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Chromophore cis/trans photoisomerization is a fundamental process in chemistry and in the activation of many photosensitive proteins. A major task is understanding the effect of the protein environment on the efficiency and direction of this reaction compared to what is observed in the gas and solution phases. In this study, we set out to visualize the hula twist (HT) mechanism in a fluorescent protein, which is hypothesized to be the preferred mechanism in a spatially constrained binding pocket. We use a chlorine substituent to break the twofold symmetry of the embedded phenolic group of the chromophore and unambiguously identify the HT primary photoproduct. Through serial femtosecond crystallography, we then track the photoreaction from femtoseconds to the microsecond regime. We observe signals for the photoisomerization of the chromophore as early as 300 fs, obtaining the first experimental structural evidence of the HT mechanism in a protein on its femtosecond-to-picosecond timescale. We are then able to follow how chromophore isomerization and twisting lead to secondary structure rearrangements of the protein β-barrel across the time window of our measurements. peerReviewed

The brown macroalga Fucus vesiculosus is a foundation species in temperate rocky shores, subjected to seasonally fluctuating environmental conditions. To obtain a more complete picture of the seasonality of F. vesiculosus ecophysiology in the northern Baltic Sea, in situ photochemistry, elemental ratios and chlorophyll a and c content of the alga were investigated in field campaigns conducted in different months throughout the year during 2017. Carbon, nitrogen, carbon to nitrogen ratio and chlorophyll a and c content of the alga varied substantially throughout the year, with highest carbon content observed in summer, and highest nitrogen content in winter. C:N ratio in F. vesiculosus apical tissue ranged from 8.6 in February to 48.3 in July. Chlorophyll a and c content followed inversely the seasonal patterns of ambient irradiance. High chlorophyll a and c content in winter was associated with higher maximum photosynthetic efficiency of energy conversion (Fv/Fm), but not with efficiency of photosynthetic energy conversion under light limitation (α). Electron transport rate correlated strongly with seawater temperature, and the highest electron transport rates were observed in summer and correlated with highest internal carbon content of the alga. Redundancy analysis conducted on measured environmental variables against physiological responses identified day of year, temperature and macronutrients in seawater as the most important variables driving the observed seasonal patterns in F. vesiculosus ecophysiology. The results suggest elevated temperatures may increase Fucus growth and photosynthesis rates in the study area.

We present a novel strategy for tailoring the fluorescent azadioxatriangulenium (KU) dye-based pH sensor to the target pH range by regulating the pKa value of the gold nanoclusters. Based on the correlation between the pKa and surface curvature of ligand-protected nanoparticles, the pKa value of the gold nanoclusters was controlled by size. In particular, three different-sized para-mercaptobenzoic acid (p-MBA) protected gold nanoclusters, Au25(p-MBA)18, Au102(p-MBA)44, and Au210–230(p-MBA)70–80 were used as the regulator for the pH range of the KU response. The negatively charged gold nanoclusters enabled the positively charged KU to bind to the surface, forming a complex and quenching the fluorescence of the KU by the energy transfer process. The fluorescence was restored after adjusting the surface charge of the gold nanocluster by controlling the solution pH. In addition, the KU exhibited a significantly different pH response behaviour for each gold nanocluster. Au210–230(p-MBA)70–80 showed a higher pH response range than Au102(p-MBA)44, which was intuitive. However, Au25(p-MBA)18 showed an unexpectedly high pH response behaviour. pKa titration measurement, molecular dynamics simulations, and essential dynamics analysis showed that small nanoclusters do not follow the scaling between the curvature and the pKa value. Instead, the behaviour is governed by the distribution and interaction of p-MBA ligands on the nanocluster surface. This work presents an effective design strategy for fabricating a range adjustable pH sensor by understanding the protonation behaviour of the ultrasmall gold nanoclusters in an atomic range. peerReviewed

The development of purely organic fluorescence emitters is of great importance for their low cost and high performance. Responding to this demand, carbazole is a promising emitter due to its extensive freedom for functionalisation, high thermal and chemical stability, as well as low cost. Herein, the effect of protonation on the fluorescence of various pyridine-functionalised carbazole-based bipolar host materials was studied both in solution and in the solid-state. The restriction of intramolecular rotation of the molecules upon protonation of the pyridyl-moiety together with easier planarization of the protonated acceptor and the donor moieties and relocalisation of the LUMO orbital on the protonated species was found to increase the fluorescence quantum yield from 16% to 80%. Additionally, in the solid-state, the J-type packing of the molecules further facilitated the increase in the fluorescence quantum yield from 1% to 49%. In both cases, the pronounced bathochromic spectral shift was observed indicating that the gap between the emissive state and the first triplet state of the molecules was diminished upon protonation. Therefore, implementing this strategy could further boost the development of future emitters. This journal is publishedVersion

Spectral cameras are traditionally used in remote sensing of microalgae, but increasingly also in laboratory-scale applications, to study and monitor algae biomass in cultures. Practical and cost-efficient protocols for collecting and analyzing hyperspectral data are currently needed. The purpose of this study was to test a commercial, easy-to-use hyperspectral camera to monitor the growth of different algae strains in liquid samples. Indices calculated from wavebands from transmission imaging were compared against algae abundance and wet biomass obtained from an electronic cell counter, chlorophyll a concentration, and chlorophyll fluorescence. A ratio of selected wavebands containing near-infrared and red turned out to be a powerful index because it was simple to calculate and interpret, yet it yielded strong correlations to abundances strain-specifically (0.85 &lt, r &lt, 0.96, p &lt, 0.001). When all the indices formulated as A/B, A/(A + B) or (A − B)/(A + B), where A and B were wavebands of the spectral camera, were scrutinized, good correlations were found amongst them for biomass of each strain (0.66 &lt, 0.98, p &lt, 0.001). Comparison of near-infrared/red index to chlorophyll a concentration demonstrated that small-celled strains had higher chlorophyll absorbance compared to strains with larger cells. The comparison of spectral imaging to chlorophyll fluorescence was done for one strain of green algae and yielded strong correlations (near-infrared/red, r = 0.97, p &lt, 0.001). Consequently, we described a simple imaging setup and information extraction based on vegetation indices that could be used to monitor algae cultures.

Interactions between an atomically precise gold nanocluster Au102(p-MBA)44 (p-MBA = para mercaptobenzoic acid) and a fluorescent organic dye molecule (KU, azadioxatriangulenium) are studied. In solution, the constituents form spontaneously a weakly bound complex leading to quenching of fluorescence of the KU dye via energy transfer. The KU can be separated from the complex by lowering pH, leading to recovery of fluorescence, which forms a basis for an optical reversible pH sensor. However, the sensor is not a stable entity, which could be delivered inside cells. For this purpose, a covalently bound hybrid is synthesized by linking the KU dye to the ligand layer of the cluster via an ester bond. Covalent linking facilitates entry of the cluster–dye hybrids into cells via endocytosis. Inside cells, the hybrids accumulate in endosomes where Au102 releases its cargo via hydrolysis of the ester bond. Changes of the local pH inside endosomes regulate reversible fluorescence due to variations in the interactions between the Au102 cluster and the dye. This work presents a concept for delivering reporter molecules into cells by using atomically precise gold nanoclusters as carriers and paves the way for future developments of cluster-reporter sensors for in vivo measurements of e.g. absolute pH values or ion concentrations. peerReviewed

Viruses need to open, i.e., uncoat, in order to release their genomes for efficient replication and translation. Especially for non-enveloped viruses, such as enteroviruses, the cues leading to uncoating are less well known. The status of the virus has previously been observed mainly by transmission electron microscopy using negative staining, cryo electron microscopy, X-ray crystallography or gradient separation (reviewed in Tuthill et al., 2010, Myllynen et al., 2016, Ruokolainen et al., 2019). However, monitoring of uncoating has been limited by the lack of methods detecting dynamic changes of the virions. Here, we present a real-time fluorescence based protocol, which detects the viral genome (RNA) during various stages of uncoating in vitro, while RNA is still inside the particle that has been expanded before the actual RNA release, and when the RNA has been totally released from the viral particle. Our method allows to explore how various molecular factors may promote or inhibit virus uncoating. peerReviewed

Fluorescent probes for sensing nucleic acids have found widespread use in the field of cell and molecular biology. However, probes combined with potential for post-synthetic conjugation, e.g. for intra-endosomal measurements of RNA, are unavailable. Herein we developed cyanine dyes that can be conjugated to viral capsid or other targets. First, we solved the crystal structure of SYBR Green II. The structural elucidation of this commonly used RNA probe provided the basis for synthesizing similar molecules with much desired function for post-synthetic conjugation. To address this need, cyanine dyes were prepared using an alternative synthesis protocol. All studied compounds showed considerable brightness upon binding to nucleic acids. However, regardless of the common chromophore on the dyes, the observed fluorescence emission intensities varied significantly, where methyl-substituted dye 1 gave values higher than SYBR Green II, whereas compounds 2–5 containing undecyl spacers had lower values. Studying the structure-activity relationship revealed the longer alkyl chains to induce slight perturbation in dye intercalation, as well as demanding larger binding area on the nucleic acid lattice, explaining these differences. To study the potential biological use of the dyes, the RNA genome of enterovirus echovirus 1 was studied in vitro with the probes. A novel method employing the low binding space requirement of 1 was developed to determine the single-to-double-stranded RNA ratio of a sample, whereas compound 4 was covalently bound to the viral capsid and used successfully to monitor the viral RNA release from within the capsid. The presented results open new possibilities for preparation and use of SYBR Green-based nucleic acid probes to further apply these compounds for increasingly demanding targeting in biological contexts. peerReviewed

Organic fluorophores, particularly stimuli-responsive molecules, are very interesting for biological and material sciences applications, but frequently limited by aggregation- and rotation-caused photoluminescence quenching. A series of easily accessible bipyridinium fluorophores, whose emission is quenched by a twisted intramolecular charge-transfer (TICT) mechanism, is reported. Encapsulation in a cucurbit[7]uril host gave a 1:1 complex exhibiting a moderate emission increase due to destabilization of the TICT state inside the apolar cucurbituril cavity. A much stronger fluorescence enhancement is observed in 2:2 complexes with the larger cucurbit[8]uril, which is caused by additional conformational restriction of rotations around the aryl/aryl bonds. Because the cucurbituril complexes are pH switchable, this system represents an efficient supramolecular ON/OFF fluorescence switch. peerReviewed

Intestinal and hepatic glucuronidation by the UDP-glucuronosyltransferases (UGTs) greatly affect the bioavailability of phenolic compounds. UGT1A10 catalyzes glucuronidation reactions in the intestine, but not in the liver. Here, our aim was to develop selective, fluorescent substrates to easily elucidate UGT1A10 function. To this end, homology models were constructed and used to design new substrates, and subsequently, six novel C3-substituted (4-fluorophenyl, 4-hydroxyphenyl, 4-methoxyphenyl, 4-(dimethylamino)phenyl, 4-methylphenyl, or triazole) 7-hydroxycoumarin derivatives were synthesized from inexpensive starting materials. All tested compounds could be glucuronidated to nonfluorescent glucuronides by UGT1A10, four of them highly selectively by this enzyme. A new UGT1A10 mutant, 1A10-H210M, was prepared on the basis of the newly constructed model. Glucuronidation kinetics of the new compounds, in both wild-type and mutant UGT1A10 enzymes, revealed variable effects of the mutation. All six new C3-substituted 7-hydroxycoumarins were glucuronidated faster by human intestine than by liver microsomes, supporting the results obtained with recombinant UGTs. The most selective 4(dimethylamino)phenyl and triazole C3-substituted 7-hydroxycoumarins could be very useful substrates in studying the function and expression of the human UGT1A10.

A trial monitoring of a typical full-scale municipal WWTP in Central Finland was aimed to explore applicability of high performance liquid chromatography – size exclusion chromatography (HPSEC) with simultaneous UV and fluorescence detection as a tool for advanced routine monitoring of wastewater treatment. High, intermediate, and low molecular weight (MW) fractions of untreated wastewater (influent) and treated wastewater (secondary effluent) were characterized in terms of UV absorbance at 254 nm (UVA254) and specific fluorescence representing tyrosine-like, tryptophan-like, and humic/fulvic-like compounds. The activated sludge treatment removed 97 ± 1% of BOD, 93 ± 2% of COD, 71 ± 7% of DOC, and 24 ± 7% of TN, while the overall reduction of UVA254 was 50 ± 6%. Total fluorescence signal declined by ∼80% for tyrosine-like, by 60–70% for tryptophan-like, and by 7–36% for humic/fulvic-like compounds. Low and intermediate MW humic/fulvic-like compounds fluorescing at λex/λem = 390/500 nm demonstrated recalcitrant behavior. Protein-like and humic/fulvic-like fractions of low MW

Photoactive yellow proteins (PYPs) make up a diverse class of blue-light-absorbing bacterial photoreceptors. Electronic excitation of the p-coumaric acid chromophore covalently bound within PYP results in triphasic quenching kinetics; however, the molecular basis of this behavior remains unresolved. Here we explore this question by examining the excitation-wavelength dependence of the photodynamics of the PYP from Halorhodospira halophila via a combined experimental and computational approach. The fluorescence quantum yield, steady-state fluorescence emission maximum, and cryotrapping spectra are demonstrated to depend on excitation wavelength. We also compare the femtosecond photodynamics in PYP at two excitation wavelengths (435 and 475 nm) with a dual-excitation-wavelength-interleaved pump–probe technique. Multicompartment global analysis of these data demonstrates that the excited-state photochemistry of PYP depends subtly, but convincingly, on excitation wavelength with similar kinetics with distinctly different spectral features, including a shifted ground-state beach and altered stimulated emission oscillator strengths and peak positions. Three models involving multiple excited states, vibrationally enhanced barrier crossing, and inhomogeneity are proposed to interpret the observed excitation-wavelength dependence of the data. Conformational heterogeneity was identified as the most probable model, which was supported with molecular mechanics simulations that identified two levels of inhomogeneity involving the orientation of the R52 residue and different hydrogen bonding networks with the p-coumaric acid chromophore. Quantum calculations were used to confirm that these inhomogeneities track to altered spectral properties consistent with the experimental results. peerReviewed

Poikkitieteelliset sovellukset ovat viime aikoina yleistyneet tieteellisessä tutkimuksessa. Tämä näkyy hyvin esimerkiksi elävän kudoksen kuvantamisen kehittymisessä, jota varten etsitään jatkuvasti parempia biologisia leimoja fysiikan ja kemian keinoin. Tässä tutkielmassa on kehitetty ensimmäinen lähi-infrapuna-alueen fluoresenssileima, joka hyödyntää hopeananopartikkeleja ja fytokromeja. Kompleksi on syntetisoitu itse ja sen rakenne on todennettu optisen spektroskopian, elektroforeesin ja elektronimikroskopian keinoin. Lupaavat fluoresenssiominaisuudet saatiin käyttämällä monomeerisen Deinococcus radiodurans -fytokromin kromoforia sitovaa domeenia (CBDmon). Hopeapartikkelien läsnäollessa CBDmonin kvanttisaanto kasvoi 20 % ja viritetyn tilan elinaika laski 30 %. Syntetisoidun kompleksin kirkkaus verrattuna CBDmonin kirkkauteen oli 470+-40% 390 nm:n ja 134+-11% 630 nm:n virityksellä. Näitä tuloksia voidaan todennäköisesti vielä kasvattaa, koska kompleksin rakennetta on mahdollista muokata monella eri tavalla. Siksi voidaan todeta, että kehitetty menetelmä on lupaava keino lähi-infrapunaalueen leimojen suunnitteluun. Applications using a cross-disciplinary approach are a common phenomenon in recent scientific research. A good example is the field of living tissue imaging where better biological labels are constantly searched with the help of physics and chemistry. In this Thesis, a near-infrared template combining silver nanoparticles and phytochromes was synthesized. The formation of the complex was confirmed by using optical spectroscopy, electrophoresis and electron microscopy. Promising fluorescent properties were obtained using monomeric chromophore binding domain (CBDmon) of a phytochrome from Deinococcus radiodurans. In the presence of silver nanoparticles, CBDmon showed 20% increased quantum yield and 30% decreased exited state lifetime. The brightness of the synthesized complex was 470+-40% at 390 nm excitation wavelength and 134+-11% at 630 nm relative to the brightness of pure CBDmon. It is likely that these promising properties could be improved by further engineering using variety of methods. Thus, the results indicate a new way to design applicable NIR-labels.

Nanotiede kasvaa edelleen tutkimusalana nopeasti, ja sille löytyy lukematon määrä sovelluskohteita. Sen vahvuutena on poikkitieteellisyys fysiikan, kemian ja biologian kesken, jotka kaikki tutkivat atomitason ilmiöitä. Näiden ilmiöiden tutkiminen ja soveltaminen vaatii paljon erilaisia, sofistikoituneita menetelmiä ja työkaluja, joilla voidaan valmistaa ja karakterisoida erilaisia nanopartikkeleita ja -rakenteita. Joissain tapauksissa vaaditaan jopa yksittäisten molekyylien tai atomien manipulointia nanometritasolla. DNA on yksi mielenkiintoisista nanotieteen tutkimuskohteista. Se on elämän kannalta merkittävä molekyyli, sillä se pitää sisällään elävän organismin rakenteellisen ohjeen. DNA:n tärkeä rooli proteiinisynteesissä vaatii sen olevan kemiallisesti erittäin vakaa ja tarkasti hallittava. Nämä ominaisuudet tekevät siitä monipuolisen ja tärkeän tutkimuskohteen. DNA:n itsejärjestyvyysominaisuuksien takia sen käyttöä erilaisina ohjelmoitavina rakennuspalikoina ja tukirakenteina on tutkittu innokkaasti. Pii-pohjaisen mikro- ja nanopiiritekniikan suorituskyvyn lähestyessä fysikaalisia rajojaan myös DNA:n sähkönjohtavuutta on tutkittu mahdollisten nanoelektroniikkaan liittyvien sovellusten takia. DNA:n hyödyntämisen mahdollistaa sen muokattavuus. DNA-oligonukleotidit ovat synteettisiä, yleensä muutaman kymmenen emäsparin pituisia yksijuosteisia DNA-molekyylejä, joihin voi valita mieleisensä sekvenssin sovelluskohteen mukaan. Molekyylin molempiin päihin on myös mahdollista liittää erilaisia funktionaalisia molekyylejä. Tämä ominaisuus on hyödyllinen erityisesti biotieteissä, joissa hyödynnetään paljon fluoresenssimikroskopiaa. Koska näytteet eivät aina ole itsessään fluoresoivia, ne täytyy tarvittaessa leimata tilanteeseen sopivilla merkkiaineilla. Lisäksi koejärjestelystä riippuen voidaan tarvita esimerkiksi erilaisia linkkeri-molekyylejä. Erilaisten molekulaaristen modifikaatioiden käyttöön kuvantamisessa liittyy kuitenkin se heikkous, että ne lisäävät näytteiden monimutkaisuutta ja hintaa. Siten kuvantamistekniikat, joiden käyttö ei vaadi erillisiä merkkiaineita, voivat joissain tapauksissa olla suureksi hyödyksi. Raman-spektroskopia, esimerkiksi, perustuu sähkömagneettisen säteilyn Raman-sirontaan fluoresenssin sijaan. Intialaiset fyysikot C. V. Raman ja K. S. Krishnan todistivat sen olemassaolon 1920-luvun alussa. Valo voi elastisen Rayleigh-sironnan lisäksi sirota Ramanaktiivisista partikkeleista epäelastisesti. Raman-moodeja on kaksi, joita kutsutaan Stokes- ja anti-Stokes-siirtymiksi. Stokes-siirtymässä systeemi siirtyy perustilalta virtuaalitilan kautta vibraatiotilalle. Siten sironneen fotonin energia on pienempi alkutilanteeseen verrattuna. Anti-Stokes-siirtymässä tapahtuu päinvastoin, jolloin fotonin loppuenergia on alkuperäistä suurempi. Raman-spektroskopiaa voidaan pitää ideaalisena menetelmänä biotieteissä, koska aineen Ramanspektri on ikään kuin sen uniikki sormenjälki. Nanopartikkelien manipulointi onnistuu esimerkiksi hyödyntämällä dielektroforeesia, jonka teorian Herbert A. Pohl esitti jo 1950-luvulla. Ilmiö tarkoittaa neutraalien tai varattujen, polarisoituvien hiukkasten indusoitua liikettä epä-homogeenisessa sähkökentässä. Esimerkkinä voi käyttää sähköistä dipolia, joka voi olla pysyvä tai indusoitu. Dipoli asettuu sähkökenttään sen kenttäviivojen mukaisesti. Kentän gradientin takia Coulombin vuorovaikutuksen suuruus dipolin eri päissä poikkeaa toisistaan, mikä johtaa nettovoimaan ja liikkeeseen kentän gradientin mukaisesti. Mikäli partikkelin sähköinen permittiivisyys on väliaineen permittiivisyyttä suurempi, liikkuu se kohti kentän maksimia, jolloin puhutaan positiivisesta dielektroforeesista. Negatiivisesta dielektroforeesista puhutaan vastaavasti silloin, kun väliaineen sähköinen permittiivisyys ylittää partikkelin permittiivisyyden. Silloin partikkeli liikkuu kohti kentän minimiä. Tässä työssä fluoresoivalla merkkiaine-molekyylillä ja tioli-ryhmällä varustettuja, neljänkymmenen emäsparin pituisia DNA oligonukleotideja vangittiin dielektroforeesin avulla kullasta valmistettujen, sadan nanometrin levyisten elektrodien väliin, jonka jälkeen näytteitä tarkasteltiin konfokaalisilla, laseriin perustuvilla fluoresenssi- ja Raman-mikroskoopeilla. Elektrodikuvio, jossa oli useita elektrodipareja, valmistettiin elektronisuihkulitografialla ja itse elektrodit elektronisuihkuhöyrystimellä. Puolijohdenäytteet tutkittiin pyyhkäisyelektronimikroskoopilla valmistusvirheiden varalta. Fluoresenssimikroskopilla varmistettiin oligonukleotidien vangitsemisen onnistuminen. Raman-spektroskopian tarkoituksena oli tutkia, voisiko sen yhdistää dielektroforeesin kanssa, jolloin lopputuloksena voisi olla suhteellisen yksinkertainen, merkkiaineeton, alle mikrometrimittakaavassa toimiva kuvantamismenetelmä, jota voi käyttää lokalisoitujen näytteiden tutkimiseen. Tutkittavien partikkeleiden keräämisellä yhteen pisteeseen, niiden tutkiminen helpottuu, mikäli konsentraatio muuten jäisi alhaiseksi. Samalla voisi yrittää selvittää partikkelien määrää. Fluoresenssimikroskooppikuvien laatu ei vastannut odotettua, mikä johtui lasereiden heikentyneestä kohdistuksesta. Riittävää Raman-spektriä ei onnistuttu mittaamaan, ainoa signaali saatiin näytteen piialustasta. Mittauksen haasteellisuus tiedettiin ennakkoon, koska käytetyn mikroskoopin spatiaalinen tarkkuus oli ainakin mikrometriluokassa, mutta ei välttämättä sadoissa nanometreissä. The purpose of this study was to see if 40 bp poly-CT DNA oligonucleotides modified with a thiol group and a Cy3 dye molecule trapped on a silicon oxide surface using dielectrophoresis could be detected under a Raman microscope. Obtaining a clear Raman signal proved to be challenging though and in the end the only peaks visible in the Raman spectra were those originating from the silicon substrate. There were problems with the confocal fluorescence microscope too. The bad alignment of its lasers made the task of acquiring proper images rather difficult. The spatial resolution of the Raman setup was known to be in the micron scale at least but not probably extending to hundreds of nano metres. Therefore the result was somewhat expected. A successful Raman detection would have been a positive note in the search of label free imaging techniques for biosciences.

Fytokromit ovat bakteerien, levien, kasvien ja sienten punaista valoa aistivia proteiineja. Keskeinen piirre fytokromien toiminnassa on valon vaikutuksesta tapahtuva siirtymä punaista valoa absorboivan (Pr) ja kaukopunaista valoa absorboivan (Pfr) tilan välillä. Deinococcus radiodurans -bakteerifytokromin valosensoriosa (CBD-PHY) on dimeerinen proteiini, joka koostuu C-terminaalisesta kromoforia sitovasta domeenista (CBD) ja fytokromiin liittyvästä domeenista (PHY). Viimeaikaiset CBD-PHY:n kristalli- ja liuosrakenteet ovat osoittaneet, että PHY-domeenien välinen etäisyys kasvaa, kun proteiini siirtyy Pr-tilasta Pfr-tilaan absorboituaan punaista valoa. Tässä työssä suunniteltiin Försterin resonanssienergiansiirtoon (FRET) perustuvat mittaukset vaihtoehtoiseksi, systeemiä häiritsemättömäksi ja kustannustehokkaaksi tavaksi mitata PHY-domeenien välimatka Pr- ja Pfr-tiloissa. CBD-PHY -proteiinia, jonka PHY-domeenien kärkiin oli lisätty kysteiini-insertiot (E373/CC/G374), ja jonka yksi pintakysteiini oli korvattu seriinillä (C93S), tuotettiin BL21 (DE3) -kannan E. coli -soluissa ja puhdistettiin korkean erotuskyvyn nestekromatografialla. Työssä kehitettiin malemidipohjainen leimausmenetelmä FRET donori (D) – akseptori (A) -parin (Alexa Fluor 488 – Alexa Fluor 546) kiinnittämiseksi inserttikysteiineihin. Leimattujen proteiininäytteiden steady-state -emissiospektrit, fluoresenssin elinajat sekä steady-state- ja aikaerotteiset anisotropiat mitattiin FRET-tehokkuuksien ja niitä vastaavien D-A-etäisyyksien määrittämiseksi Pr- ja Pfr-tiloissa. DA-leimatun CBD-PHY:n Pr- ja Pfr-tilojen emissiospektreissä nähtiin FRET:iin viittaava intensiteetin muutos sekä D:n että A:n emissiossa. Aikaerotteisista tuloksista FRET-signaalia ei yllättäen havaittu. Tämän lisäksi eri mittaustekniikat eivät paljastaneet eroa Pr:n ja Pfr:n välillä, eivätkä emissiospektreistä määritetyt D-A -etäisyydet vastanneet aiemmista liuosrakenteista arvioituja etäisyyksiä. Odottamattomille tuloksille voidaan esittää useita mahdollisia syitä. On ilmeistä, että työssä suunniteltu leimausprotokolla vaatii edelleen kehittämistä. Toisaalta on myös mahdollista, että systeemi on todellisuudessa ennakkokäsityksiä monimutkaisempi esimerkiksi väriainemolekyylien sammutuksen tai väriaineiden ja CBD-domeeneihin sitoutuneiden biliverdiini-molekyylien välisen FRET:in vuoksi. Phytochromes are red-light sensing proteins of bacteria, algae, plants, and fungi. A central feature of the function of phytochromes is a light-induced conversion between a red absorbing (Pr) state and a far-red absorbing state (Pfr). The photosensory unit (CBD-PHY) of Deinococcus radiodurans bacteriophytochrome is a dimeric protein consisting of a C-terminal chromophore binding (CBD) domain and a phytochrome-associated (PHY) domain. Recent crystal and solution structures of CBD-PHY have shown that the separation between the two PHY domains increases when the protein is converted from Pr to Pfr state as a result of red-light absorption. Förster resonance energy transfer (FRET) based measurements were designed in this work to be used as an alternative, non-invasive, and cost-efficient way for probing the PHY domain separation in Pr and Pfr. CBD-PHY engineered with cysteine insertions at the ends of the PHY domains (E373/CC/G374) and one surface cysteine substituted with serine (C93S) was expressed in E. coli strain BL21 (DE3) cells and purified with high-pressure liquid chromatography. A maleimide-based labeling scheme was developed for attaching a FRET donor (D) – acceptor (A) pair (Alexa Fluor 488 – Alexa Fluor 546) to the cysteine insertions. Steady-state fluorescence emission spectra, fluorescence decays, and steady-state and time-resolved fluorescence anisotropies were measured for determining FRET efficiencies and corresponding D-A distances in Pr and Pfr. Emission spectrum of DA-labeled CBD-PHY in Pr and Pfr revealed FRET-associated intensity changes in both D and A emission. Surprisingly, FRET signal was absent from time-resolved results. Furthermore, none of the measurement techniques revealed a difference between Pr and Pfr, and there was discrepancy between D-A distances determined from steady-state results and those evaluated from earlier solution structures. Multiple explanations can be proposed for the unexpected results. It is evident that further development of protein labeling protocols is still needed. There is also a possibility that the system is more complicated than what was initially expected because of processes such as quenching of the dyes or FRET between the dyes and biliverdin molecules bound to the CBD-domains.

Pyrophosphate (PPi) anion is an integral part of our physiology. It partakes in the crucial energy transactions in cells and proffers energy for the harmonious functioning of the cell machinery. Discrepancies in PPi concentrations have been associated with several pathological aberrations such as chronic pyrophosphate arthropathy, calcium pyrophosphate deposition disease, cancer etc. However, there still exists a strong demand for an efficient and specific cell based assay for PPi in order to tap its importance to full potential. Terpyridine-Zn2+ complex (ZnCl2L probe) binds with PPi in 1:3 stoichiometry. This association engenders an emission band (λem) at ~591 nm when excited at 440 nm with spectrophotometer. It has lowest detection limit (LOD) of 0.8 nM. Water media based, ultra sensitive detection attribute of fluorescent chemosensor ZnCl2L probe was implemented to investigate pyrophosphate concentrations. In addition to that available commercial kits from Life Technologies, Lonza & Abcam manufacturers were also used for comparison. The probe showed remarkable detection ability for PPi. It generated a linear curve from 0-13.32 μM concentration. Higher concentration (>50 μM) of probe solution should be implemented in order to test PPi concentrations higher than 13.32 μM. The measured pyrophosphate content with the probe in cells was found to be in micromolar range. Contribution of interference from other molecules still remains to be tested. The stability of the PPi-probe-complex was tested against several time points, and was found stable until 24 hr time point. Intense reddish-orange fluorescence in cells stained with ZnCl2L probe under confocal microscope setup confirmed the phenomenon. In conclusion, the ZnCl2L probe proved to offer a reliable method to measure PPi. It could hopefully be used to make measurements from various body fluids. Thus, PPi analysis may assist in diagnosis of diseases where increased levels of PPi are present.

Phytochromes are multi-domain red light photosensor proteins, which convert red light photons to biological activity utilizing the multitude of structural and chemical reactions. The steady increase in structural information obtained from various bacteriophytochromes has increased understanding about the functional mechanism of the photochemical processes of the phytochromes. Furthermore, a number of spectroscopic studies have revealed kinetic information about the light-induced reactions. The spectroscopic changes are, however, challenging to connect with the structural changes of the chromophore and the protein environment, as the excited state properties of the chromophores are very sensitive to the small structural and chemical changes of their environment. In this article, we concentrate on the results of ultra-fast spectroscopic experiments which reveal information about the important initial steps of the photoreactions of the phytochromes. We survey the excited state properties obtained during the last few decades. The differences in kinetics between different research laboratories are traditionally related to the differences of the studied species. However, we notice that the variation in the excited state properties depends on the subunit composition of the protein as well. This observation illustrates a feedback mechanism from the other domains to the chromophore. We propose that two feedback routes exist in phytochromes between the chromophore and the remotely located effector domain. The well-known connection between the subunits is the so-called tongue region, which changes its secondary structure while changing the light-activated state of the system. The other feedback route which we suggest is less obvious, it is made up of several water molecules ranging from the dimer interface to the vicinity of the chromophore, allowing even proton transfer reactions nearby the chromophore. peerReviewed

Echovirus 1 (EV1) kuuluu Pikornavirusperheeseen ja tarkemmin luokiteltuna Enterovirusten sukuun. EV1 on pieni ja vaipaton RNA-virus, jonka proteiinikuori koostuu neljästä proteiinista VP1-VP4. Infektion on osoitettu alkavan, kun EV1 tarttuu α2β1-integriiniin solun pinnalla, jonka jälkeen virus otetaan solun sisään kaveoliinivälitteistä endosytoosia sekä makropinosytoosia muistuttavalla mekanismilla. Toisaalta sitä, kuinka virus avautuu sekä vapauttaa RNA:nsa, ei vielä tarkalleen tiedetä. Tässä tutkimuksessa EV1 puhdistettiin ensin sakkaroosigradientilla, jonka jälkeen virus leimattiin fluoresoivalla amiinireaktiivisella värillä. Tulokset osoittivat, että leimauksen onnistumiseksi tarvitaan suhteellisen vähäinen leimamäärä, ja että vähäinen leimamäärä riittää fluoresenssin havaitsemiseksi sekä on tärkeä viruksen infektiivisyyden säilyttämiseksi. Leimattua EV1:stä käytettiin solukokeissa, joissa Fosfolipaasi C inhibiittoria (PLC-inhibiittori) käytettiin viruksen endosytoosin estämiseen, jolloin asettamamme hypoteesin mukaan endosytoosia ei tapahdu, vaan virus jää solun pinnalle. Immunofluoresenssikokeiden ja kvantitatiivisen analysoinnin perusteella endosytoosi estyi, vaikkakin ero kontrollisoluihin oli ainoastaan 10 %. Lisäksi toinen immunofluoresenssikoe osoitti, että EV1:n infektiivisyys laski käytettäessä PLC-inhibiittoria. Immunofluoresenssikokeissa ongelmia aiheuttivat tyhjät ja rikkinäiset viruspartikkelit, jotka havaittiin EV1:n karakterisoinnin aikana läpäisyelektronimikroskoopilla. Leimautuneet tyhjät ja rikkinäiset partikkelit aiheuttivat todennäköisesti väärää signaalia immunofluoresenssikokeissa, joiden perusteella voitiin todeta, että viruksen puhdistaminen ja leimaaminen vaativat lisää optimointia. Suora kapsidileima voisi onnistuessaan toimia työkaluna tulevissa viruksen avautumiskokeissa ja livetutkimuksissa, jolloin vasta-aineleimauksiin liittyvä taustafluoresenssi saataisiin minimoitua. Lisäksi PLC-inhibiittori voisi mahdollistaa viruksen avautumisen tutkimisen solun pinnalla endosomaalisten rakenteiden sijaan. Echovirus 1 (EV1) belongs to the family of Picornaviridae and more specifically to the genus Enterovirus. EV1 is a small, non-enveloped RNA-virus which has a capsid consisting of four proteins VP1-VP4. EV1 has been shown to bind to α2β1-integrin on the cell surface and then endocytosed inside the cell by a mechanism that resembles both caveolin-mediated endocytosis and macropinocytosis. Instead, uncoating and RNA release are poorly understood. Here, we have successfully purified EV1 with sucrose gradient and labeled the virus with an amine reactive dye. The results showed that for successful labeling, considerably low amount of dye is sufficient for signal detection, and according to end-point dilution assay, is necessary for maintaining virus infectivity. Capsid labeled EV1 was used in cellular studies where Phospholipase C-inhibitor (PLC-inhibitor) was tested as an endocytosis blocker. According to our hypothesis PLC-inhibitor blocks endocytosis, and as a result, virus remains on the cell surface. Indeed, based on immunofluorescence studies and quantification with Bioimage XD software, endocytosis was blocked, although difference to control cells was only 10%. Additionally, another immunofluorescence study showed that PLC-inhibitor decreased EV1 infection. Immunofluorescence studies were hindered by empty and disassembled virus particles, which were detected during EV1 characterization with transmission electron microcopy. Labeled empty and disassembled particles probably caused false signal in immunofluorescence studies, and thus, it was concluded that virus purification and labeling has to be more optimized for these kinds of studies. Fluorescent capsid label could provide us a tool for future uncoating and live imaging studies by minimizing non-specificity that is usually related to immunolabeling. In addition, PLC-inhibitor could enable uncoating studies to be carried out on the cell surface instead of endosomes.

The number of fluorescence applications and methods of analysis is growing in the biological and related sciences. The aim of this work was to accurately determine the diffusion coefficient in nucleus, diffusion in the cytoplasm and nucleo-cytoplasmic translocation should be considered as well. Fluorescence recovery after photobleaching (FRAP) experiments were used in order to study protein diffusion. A numerical model for FRAP was used to determine the diffusion properties of proteins. The present method removed the difficulties of the conventional analysis, could produce, interesting results for protein diffusion in the cell.

The unique nature of optical properties of single-walled carbon nanotubes (SWCNT), together with their promising potential applications, have created enormous interest towards the photophysics of SWCNT. Many aspects of carbon nanotubes originate from the electronic structure of carbon honeycomb lattice and one-dimensionality. SWCNTs exist in various chiral structures and diameters, which the optical and electrical properties are dependent on. It has been discovered that SWCNT excited states are excitonic with strong Coulomb interaction between the electron and the hole. However, many features of excitons are not yet well defined, such as absorption cross-sections, fluorescence quantum yields, exciton lifetimes, and the nature of exciton mobility. The cylindrical shape with all atoms on the surface makes SWCNTs highly sensitive to environment which is a significant factor affecting the optical properties. First part of this thesis concentrates on the temperature dependence of excitonic absorption transitions. The importance of this study lies in the complexity of environmentally induced mechanisms to alter the energy gap and shift the electronic absorption transitions as a function of temperature. The results revealed abrupt shift in the energy gap interpreted to originate from interactions with water. The second part discusses the processes occurring after absorption: exciton mobility and dynamics. By analyzing reactions between single-molecule diazonium salt and individual nanotubes, the distance travelled by exciton during its lifetime, in different surfactants and for different chiral structures was determined. The results indicate that localized excitons have diffusional motion along the nanotube axis. The large diffusion ranges obtained (160 - 340 nm) explain the sensitivity of SWCNT emission to changes in local environment. The dynamics of excitons were studied with excitation dependence measurements. Results revealed the existence of long lived states that act as quenchers of emission even at relatively low excitation intensities.

To obtain data on phytoplankton dynamics with improved spatial and temporal resolution, and at reduced cost, traditional phytoplankton monitoring methods have been supplemented with optical approaches. In this thesis, I have explored various fluorescence-based techniques for detection of phytoplankton abundance, taxonomy and physiology in the Baltic Sea. In algal cultures used in this thesis, the availability of nitrogen and light conditions caused changes in pigmentation, and consequently in light absorption and fluorescence properties of cells. In the Baltic Sea, physical environmental factors (e.g. mixing depth, irradiance and temperature) and related seasonal succession in the phytoplankton community explained a large part of the seasonal variability in the magnitude and shape of Chlorophyll a (Chla)-specific absorption. The variability in Chla-specific fluorescence was related to the abundance of cyanobacteria, the size structure of the phytoplankton community, and absorption characteristics of phytoplankton. Cyanobacteria show very low Chla-specific fluorescence. In the presence of eukaryotic species, Chla fluorescence describes poorly cyanobacteria. During cyanobacterial bloom in the Baltic Sea, phycocyanin fluorescence explained large part of the variability in Chla concentrations. Thus, both Chla and phycocyanin fluorescence were required to predict Chla concentration. Phycobilins are major light harvesting pigments for cyanobacteria. In the open Baltic Sea, small picoplanktonic cyanobacteria were the main source of phycoerythrin fluorescence and absorption signal. Large filamentous cyanobacteria, forming harmful blooms, were the main source of the phycocyanin fluorescence signal and typically their biomass and phycocyanin fluorescence were linearly related. Using phycocyanin fluorescence, dynamics of cyanobacterial blooms can be detected at high spatial and seasonal resolution not possible with other methods. Various taxonomic phytoplankton pigment groups can be separated by spectral fluorescence. I compared multivariate calibration methods for the retrieval of phytoplankton biomass in different taxonomic groups. Partial least squares regression method gave the closest predictions for all taxonomic groups, and the accuracy was adequate for phytoplankton bloom detection. Variable fluorescence has been proposed as a tool to study the physiological state of phytoplankton. My results from the Baltic Sea emphasize that variable fluorescence alone cannot be used to detect nutrient limitation of phytoplankton. However, when combined with experiments with active nutrient manipulation, and other nutrient limitation indices, variable fluorescence provided valuable information on the physiological responses of the phytoplankton community. This thesis found a severe limitation of a commercial fast repetition rate fluorometer, which couldn t detect the variable fluorescence of phycoerythrin-lacking cyanobacteria. For these species, the Photosystem II absorption of blue light is very low, and fluorometer excitation light did not saturate Photosystem II during a measurement. This thesis encourages the use of various in vivo fluorescence methods for the detection of bulk phytoplankton biomass, biomass of cyanobacteria, chemotaxonomy of phytoplankton community, and phytoplankton physiology. Fluorescence methods can support traditional phytoplankton monitoring by providing continuous measurements of phytoplankton, and thereby strengthen the understanding of the links between biological, chemical and physical processes in aquatic ecosystems. Optiset automaattilaitteet tarjoavat kustannustehokkaan ja ajallisesti ja paikallisesti hyvin kattavan tavan seurata planktonlevien dynamiikkaa. Tässä väitöskirjatyössä olen selvittänyt erilaisten fluoresenssimenetelmien käyttöä planktonlevien määrän, levälajiston vaihtelun ja fysiologisen tilan mittaamisessa Itämerellä. Leväviljelmiä käyttäen havaitsin, että typen saatavuus ja valaistus vaikuttivat leväsolujen pigmentaatioon, sekä absorptio- ja fluoresenssiominaisuuksiin. Itämeressä fysikaaliset ympäristötekijät (sekoittuvan vesikerroksen syvyys, auringon säteilyn määrä, ja lämpötila) ja kasviplanktonyhteisöjen vuosisukkessio selittivät suuren osan a-klorofyllin spesifisen absorption voimakkuudesta ja spektraalisesta vaihtelusta. Havaittua a-klorofylli-spesifisen fluoresenssin vaihtelua selittivät syanobakteerien osuus planktonlevien biomassasta, planktonleväyhteisöjen kokojakauma ja planktonlevien absorptio-ominaisuudet. Elävien syanobakteerien sisältämän a-klorofyllin fluoresenssi on hyvin alhainen. Muiden planktonlevien läsnäollessa a-klorofyllin fluoresenssia ei voikaan käyttää syanobakteerien esiintymisen selvittämiseen. Syanobakteerikukintojen aikaan Itämerellä fykosyaniinin fluoresenssi selitti suuren osan a-klorofyllin pitoisuuksien vaihtelusta. Tutkimusteni mukaan, luotettavan arvion saaminen Itämeren levämäärästä kesäaikaan vaatiikin a-klorofyllin fluoresenssin lisäksi fykosyaniinin fluoresenssin mittaamista. Fykobiliinit ovat syanobakteerien tärkeimmät fotosynteettiset pigmentit. Avoimella Itämerellä fykoerytriinin fluoresenssin ja absorption määrä liittyi pienikokoisten syanobakteerien esiintymiseen. Fykosyaniinin fluoresenssi oli puolestaan peräisin isoista rihmamaisista syanobakteereista, jotka muodostavat haitallisia kukintoja. Solulaskentojen perusteella arvioidun rihmamaisten syanobakteerien biomassan ja fykosyaniinin fluoresenssin välille saatiin lineaarinen riippuvuussuhde. Fykosyaniinin fluoresenssin sen avulla voidaan arvioida syanobakteerikukinnan kehitystä aika- ja tilaskaalalla, mikä ei aikaisemmin ole ollut mahdollista. Taksonomisia planktonleväryhmiä voidaan tunnistaa fluoresenssisormenjälkien avulla. Väitöskirjatyössäni vertailin monimuuttujamenetelmien soveltuvuutta spektraalisten aineistojen tulkinnassa. Tilastollinen korrelaatiomenetelmä (PLS, partial least squares) osoittautui paremmaksi kuin perinteisemmät regressiomenetelmät ja PLS menetelmän avulla saadaan luotettava kuva eri leväryhmien vuosisukkessiosta ja leväkukinnoista. Fluoresenssin kvanttisaannon muutosten avulla voidaan tutkia planktonlevien fysiologista tilaa. Tulokseni osoittivat, että Itämerellä tämä menetelmä yksinään ei anna luotettavaa kuvaa planktonlevien ravinnerajoitteisuudesta. Yhdistettynä aktiivisiin ravinnemanipulaatiokokeisiin, ja muihin ravinnerajoitteisuuden indikaattoreihin, menetelmä tarjoaa kuitenkin arvokasta tietoa planktonleväyhteisön fysiologisesta vasteesta ravinnelisäyksiin. Työssä havaittiin lisäksi, että yleisesti käytössä oleva kaupallinen FRR fluorometri ei sovellu Itämeren kukintoja muodostavien rihmamaisten syanobakteerien fysiologian tutkimiseen niiden erikoisen pigmentaation vuoksi. Väitöskirjatyöni osoitti elävien planktonlevien fluoresenssimittausten olevan hyödyllisiä silloin kun halutaan jatkuvatoimisesti havainnoida planktonlevien määrää, syanobakteerien määrää, taksonomisten leväryhmien esiintymistä ja planktonleväsolujen fysiologista tilaa. Automatisoidut fluoresenssimenetelmät tukevat kustannustehokkaasti perinteistä planktonleväseurantaa ja niiden avulla voidaan saada entistä tarkempaa tietoa vesiekosysteemien toiminnan syy-seuraussuhteista.