suuppar resolution microscopy

prakaashatthinte vyathichalanathaal defraction paridhi chumathunnathinekkaal uyarnna resolutionukal atharam chithrangalkk anuvadikkunna opttikkal microscopy saankethikavidyakalude oru parambarayaanu suuppar-resolution microscopy. suuppar-resolution imaging teknikkukal niyar-feeldineo (phottoon-tunneling microscopy, pendry superlance niyar feeld scanning opttikkal microscopy upayogikkunnava) allengil vidoora feeldineo aasrayikkunnu. clossed pinholulla konfokkal microscopy allengil deconvalution allengil dittaktar adhishtitha pixel resainment (udha.jii. ree-scan microscopy, pixel ree assainment), samyojichu microscope, siim, smi polulla structured-illumination microscopy saankethikavidyakal.
vidoora feeldil suuppar-resolution microscopic rund pradhana grouppukal undu, avaykku valare valia gadakathiloode resolution mechappeduthaan kazhiyum
2014 october october rasathanthrathinulla nobal sammanam eric bettsig, dablyu.iii. nanodimensionilekku opttikkal microscopy" konduvarunna "suuppar-resolution fluorescence microscopy" yude vikasanathinaayi morner stephaan narakavum. suuppar-resolution microscopy vyathyastha reethikal biomedical research community kooduthalaayi sweekarikkunnu, eee saankethikavidyakal tanmatra thalatthil jaiva pravartthanam manasilakkunnathinulla anivaryamaaya upakaranangalayi maarukayaan.
charithram.
1978 aayappozhekkum, abeh paridhi thakarkkunnathinulla aadyathe saidhaanthika ashayangal vikasippicheduthu, ithu oru samyojichu microscope oru konfokkal lazer-scanning fluorescence microscopy upayogikkendathundu, avide prakasham ellah vashathuninnum oru pothu focusilake kendreekarikkunnu, athu 'pooyint-bye-pooyint' eksaitteshanum 'pooyint-bye-pooyint' kandethalum chernnu vasthu scan cheyyaan upayogikkunnu.
ennirunnaalum 1978 muthalulla prasidheekaranam anuchithamaya bhauthika nigamanathiletthiyirunnu (athaayath.iii. prakaashatthinte oru pooyint poleyulla sthaanam), kharakonile maruvashathe cherkkunnathinte yadhaartha nettamaayi aksheeya resolution vardhana poornnamaayum nashtappettu.
sub-defraction microscopy teknikkukalekkurichulla oru kemistry bloginte avalokanathil ninnu (anumathiyode) inipparayunna chila vivarangal shekharichu.
1986ll uthejippicha udvamanathe adisthaanamaakkiyulla oru suuppar resolution opttikkal microscopinu okhonin pettant nedi.
suuppar resolution teknikkukal.
niyar feeld opttikkal random mapping (norm) microscopy.
oru immersion draavakathil naanoparttikkilukale brouniyan chalanam nireekshikkunnathiloode oru vidoora feeld microscope vazhi opttikkal niyar feeld ettedukkunna oru reethiyaanu niyar-feeld opttikkal random mapping (norm) microscopy.
yaadruchikamaayi neengunna naanopaarttikkilukaliloo vasthuvinte uparithala scanning upayogikkunnu. sookshumadarshiniyiloode, naanoparttikkilukal samamithamaaya vrithaakrithiyilulla paadukal pole kaanapedunnu. spott veethi pooyint spread functionu (~ 250 enm) thulyamaanu, ithu microscope resolution nirvachikkunnu. thannirikkunna kanikayude parshwa kordinettukal microscopinte resolutionekkaal valare uyarnna krithyathayode vilayiruthaan kazhiyum. niravadhi frameukalil ninnu vivarangal shekharikkunnathiloode microscopinte muzhuvan kazhcha mekhalayiludaneelamulla feeld theevrathayude vitharanam mapp cheyyaan kazhiyum. ensome, ansome ennivayumaayi thaarathamyappeduthumbol eee reethikk tipp positioning cheiyunnathinu prathyeka upakaranangalonnum aavasyamilla, koodaathe valia kazhchappadum aazhathilulla focus undu. valia ennam scanning "censorukal" ullathinaal oraalkk churungiya samayathinullil imej ettedukkal nedaan kazhiyum.
samyojichu.
mechappetta axial resolutionulla lazer scanning fluorescence microscopanu samyojichu microscope. 500-700 nm-nte saadhaarana moolyam 100-150 nm aayi mechappeduthaam, ithu saadhaarana konfokkal microscopic 5 muthal 7 madangu vare kuranja voliyamulla eathaandu golaakrithiyilulla phokkal spottumaayi poruthappedunnu.
ore jyaamitheeya sthaanathekku focus cheytha rund ethir objective lensukal upayogichaanu resolutionile mechappeduthal kaivarikkunnath. koodaathe, rund objective lensukaliloodeyum opttikkal paathayude neelatthile vyathyasam shradhaapoorvvam kuraykkunnu. ithiloode, rund lakshyangaludeyum pothuvaaya kendra pradeshathu vasikkunna thanmaathrakale iruvashathuninnum yojicha reethiyil prakaashippikkaanum prathifalippikkunnatho purappeduvikkunnatho aaya prakasham yojicha reethiyil shekharikkaanum kazhiyum.iii. dittaktaril puranthallappedunna prakaashatthinte yojicha superposition saadhyamaan. prakaashatthinum kandethalinum upayogikkunna soolid angle formula _ 1 vardhippikkukayum anuyojyamaaya kesilekk adukkukayum cheiyunnu, avide sample oresamayam prakashikkukayum ellah vashathuninnum kandethukayum cheiyunnu.
ithuvare, sthirakoshangalile sted microscopy, jeevakoshangalile maattaavunna proteinukalulla resolft microscopy ennivayumaayi samyojichu samyojichu microscopyle ettavum mikacha gunanilavaarathiletthiyittundu.
structured illumination microscopy (siim).
structured illumination microscopy (siim) nireekshikkaavunna pradeshathinu purathulla freekwansi spacil ninnu vivarangal shekharichukondu speshyal resolution vardhippikkunnu. eee prakriya cheiyunnathu reciprocal speysilaanu oru sii imejinte forrier transformil (eph. tii) reciprocal spacinte vividha mekhalakalil ninnulla superimposed adhika vivarangal adangiyirikkunnu; prakasham chila ghattangaliloode mattunna niravadhi frameukal ullathinaal, kooduthal resolution vivarangalulla eph. tii imej computationalaayi verthirikkaanum punarnirmmikkaanum kazhiyum. reverse adii punarnirmmicha chithram suuppar resolution chithrathilekku thirike nalkunnu.
chila medical roganirnayathinulla oru upakaranamaayi electron microscopic pakaram siim microscopic kazhiyum. vrukkasambandhamaaya asukhangal, vrukka arbudam, raktharogangal enniva kandethunnathu ithil ulppedunnu.
structured illumination microscopy" enna padam pilkkala varshangalil mattullavar upayogichirunnuvenkilum, gyoora (1995) aadyamaayi prasidheekaricha phalangalil 50 enm pitch grating upayogichu roopakalppana cheytha prakasham 50 enm pichinte randaamathe grettingine prakaashippichu, graattingukal parasparam aavarthikkukayum magnification nedunnathinu aavashyamaaya koneeya alavu upayogichu karakkukayum cheythu. prakasha tharamgadairkhyam 650 enm aayirunnengilum 50 enm grating eluppathil pariharichu. ithu 232 enm enna abeh resolution paridhiyekkaal ekadesham 5 madangu mechappeduthal kaanichu, ithu upayogicha samkhyaa apperturinum tharamgadairkhyathinum labhicha ettavum cheriyathaanu. eee kruthiyude kooduthal vikasanathil, ivaanasent feeld ghattam ghattamayi maattunnathiloode suuppar-resolution laattaral topography kaivarikkamennu gyoora kaanichu. niravadhi yuu.ezz. gueraic vyakthigathamaayo sahapravarthakarudeyo pettantukal nalkukayum polaroid korppareshanu nalkukayum cheythu. dier energy systems, calimetrics incorporation aanu eee saankethikavidyaykkulla licensukal nediyathu., koodaathe nanopec corp. opttikkal daatta sambharanathilum microscopy eee suuppar resolution saankethikavidya upayogikkunnathinaayi.
spatially modulated illumination (smi).
gadanaaparamaaya prakaashatthinte oru nadappaakkal spatially modulated illumination (smi) ennariyappedunnu. staanderd structured illumination pole, smi technic oru microscopinte pooyint spread function (psf) uchithamaaya reethiyil parishkkarikkunnu. ennirunnaalum, eee saahacharyathil, "opttikkal resolution thanne mechappeduthiyittilla"; pakaram fluorescent vasthukkalude dhoora alavukalude krithyatha paramaavadhiyaakkaan gadanaaparamaaya prakasham upayogikkunnu, "kurachu pathinaayirakkanakkin naanometerukalude tanmatra alavukalil valippam alakkunnathu prapthamaakkunnu".
vertico smi microscope achuthandil onno rando ethir interferring lazer beemkal upayogichu gadanaaparamaaya prakasham kaivarikkunnu. imej cheiyunna vasthu taranga mandalatthiloode uyarnna krithyathayulla ghattangaliloode neekkunnu, allengil taranga mandalam thanne ghattam shifttukaliloode vasthuvumayi thaarathamyappeduthumbol neekkunnu. ithu mechappetta akshaakrithiyilulla valippathinum dooraprathisandhikkum kaaranamaakunnu.
smi mattu suuppar resolution saankethikavidyakalumaayi samyojippikkaam, udaaharanathinu cheiyunnu limoon allengil els-tirfumaayi paarshwasthamaaya gadanayulla prakaashathodukoodiya motham aantharika prathiphalana interferometeraayi (eee avasana upakaranavum saankethikavidyayum adisthaanaparamaayi oru ghattam-marria phottoon tunneling microscopanu, ithu ghattam-marria ivaanasent feeldullah (gyoora, 1996) motham aantharika prathiphalana laitt microscope upayogikkunnu). munbu samaanathakalillaatha opttikkal resolutionilulla manushya nethrakoshangalil ninnulla vibhagangalil autofluorophore vitharanagalude laitt-opttikkal chithrangal nedaan eee smi saankethikatha oraale anuvadikkunnu. moonnu vyathyastha eksaitteshan tharamgadairkhyangalude (488,568,647 enm) upayogam autofluorescence signalinekkurichulla spectral vivarangal shekharikkaan oraale prapthamaakkunnu. macular degeneration baadhicha manushyante nethrakoshangal parisodhikkaan ithu upayogichittundu.
nirnnayakamaaya pravarthanaparamaaya saankethikavidyakal.
parambaraagathamo konfokkal microscopy upayogichu chithreekarikkaan kazhiyaatha saambilukalile vishadaamshangal chithreekarikkaan kazhiyunna valare uyarnna resolutionulla oru opttikkal microscopy reversible saturable opttikkal fluorescence traansishanukal (resolft) microscopy. resulftinullil sted microscopy, gsd microscopy ennivayude thathwangal saamaanyavalkkarikkappedunu. koodaathe, resolft allengil siim ozhikeyulla mattu ashayangalulla saankethikavidyakalum undu. udaaharanathinu, naitrajan-shoonyatha kendratthinte opttikkal, gettu property upayogikkunna fluorescence microscopy, allengil taapa vikiranathinte uthejippicha emission (settar) vazhi suuppar resolution, ithu black body vikiranathinte aantharika suuppar-linerykal upayogikkukayum microscopic appurathu suuppar-resolution enna ashayam vikasippikkukayum cheiyunnu.
uthejippicha malineekarana kuravu (ezz. tii. iii. dii).
stimulated emission depletion microscopy (ezz. tii. iii. dii) rund lazer pulsukal upayogikkunnu, fluoroforukale avayude fluorescent avasthayilekku uthejippikkunnathinu eksaitteshan pulse, stimulated emission vazhi fluoroforukalude dii-eksaitteshanaayi ezz. tii. iii pulse. praayogikamaayi, eksaitteshan lazer pulse aadyam prayogikkunnu, athinusesham oru sted pulse udan varum (thudarchayaaya taranga laserukal upayogichu palsukalillaathe sted upayogikkunnu). koodaathe, eksaitteshan phokkal spottumaayi poruthappedunna seero-intensity spott avatharippikkunna vidhathilaan sted pulse parishkkarikkunnath. sted beam theevrathaye uthejippicha udvamana nirakkinte noonn-leanier aashrithathvam kaaranam, phokkal eksaitteshan spottinu chuttumulla ellah fluoroforukalum avayude oof avasthayilaayirikkum (fluoroforukalude graundu stettu). eee phokkal spott scan cheiyunnathiloode oraal chithram veendedukkunnu. eksaitteshan phokkal spottinte pooyint spread function (pii. ezz. eph) nte paramavadhi pakuthi (eph. dablyu. echu. emm) yill poornna veethi saidhaanthikamaayi sted pulsinte theevratha uyarthunnathiloode ekapaksheeyamaaya veethiyilekku compress cheyyaan kazhiyum.
vyapakamaaya upayogam thadanja stedinte pradhana poraayma yanthrangal sankeernamaanu ennathaanu. oru vashath, oru chithram veendedukkunnathinu sample scan cheyyendathinte aavasyakatha kaaranam valia feeld kaazhchakalkk imej ettedukkal vegatha thaarathamyena mandagathiyilaanu. maruvashathu, cheria feeld kaazhchakalkk ithu valare vegathilaakaam secondil 80 frameukal vare recordingukal kaanichittundu. stedumaayi bandhapetta oru valia "ees" moolyam kaaranam, uyarnna theevrathayulla eksaitteshan pulsinte aavasyamundu, ithu saambilinu kedupaadukal varuthiyekkaam.
graundu stettu depletion (gsd).
graundu stettu depletion microscopy (gsd microscopy) oru fluoroforinte triple avasthaye oof-stateayum single stettu oonn-stateayum upayogikkunnu, athil single stettu thanmaathrayude paridiyilulla fluoroforukale trippilt avasthayilekku nayikkaan oru eksaitteshan lazer upayogikkunnu. ithu sted poleyaanu, avide oof-stettu fluoroforukalude graundu stettu aanu, athinaalaan eee kesil samavaakyam () baadhakamaakunnath. formula _ 3 moolyam stedinekkaal cheruthaanu, ithu valare cheria lazer theevrathayil suuppar resolution imaging saadhyamaakkunnu. stedumaayi thaarathamyappeduthumbol, gsdyil upayogikkunna fluoroforukal saadhaaranayaayi photostable kuravaanu; trippilt avasthayude saachureshan thirichariyaan budhimuttaayirikkum.
pooritha gadanaaparamaaya prakasha microscopy (ezz. ezz. aii. emm).
pooritha structured-illumination microscopy (ezz. ezz. aii. emm) eksaitteshan laserinte theevrathayil fluoroforukalude udvamana nirakkinte nonleeniyar aashrithathvam chooshanam cheiyunnu. fluoroforukale avayude fluorescent avasthayil poorithamaakkunnathinu aavashyamaaya theevrathayodu aduthulla oru sinusoidal prakasha paatten prayogikkunnathiloode, oraal moyer arikukal veendedukkunnu. computational teknikkukal upayogichu verthirichedukkaan kazhiyunna uyarnna oordar speshyal vivarangal arikukalil adangiyirikkunnu. vivarangal verthiricheduthukazhinje, oru suuppar resolution chithram veendedukkunnu.
ezz. sii. aii. emm.-nuu prakasha paatten onniladhikam thavana maattendathundu, ithu saankethikavidyayude thaalkkaalika resolution phalapradamaayi parimithappedutthunnu. koodaathe, saambilinu vikirana kedupaadukal varuthukayum siim upayogikkaavunna saadhyamaaya prayogangal niyanthrikkukayum cheiyunna saturating avasthakal kaaranam valare photostable fluoroforukalude aavasyamundu.
eee microscopy udaaharanagal sekshan structured illumination microscopy (siim) yill kaanichirikkunnu sell nucleusinteyum nucleusinteyum microscopy upayogichu record cheytha maittotik ghattangaludeyum chithrangal.
yaadruchikamaaya pravartthana saankethikavidyakal.
praadeshikavalkkarana microscopy.
emittarukale verthirikkukayum avayude chithrangal pooyint spread function (pii. ezz. eph) upayogichu gadippikkukayum cheythukondu suuppar resolution nedunna ellah microscopy saankethikavidyakalum single molecule localisation microscopy (ezz. emm. ell. emm) samgrahikkunnu. saadhaaranayaayi, pooyint spread functionte veethi (~ 250 enm) resolution parimithappedutthunnu. ennirunnaalum, oru otappetta emittar nalkiyaal, oraalkk () enna samavaakyam anusarichu athinte theevratha parimithappedutthiyaal maathrame athinte sthaanam krithyathayode nirnnayikkaan kazhiyoo.
eee phitting prakriya otappetta emittarukalkku maathrame vishwasaneeyamaayi nadatthaan kazhiyoo (deconvalution kaanuka), rasakaramaaya jaiva saambilukal emittarukal upayogichu valare saandramaayi label cheythirikkunnathinaal ellah emittarukalum ore samayam sajeevamaakumbol phitting asaadyamaanu. ore samayam oru viralamaaya emittarukal maathram sajeevamaakkukayum eee kurachu emittarukale valare krithyamaayi praadeshikavalkkarikkum avaa nirjjeevamaakkukayum mattoru upasett sajeevamaakkukayum cheythukondu smlm saankethikavidyakal eee ashayakkuzhappam pariharikkunnu.
pashchaathalavum camera pixalation pariganikkukayum oru saadhaarana microscopinte pooyint spread functionaayi (eyari disk) gausiyan ekadesha kanakk upayogikkukayum cheiyunna saidhaanthika resolution thompson mattullavarum nirddeshikkunnu. mortenson thudangiyavar nannayi tyoon cheythu.:
saadhaaranayaayi, fluoroforukal upayogichaanu localisation microscopy nadathunnathu. anuyojyamaaya fluoroforukal (udha.jii. kodunkaattine sambandhichidatholam) mikka samayathum fluorescent allatha irunda avasthayil vasikkukayum saadhaaranayaayi kuranja theevrathayilulla eksaitteshan lazer upayogichu yaadruchikamaayi sajeevamaakkukayum cheiyunnu. oru reedt lazer fluorescencine uthejippikkukayum bleachukal allengil photosvichukal fluoroforukale oru irunda avasthayilekku thirike konduvarikayum cheiyunnu, saadhaaranayaayi 10-100 emssinullil. nanoscale topography (peyint) imajinginaayi pooyintukal shekharikkunnathil, fluoroforukal bandhippikkunnathin munbu noonn fluoresentum athinusesham fluoresentum aanu. fluorescent ghattathil purathuvidunna photonukal oru camera upayogichu shekharikkukayum falamaayundaakunna fluoroforinte chithram (pii. ezz. eph. valachodikkunnu) kurachu angstrom kramatthil polum valare uyarnna krithyathayode gadippikkaan kazhiyum. eee prakriya aayirakkanakkin thavana aavarthikkunnath ellah fluoroforukalkkum thilakkamulla avasthayiloode kadannupokaanum record cheyyaanum kazhiyumennu urappaakkunnu. oru computer suuppar-resolution chithram punarnirmmikkunnu.
eee reethikalkku upayogikkunna fluoroforukalude abhikaamyamaaya swabhaavasavisheshathakal, resolution paramavadhi vardhippikkunnathinu, avaa thilakkamullathayirikkanam ennathaanu. athaayath, avaykku uyarnna vamshanasha gunakavum uyarnna alavil vilavum undaayirikkanam. avaykku uyarnna kontraast anupaathavum undaayirikkanam (prakaashaavasthayil purappeduvikkunna photonukalude ennavum irunda avasthayil purappeduvikkunna photonukalude ennavum thammilulla anupatham). koodaathe, naikvist maanadandamanusarichu idathoornna label cheytha sample abhikaamyamaanu.
praadeshikavalkkarana microscopy reethikalude ennam kooduthalum upayogikkunna fluoroforukalude tharatthil vyathyaasappettirikkunnu.
spectral presition disttans microscopy (ezz. pii. dii. emm).
oru microscopinte resolutionekkaal valare mikacha reethiyil orotta, cheria prakasha srothass kandethaan kazhiyum prakasham oru mangiya spott srishtikkumenkilum, microscopinte pooyint spread function, dittaktarinte shabda savisheshathakal muthalaayava kanakkileduthu mangiya spottinte kendram krithyamaayi kanakkakkaan computer algorithangal upayogikkaam. ennirunnaalum, parasparam valareyadhikam srothassukal ullappol eee sameepanam pravartthikkilla uravidangal ellaam orumichu mangunnu.
oro srothassukalum mattullavaril ninnu "optikkalaayi otappetta" aayirikkunnathinayi oru samayat kurachu srothassukal maathram alakkunnathiloode niravadhi srothassukal ulla prashnathe marikadakkunna fluorescence microscopy praadeshikavalkkarana saankethikavidyakalude oru kudumbamaanu spectral presition disttans microscopy (ezz. pii. dii. emm).iii.sookshumadarshiniyude resolutionekkaal kooduthal, saadhaaranayaayi ~ 200-250 nm) kondu verthirikkappedunnu, parisodhanayilulla kanangalkk vyathyastha spectral signecharukalundenkil, uchithamaaya prakasha srothassukalum filterukalum upayogichu oru samayam kurachu thanmaathrakalil ninnu prakasham nokkaan kazhiyum. phalapradamaaya pooyint imej functionte pradhana paramavadhi pakuthiyolam veethiyil prathinidheekarikkunna parambaraagatha opttikkal resolutionekkaal niravadhi madangu mikacha phalapradamaaya opttikkal resolution ithu kaivarikkunnu.
vyathyastha spectral swabhaavasavisheshathakalulla ("topological resolution") rund panchiphom kanikakal thammilulla alakkaavunna ettavum cheria dooratthinte adisthaanathil ezz. pii. dii. emm upayogichu kaivarikkaan kazhiyunna gadanaaparamaaya resolution prakadippikkaan kazhiyum. praadeshikavalkkaranam, kanika saandrata muthalaayavayude krithyathayekkurichu anuyojyamaaya saahacharyangalil modeling theliyichittundu.topological resolution" oru "space freekwansi" yumayi poruthappedunnu, athu classical nirvachanatthinte adisthaanathil, valare mechappetta opttikkal resolution thulyamaanu. fluorescent ayussum mattu saankethikavidyakalum adisthaanamaakki thanmaathrakale kooduthal sookshumamaaya reethikalil verthirichariyaan kazhiyum.
oru pradhana prayogam genome gaveshanathilaanu (genome pravarthanaparamaaya samghadanayekkurichulla patanam). upayogathinte mattoru pradhana mekhala membranukalude gadanayekkurichulla gaveshanamaanu.
spdmphymod.
chila foto-physical avasthakal undengil jii. eph. pii, alexa dye, fluorescine moleculukal thudangiya niravadhi staanderd fluorescent daikalkkulla praadeshikavalkkarana microscopy saadhyamaan. physically modifiable fluoroforukal (spdmphymod) saankethikavidya ennu vilikkappedunna eee saankethikavidya upayogichu, prathyeka foto-switch cheyyaavunna/foto-aaktivettabil fluorescence thanmathrakal upayogikkumbol rund lazer tharamgadairkhyangal aavasyamulla mattu praadeshikavalkkarana microscopy saankethikavidyakalil ninnu vyathyasthamayi anuyojyamaaya theevrathayude orotta lazer tharamgadairkhyam naano imajinginu mathiyakum. pukayila mosaik vairas (tmv) kanangalekkurichulla vishakalanam allengil vairas-sell edapedalinekkurichulla padanamaanu ezz. pii. dii. emm. fimode upayogathinte mattoru udaaharanam.
single-trippilt avastha parivartthanangale adisthaanamaakki eee prakriya thudarukayum orotta thanmatra valare deerghakaalam jeevikkunna reversible dark stettilekku (niravadhi secondukal vare ardhaayussulla) aadyam varunnathilekku nayikkukayum cheiyunna eee prakriya sambandhichidatholam spdmphymod-nuu nirnaayakamaan, athil ninnu athu valare deerghakaalam jeevikkunna, irrervirsible dark stettilekku madangunnathinumumbu niravadhi millie secondukalkku niravadhi photonukal purappeduvikkunna oru fluorescent avasthayilekku madangunnu. spdmfimode microscopy ore spectral prakasha aavruthi purappeduvikkunna fluorescent thanmathrakal upayogikkunnu, pakshe minnunna swabhaavasavisheshathakale adisthaanamaakki vyathyastha spectral signecharukalundu. ore sellinte randaayiram chithrangal samyojippikkunnathiloode, lazer opttikkal krithyatha alavukal upayogichu, ganyamaayi mechappetta opttikkal resolutionodukoodiya praadeshikavalkkarana chithrangal record cheyyaan kazhiyum.
jii. eph. pii, aarr. eph. pii, vaii. eph. pii, alexa 488, alexa 568, alexa 647, 405, allengil, aatto 488, fluorescen ennivayaanu ithinakam thanne ezz. pii. dii. emm. fimode saankethikavidyayil vijayakaramaayi upayogichirikkunna saadhaarana fluorescent chayangal.
3D (thanutha) yill cryogenic opttikkal localisation.
cheiyunnu (colled) yile cryogenic opttikkal localisation ennathu angstrom skeyil resolutionodukoodiya cheruthum idatharavumaaya orotta biomoleculinullil onniladhikam fluorescent sittukal praadeshikavalkkarikkaan anuvadikkunna oru reethiyaanu. kuranja thapanilayil mandagathiyilulla photochemistry photobleachinginu munbu oro fluorophoril ninnum puranthallaan kazhiyunna uyarnna ennam photonukalilekku nayikkunnathinaal eee sameepanathile praadeshikavalkkarana krithyatha vardhippikkunnu. thalphalamaayi, cryogenic stokkaastik localisation microscopy oru cheria proteinil gadippichirikkunna niravadhi fluoroforukalude 3D sthaanangal pariharikkunnathin aavashyamaaya sub molecular resolution kaivarikkunnu. electron microscopy ninnu ariyappedunna algorithangal upayogikkunnathiloode, fluoroforukalude 2 dii projectionukal oru 3 dii konfigareshanaayi punarnirmmikkunnu. labelinte valuppathe aashrayich jaladosham fluorescence microscopy athinte adisthaana paridhiyilekku konduvarunnu. ex-reey christalography, magnetic resonance spectroscopy, electron microscopy thudangiya mattu gadanaaparamaaya jeevashaasthra saankethikavidyakalumaayi samyojippichu moolyavathaaya pooraka vivarangalum prathyekathayum nalkaanum eee reethi upayogikkaam.
binding-activated localisation microscopy (baam).
single molecule localisation microscopy (smlm) ykkulla oru pothu aashayamaanu binding-activated localisation microscopy (baam): dnaud oru daiyudeyum gunangal parishkkarikkunnathine adisthaanamaakki dna-binding daikalude suuppar-resolution imaging. raasa paristhithiyude shradhaapoorvvam krameekarikkunnathiloode-fluorescence signalil praadeshikavum reversible dna urukunnathilekkum sankaravalkkarana niyanthranathilekkum nayikkunnathiloode-dna-binding dye thanmathrakal avatharippikkan kazhiyum. oru samayat kurachu dna-binding dye signalukal maathram rajisttar cheyyaanum optikkalaayi verthirikkaanum intercalating, minor-grove binding dna daikal upayogikkaam. ekadesham 50 enm pratheekshicha gadanaaparamaaya resolutionodukoodiya aanava vaasthuvidyayile nanoscale vyathyaasangalkk dna gadanayile ettakkurachilukal-assisted baam (efbalm) upayogichittundu. dna gadanayile ettakkurachilukale adisthaanamaakki binding-activated localisation microscopy upayogichu kromaattin nanostructure imaging cheyyuka. aduthide, oru amyloyidumaayi bandhippikkumbol niyad-4-nte fluorescence kwaandam vilavu ganyamaayi vardhippichathu amyloid fibrilukaludeyum oligomarukaludeyum baam imajinginaayi upayogichu.
kodunkaattu, eenthappana, eph. paalm.
stokkaastik opttikkal reconstruction microscopy (stoom), foto activated localisation microscopy (paam), fluorescence foto-aaktiveshan localisation microscopy (eph. pii. ell. emm) enniva suuppar resolution imaging teknikkukalaanu, avaa thudarchayaaya aaktiveshanum photoswichable fluoroforukalude samaya-pariharikkapetta praadeshikavalkkaranavum upayogichu uyarnna resolution chithrangal srishtikkunnu. imaging samayat, oru prathyeka fluoroforinte eaka-tanmatra chithrangalude centroid sthaanangal kandethunnathiloode oro fluoroforinteyum sthaanam uyarnna krithyathayode nirnnayikkaan kazhiyunna vidhathil, eathu nimishavum fluoroforukalude optikkalaayi pariharikkaavunna oru upavibhagam maathrame fluoroforukal sajeevamaakoo. fluoroforukalude oru upavibhagam pinneedu nirjjeevamaakkukayum mattoru upavibhagam sajeevamaakkukayum imej cheyyukayum cheiyunnu. eee prakriyayude aavarthanam niravadhi fluoroforukale praadeshikavalkkarikkaanum imej daattayil ninnu oru suuppar resolution chithram nirmmikkanum anuvadikkunnu.
eee moonnu reethikalum churungiya samayathinullil swathanthramaayi prasidheekarikkappettu, avayude thathwangal samaanamaan. nuclic aasidukalumaayo proteinukalumaayo gadippichirikkunna roopappeduthunnathiloode, allengil chayangal upayogichaanu kodunkaattine aadyam vivarichirunnathu, athesamayam eentappanayum efpalm photoswichable fluorescent proteinukal upayogichaanu vivarichirunnathu. thathwathil photoswichable fluorophore upayogikkaan kazhiyum, koodaathe vividha probukalum labeling thanthrangalum upayogichu kodunkaattu pradarshippichittundu. roopappeduthunnathiloode polulla single fluoroforukalude yaadruchika photoswiching upayogichu, orotta chuvanna lazer eksaitteshan uravidam upayogichu kodunkaattu nadatthaam. chuvanna lazer randum oru adakt roopappeduthunnathiloode roopappeduthunnathiloode fluoroforine irunda avasthayilekku maattukayum pinneedu thanmatra fluorescent avasthayilekku thirike nalkukayum cheiyunnu. mattu niravadhi chaayangalum kodunkaattinoppam upayogichittundu.
single fluoroforukalkku purame, oru aaktivettar fluorophore (alexa 405, 405, allengil allengil polullava), photoswichable reporter dye dye, alexa 647, cy5.5, allengil allengil polullava) enniva ulkkollunna dye-jodikal kodunkaattinoppam upayogikkaam. eee padhathiyil, aaktivettar fluorophore, athinte agiranam paramavadhi aduthu aaveshabharithamaakumbol, photoswichable dye fluorescent avasthayilekku veendum sajeevamakkaan sahayikkunnu. upayogikkunna aaktivettar fluoroforine aashrayich dye-jodikale verthirichariyaan vyathyastha aaktiveshan tharamgadairkhyangal upayogicho allengil aaktivettar fluoroforukalo allaatheyo spectrally vyathirikthamaya photoswichable fluoroforukal upayogicho multikalar imaging nadathiyittundu. photoswichable fluorescent proteinukalum upayogikkaam. antibody staining, proteinukalude nerittulla samyojanam, janithaka encoding enniva upayogichu photoswichable probukal upayogichu jaiva gadanakalude valare nirddishta labeling nediyittundu.
opttikkal aastigmattisam upayogichu trimana imajingilekkum kodunkaattu vyaapippichittund, athil pooyint spread functionte deerghavrithaakrithiyilulla aakruthi niravadhi micrometer kattiyulla saambilukalkkaayi x, y, z sthaanangale encode cheiyunnu, koodaathe jeevanulla koshangalil ithu pradarshippichittundu. innuvare, eee saankethikavidya kaivaricha speshyal resolution laattaral alavukalil ~ 20 enm umm axial alavukalil ~ 50 enm umm aanu; koodaathe temporal resolution 0.1-0.33s pole vegathayullathaanu.
nanoscale topography (peyint) imajinginaayi pooyintukal shekharikkuka.
nanoscale topography (peyint) imajinginulla pooyintukal shekharikkunnathu molecular adasorption/obsorption, photobleaching/desorption ennivayiloode stokkaastik single molecule fluorescence kaivarikkunna oru molecule localisation reethiyaanu. nail redu aayirunnu aadyam upayogicha chayam, ithu jaleeya laayaniyil fluorescent allaathathum ennaal mysellukal allengil jeevanulla koshabhithikal polulla hydrophobic parithasthithiyil cherkkumbol fluorescent aanu. athinaal, nanamolar thalatthil chaayatthinte saandrata cheruthaakkunnu, angane defraction-limittad eriyayilekkulla thanmaathrayude sorption nirakk millisecond mekhalayilaanu. nischalamaaya oru lakshyathilekku single-dye thanmaathrakalude (probukal) yaadruchikamaaya bandhanam oru saadhaarana widefield fluorescence microscopinu keezhil praadesikamaayum thaalkkaalikamaayum pariharikkanaakum. feeld irunda avasthayilekku thirike konduvarunnathinu oro chayavum photobleach cheiyunnu, athinaal adutha chayam bandhippikkanum nireekshikkaanum kazhiyum. mattu yaadruchika reethikalumaayi thaarathamyappeduthumbol eee reethiyude prayojanam, nischitha lakshyathinte suuppar-resolution imej nedunnathinupurame, defusing probe moleculukalude dynamic binding kainattiks alakkaan kazhiyum ennathaanu.
cheiyunnu suuppar resolution saankethikavidya samyojippikkuka (udha.jii. eratta-helix pooyint spread function mornerude groupil vikasikkunnu), foto-activated daikal, power-dependent active intermittansi, nanoscale topography imajinginaayi pooyintukal shekharikkal, spripaint (spry = power-dependent active intermittansi vazhi suuppar resolution) ennivaykku live-sell mathilukal suuppar-resolute cheyyaan kazhiyum. dye agiranam/agiranam, photobleaching/desorption nirakkukal enniva thammilulla santhulithaavastha nilanirthunnathiloodeyaanu peyint pravartthikkunnathu. sthithivivarakkanakku thathwangal upayogichu eee balance kanakkakkaam. oru vaathakathilo draavaka layaniyilo oru uparithalathilekko interfasilekko oru nerppicha laayaniyude agiranam allengil agiranam cheiyunna nirakk fikkinte vyaapana niyamangal upayogichu kanakkakkaam. oru nischitha laayaniyude avasthaykkum prakaashashakthiyude saandrathaykkum photobleaching/desorption nirakk alakkaan kazhiyum.
saadhaarana chayangal upayogikkunnathinaayi dna-peyint kooduthal vipuleekarichu, avide oru dye-label cheytha dna probinte dynamic binding unbinding oru nischitha dna origaamiyilekku upayogikkunnu, ithu yaadruchikamaaya single-molecule imaging nedunnu. dna-peyint inii paristhithi-sensitiivu chayangalil maathram parimithappedutthiyittilla, mathramalla probukalude adasorption desorption kainattiksum lakshyathilekku alakkaan kazhiyum. chalikkunna chaayangalude camera blurring ephakt eee reethi upayogikkunnu. oru saadhaarana chayam laayaniyil vyaapikkumbol, thaarathamyena vegathayeriya vegathayum thaarathamyena dairkhyameriya camera exposure samayavum kaaranam oru saadhaarana ccd camerayile athinte chithram mangunnu, ithu fluorescence pashchaathalatthinu kaaranamaakunnu. ennirunnaalum, athu oru nischitha lakshyavumayi bandhippikkumbol, dye neengunnathu nirtthunnu; pooyint spread functionilekku vyakthamaaya inputt nedaan kazhiyum.
eee reethiyude padam emm. bii. peyint (chalana mangaline soochippikkunna "emm. bii") aanu. imajinginaayi oru motham aantharika prathiphalana fluorescence microscope (tii. aarr. eph) upayogikkumbol, eksaitteshan depth substratil ninnu ~ 100 enn. emm aayi parimithappedutthunnu, ithu substratinu sameepamulla mangiya chayangalil ninnulla fluorescence pashchaathalatheyum bulk laayaniyile pashchaathalatheyum kuraykkunnu. thaarathamyena mithamaaya foto eksaitteshan theevrathakalil 20 enm enna saadhaarana single frame speshyal resolutionukalum 20 ems enna single molecule kainettik temporal resolutionukalum nalkunna emm. bii. paintinaayi valare thilakkamulla chayangal upayogikkaam, ithu single proteinukalude molecular seppareshan padikkaan upayogapradamaanu.
daatta ettedukkunna samayat forrier playinil sthaapichirikkunna oru bhramana ghatta mask upayogichu temporal resolution kooduthal mechappeduthukayum (20 thavana) temporal vivarangal adangiyirikkunna valachodicha pooyint spread function pariharikkukayum cheythu. suuppar temporal-resolution microscopy (strem) ennaanu eee reethikk peru nalkiyathu.
label rahita praadeshikavalkkarana microscopy.
label rahita cellukalil polum, localisation microscopy ezz. pii. dii. emm upayogichu ekadesham 50 enn. emm. paridiyilulla sellular gadanakalude opttikkal resolution nedaan kazhiyum.
rund vyathyastha lazer tharamgadairkhyangal upayogikkunnathiloode, autofluorasent gadanakalude ganyamaaya resolution mechappeduthalinu purame, parambaraagatha fluorescence wide feeld imaging saahacharyangalil kandethaan kazhiyaatha sellular vasthukkale ezz. pii. dii. emm velippeduthunnu.
oru niyanthranamenna nilayil, praadeshikavalkkarana chithratthile kandethiya vasthukkalude sthaanangal braitt feeld chithravumaayi poruthappedunnu.
valare ekeekrithamaaya plasmonic mettaasarfeysile uparithala mechappeduthiya raman scattering signalinte ettakkurachilukal upayogichu label rahita suuppar resolution microscopy pradarshippichittundu.
direct stochaastik opttikkal reconstruction microscopy (dii stoom).
otta fluoroforinte photoswiching aanu kodunkaattu upayogikkunnathu. kodunkaattil, fluoroforukal oru reducing aandu ooksidaising buffering sisttathil (rox) ulchernnirikkunnu, koodaathe fluorescence aaveshabharithamaanu. chilappol, yaadruchikamaayi, fluorophore bafarinte ooksideshan avasthayodu sensitiivu aaya oru triple allengil mattethenkilum irunda avasthayilekku praveshikkum, athil ninnu avaye fluores acam, angane orotta thanmaathrayude sthaanangal rekhappeduthaanaakum. ekadesham ore samayam 3 swatantra laborattarikalil kodunkaattu reethiyude vikasanam sambhavichu, ithine "reversible photobleaching microscopy" (rpm), "graundu stettu depletion microscopy thudarnnu vyakthigatha molecule ritten" (gsdim), athupole ippol pothuve amgeekarikkappetta monicker distom ennum vilikkunnu.
praadeshikavalkkarana microscopic vendiyulla software.
eathaanum minitukalkkullil sajeevamaaya fluoroforukalude dasalakshakkanakkinu chithrangalumaayi pooyint spread function (pii. ezz. eph) krithyamaayi poruthappedutthaan kazhiyunna softweyarine praadeshikavalkkarana microscopy valareyadhikam aasrayichirikkunnu. prakrithishaasthrathil upayogikkunna classical vishakalana reethikalum software syoottukalum eee prasnangal computationalaayi pariharikkan valare mandagathiyilullathinaal, palappozhum minittukalil alakkunna daatta prosess cheiyunnathinu manikorukal edukkunna kanakkukoottal, prathyeka software prograamukal vikasippicheduthittundu. eee praadeshikavalkkarana software paakkejukalil palathum ooppan sozhs aanu; avaa ezz. emm. ell. emm software maanadandatthil pattikappedutthiyittundu. thanmaathrakalude sthaanam nirnnayichukazhinjaal, lokkeshanukal pradarshippikkukayum pradarshanathinaayi niravadhi algorithangal vikasippikkukayum venam.
suuppar resolution opttikkal fluctuation imaging (sophi).
pixalukalude thaalkkaalika autocorilation nerittu kanakkukoottunnathiloode single molecule localisation microscopy (smlm) yill antharleenamaaya psf fittinginte aavasyakatha ozhivakkaan kazhiyum. eee saankethikavidyaye suuppar-resolution opttikkal fluctuation imaging (sophi) ennu vilikkunnu, oresamayam sajeevamaaya fluoroforukalude saandrata valare kooduthalaayirikkumbol ithu smlminekkaal krithyamaanennu theliyikkappettittund.
sarvvyaapiyaaya praadeshikavalkkarana microscopy (oolm).
omnipresent localisation microscopy (oo. ell. emm) ennathu eaka thanmatra microscopy (ezz. emm. ell. emm) saankethikavidyakalude oru vipuleekaranamaanu, ithu oru poruthappedaattha prakasha srothass (merkuri-arck lamp polullava), parambaraagatha epiphlurescence microscope sajjeekaranam enniva upayogichu uyarnna saandrathayulla eaka thanmatra imaging anuvadikkunnu. aazhathilulla neela niratthilulla oru cheria spodanam (405 enm laserinu pakaram oru 350-380 enm upayogichu) pareekshana saambilukalil 90 enm resolutionaayi thanmaathrakalude deerghakaala reactivation prapthamaakkunnu. avasaanamaayi, correlative sted, smlm imaging enniva oru lalithamaaya imaging meediyam upayogichu ore biological saambilil nadatthaan kazhiyum, ithu kooduthal mechappetta resolution oru adisthaanam nalkum. eee kandethalukalkk suuppar resolution imajingine janaadhipathyavalkkarikkaanum parimithamaaya bajattil polum uyarnna saandrathayulla single molecule emejukal srishtikkaan eathoru shaasthranjaneyum sahayikkaanum kazhiyum.
sequential imaging (ressy) vazhi resolution vardhippikkuka.
saidhaanthikamaayi paridhiyillaattha resolution nedaan kazhiyunna dna-paintinte vipuleekaranamaanu sequential imaging (ressy) vazhi resolution mechappeduthal. oru nischitha taargett speesheesine thirichariyaan oru label tharam upayogikkunnathinu pakaram, athe taargettinte pakarppukal oorthoganal dna sequenceukal upayogichu label cheythirikkunnu. thudarchayaayi (i.iii. verthiricha) imaging, parambaraagatha ezz. emm. ell. emmile ovarlaappu cheiyunna praadeshikavalkkarana meghangal (1) pariharikkanum (2) orotta "suuppar" praadeshikavalkkaranathilka samyojippikkaanum kazhiyum, eathu alavukalude krithyathayum praadeshikavalkkaranangalu adisthaana ennavum. dna-peyintil nedaan kazhiyunna praadeshikavalkkaranangalu ennam paridhiyillaathathinaal, resiyude saidhaanthika prameyavum angane thanne. adisthaana imaging roundukalil ninnulla ressy localisationukal pothinju oru samyojithavum uyarnna pariharamullathumaaya chithram srishtikkunnu.
saankethikavidyakalude samyojanam.
cheiyunnu laitt microscopic nanocysing (limoon) microscopy.
vertico smi microscope upayogichu laitt microscopy nanocysing microscopy microscopy limoon) chithrangal smi, sspdm ennivayude samyojanathiloode saadhyamaakkunnu, athuvazhi aadyam smi, thudarnnu sspdm prakriya prayogikkunnu.
smi prakriya kanangalude kendravum avayude vyaapanavum microscope aaksisinte dishayilekku nirnnayikkunnu. kanangalude/thanmaathrakalude kendram 1-2 enm krithyathayode nirnnayikkaan kazhiyumenkilum, eee pooyintinu chuttumulla vyaapanam ekadesham 30-40 enm akshavyasam vare nirnnayikkaan kazhiyum.
thudarnnu, oro kanikayudeyum/thanmathrayudeyum paarshwastha sthaanam ezz. pii. dii. emm upayogichu nirnnayikkukayum kurachu naanomeettar krithyatha kaivarikkukayum cheiyunnu.
cheiyunnu duval kalar modil oru biological aaplikkeshan enna nilayil, nilayil, herr2/nyoo klasttarukalude speshyal krameekaranangal kaivarichu. protein klasttarukalude moonnu dishakalileyum sthaanangal ekadesham 25 enm krithyathayode nirnnayikkaan kazhiyum.
samyojitha parasparabandhitha prakashavum electron microscopy.
oru suuppar resolution microscope oru electron microscope samyojippikkunnath fluorescence markerukal nalkunna labeling upayogichu sandarbhochitha vivarangal drishyavalkkarikkaan prapthamaakkunnu. oru laitt microscope maathram upayogikkumbol gaveshakanu avasheshikkunna karutha pashchaathalatthinte prashnathe ithu marikadakkunnu. oru samyojitha samvidaanathil, sample oresamayam rund microscopukalum upayogichu alakkunnu.
neural netvarkkukal upayogichu saankethikavidyakal mechappeduthuka.
aduthide, artificial intelligence computingile purogathi kaaranam, opttikkal microscope photographic emejukalude suuppar-resolution vardhippikkunnathinu deep-laining neural netvarkkukal (ganz) upayogichu, opttikkal microscope upayogichu 20x muthal 1500x vare, oru scanning electron microscopumaayi thaarathamyappeduthumbol, oru neural lense vazhi, poositroon-emission tomography, fluorescence microscopy enniva upayogichu.

