogichu, jeevajaalangalile poost synaptic saandrathayude naano ghadana pariharikkanum kazhiyum.
viidiyoo nirakkukalilum athinappurathum nilayurappichu.
suuppar-resolution cheria pixelukal aavasyamaanu, athaayath oru saambilil ninnu kooduthal sthalangal neduka, ithu kooduthal ettedukkal samayathilekku nayikkunnu. ennirunnaalum, phokkal spottinte valuppam kshayathinaayi upayogikkunna laserinte theevrathayeyaanu aashrayichirikkunnath. thalphalamaayi, eee spott sais tyoon cheyyaan kazhiyum, valupavum imaging vegathayum maattunnu. oro nirddishta imaging jolic eee rund gadakangalkkidayil oru vittuveezhchayil ethaan kazhiyum. secondil 80 frameukalude nirakk rekhappeduthiyittundu, phokkal spottukal ekadesham 60 enm aanu. cheria feeld kaazhchaykkaayi secondil 200 frameukal vare athicheraam.
prasnangal.
eksaitteshanil ninnu athilum uyarnna excited avasthayilekko allengil trippilt avasthayile eksaitteshanil ninno photobleaching sambhavikkam. oru uttejita electron mattoru uyarnna uthejithaavasthayilekku aaveshabharithamaakunnathu thadayunnathinu, badal udvamanam uthejippikkunnathinu aavashyamaaya photoninte oorjjam aaveshabharithamaaya oru avasthayil ninnu mattonnilekku aaveshathinte oorjjathe ovarlaappu cheyyaruthu. fluoroforukalumaayi sanbarkkam pulartthunna oro lazer photone uthejippicha udvamanathinu kaaranamakumennum electron mattoru uyarnna oorjjaavasthayilekku aaveshabharithamaakillennum ithu urappakkum. triple avasthakal single avasthakalekkaal valare kooduthal kaalam nilanilkkunnu, triple avasthakal aaveshakaramaakaathirikkaan, lazer pulsukal thammilulla samayam mattoru shamana reethiyiloode electronine vishramikkaan anuvadikkunnathinu mathiyaya dairkhyamullathaayirikkanam, allengil triple avasthaye samippikkaan oru raasa samyuktham cherkkanam.

