Browsing by Subject "fluoresenssi"

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  • Seppälä, Jukka (Finnish Environment Institute, 2009)
    Monographs of the Boreal Environment Research 34
    To obtain data on phytoplankton dynamics (abundance, taxonomy, productivity, and physiology) with improved spatial and temporal resolution, and at reduced cost, traditional phytoplankton monitoring methods have been supplemented with optical approaches. Fluorescence detection of living phytoplankton is very sensitive and not disturbed much by the other optically active components. Fluorescence results are easy to generate, but interpretation of measurements is not straightforward as phytoplankton fluorescence is determined by light absorption, light reabsorption, and quantum yield of fluorescence - all of which are affected by the physiological state of the cells. 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. The variation of absorption and fluorescence properties of natural phytoplankton populations in the Baltic Sea was more complex. 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. Subsequent variations in the variables affecting fluorescence were large; 2.4-fold for light reabsorption at the red Chla peak and 7-fold for the spectrally averaged Chla-specific absorption coefficient for Photosystem II. In the studies included in this thesis, Chla-specific fluorescence varied 2-10 fold. This 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. It was shown that for reliable phycocyanin detection, instrument wavebands must match the actual phycocyanin fluorescence peak well. In order to initiate an operational ship-of-opportunity monitoring of cyanobacterial blooms in the Baltic Sea, the distribution of filamentous cyanobacteria was followed in 2005 using phycocyanin fluorescence.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. During a mesocosm experiment, a partial least squares regression method gave the closest predictions for all taxonomic groups, and the accuracy was adequate for phytoplankton bloom detection. This method was noted applicable especially in the cases when not all of the optically active compounds are known.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.
  • Huotari, Jussi; Ketola, Mirva (Suomen ymäristökeskus, 2014)
    Ympäristöhallinnon ohjeita 5/2014
    Kenttäkäyttöisten antureiden ja langattoman tiedonsiirron kehittymisen myötä jatkuvatoiminen, fluoresenssiin perustuva levämäärien mittaus on yleistynyt. Mittaustapaan liittyy kuitenkin epävarmuustekijöitä, jotka on tunnettava luotettavien tulosten saamiseksi. Tämän oppaan tarkoitus on auttaa jatkuvatoimista levämäärän mittausta suunnittelevia tai jo toteuttavia tahoja huomioimaan ne moninaiset seikat, joita mittaamiseen ja aineiston laadunvarmistukseen liittyy. Opas pyrkii antamaan lukijalle kuvan fluoresenssiin perustuvan jatkuvatoimisen levämäärien mittauksen perusteista, lähtien levien pigmenttikoostumuksesta ja fluoresenssista ilmiönä, edeten fluoresenssiin vaikuttaviin tekijöihin sekä mittaustavan rajoitteisiin. Opas antaa ohjeita mittauslaitteiston valintaan ja hankintaan, sekä sijoittamiseen, käyttöön ja huoltoon tuoden esille yleisimpiä ongelmakohtia. Lopuksi keskitytään aineiston laadunvarmistukseen ja annetaan ohjeita laitteiston ja aineiston kalibrointia sekä aineiston käsittelyä varten. Tämä opas on käyttäjiltä käyttäjille suunnattu teos, johon on koottu eri tavoin fluoresenssimittausten parissa työskennelleiden henkilöiden arvokkaita kokemuksia ensimmäistä kertaa yksiin kansiin.
  • el-Khouri, Hanna (Helsingin yliopisto, 2015)
    Ilmaston muuttuessa puiden kasvuolosuhteet muuttuvat lämpötilan ja hiilidioksidipitoisuuden kasvaessa. Vaikka puiden fysiologiset vasteita on tutkittu jo pitkään, tarkempi tieto puiden vuosirytmin muutoksista ja mahdollisista vaikutuksista vuosittaiseen kasvuun on kiinnostavaa. Hiilidioksidin ja lämpötilan vaikutuksia keväiseen lehtien kehitykseen tutkittiin keväällä rauduskoivulla (Betula pendula roth). Tavoitteena oli selvittää tarkemmin silmujen puhkeamisen jälkeistä fotosynteesikapasiteetin kehittymistä ja kohotetun hiilidioksidipitoisuuden ja lämpötilan vaikutusta siihen kaasunvaihtomittausten avulla. Toisena työn tavoitteena on verrata fluoresenssimittauksilla mitattua PSII:n valoreaktioiden maksimitehokkuutta kaasunvaihtomittauksiin. Koe-asetelma toteutettiin kasvihuoneolosuhteissa neljällä eri huoneella. Nykyilmastoa vastaavissa kontrollihuoneissa ilman hiilidioksidipitoisuuden tavoitteeksi asetettiin 380 ppm, ja lämpötila +2 °C ulkolämpötilaan verrattuna. Loppi2100-olosuhteet kuvaavat tulevaisuuden skenaariota, hiilidioksidipitoisuus tasolla 700 ppm ja lämpötila kontrolliin verrattuna +2 °C. Jokaisessa huoneessa kasvatettiin viittä rauduskoivua, joista mitattiin viikoittain kaasunvaihtomittausten avulla mm. hiilen assimilaatiota ja muita fotosynteesiin liittyviä tunnuksia. Eri huoneiden erilaisen lämpötilakehityksen ja eri mittauspäivien takia erot tasattiin laskemalla huonekohtainen lämpösumma, jonka avulla tulokset saatiin vertailukelpoisiksi eri huoneiden välillä. Tulosten mukaan hiilen assimilaatio on suurempaa ja kehittyy nopeammin Loppi2100-olosuhteissa verrattuna kontrolliin. Lämpösumman avulla aineistosta muodostettiin ennustemalli, jonka residuaalien avulla voidaan todeta, että ero käsittelyjen välillä on tilastollisesti merkittävä. PSII:n maksimitehokkuus korreloi kaasunvaihtomittausten tulosten kanssa, vahvistaen käsitystä siitä, että valoreaktioiden tehokkuus antaa hyvän kuvan koko fotosynteesikoneiston kapasiteetista.
  • Rojalin, Tatu (Helsingfors universitet, 2015)
    Raman spectroscopy is based on vibrations that occur between the atoms of a compound. The overall structural energy is derived from the electronical energy as well as vibrational, rotational and translational energy. In Raman spectroscopy the vibrational and rotational energies are essential. Usually the excitation energy used in Raman spectroscopy can be either in the region of visible light or NIR. The sample absorbs the energy and energy is also scattered back to all possible directions. Elastic scattering is called the Rayleigh scattering. In that case the back-scattered photons have an equal energy as the original excitation energy. However, some of the scattering happens inelastically and it forms the basis of Raman-phenomena. If the detected photons have smaller energy than the original, it is called the Stokes scattering. If the energy is bigger, it is anti-Stokes scattering. Raman is typically very rare and weak phenomenon. The spectral features in Raman spectra consist of the intensities and energies of the back scattered photons. Raman spectroscopy provides very accurate and detailed structural information on the molecule. It is basically a label-free technique with minimal need for sample preparation and the measurements can also be carried out e.g. through container walls. Further, Raman is quite insensitive to hydrous samples and it is suitable to solutions and biological assessments. However, there are some drawbacks that are formed by the luminescence phenomena i.e. fluorescence. Strong fluorescent backgrounds can mask the relevant Raman features in spectra because Raman and fluorescence are competetive processes. For instance many drug molecules have such structures that they cause strong fluorescence. It is also one of the reasons that pharmaceutical applications and measurements have been partly limited due to this problem. There are applications to improve and enhance a Raman signal. For example resonance phenomena and SERS are favored. To solve the fluorescence-related problems there are also means; one can change the laser wavelenght, photobleach the sample or apply different kinds of data manipulation techniques to the spectral data achieved. There are drawbacks with these methods. They can be slow, complex, damage the samples and still insufficient fluorescence suppression is a problem. In this study a novel time-gated CMOS-SPAD detection technique is applied to non-fluorescent and fluorescent drug measurements. The new detection system has a programmable on-chip delay time and it is synchronized with a picosecond pulsed laser. The scattered photons can be measured in the time scale when they are simultaneously measured in traditional energy and intensity wise. Raman scattering occurs in the timescale of sub-picoseconds while the fluorescence phenomena happen typically in the order of nanoseconds. This time difference can be exploited effectively to suppress the fluorescence. In the literature review of this study the basis of vibrational spectroscopy is introduced - especially Raman spectroscopy. The techniques related, as well as the novel time-resolved technique are covered. Further, different kinds of applications in the field of Raman spectroscopy are reviewed, mainly pharmaceutics-related and biologically relevant applications. In the experimental work the focus was to compare a continuous-wave 785 nm laser setup coupled with the CCD-detector to the pulsed picosecond 523 nm laser coupled with the CMOS-SPAD-detector. The measurements were performed on different kinds of drugs, both non-fluorescent and fluorescent. The aim was to obtain information on the effectiveness of CMOS-SPAD-technique on fluorescence suppression for solid drugs and solutions. Secondary goals were to collect knowledge on the similarities and differences between the Raman setups used for solution measurements, to optimize and discuss the key elements of setups for solids and solutions and to show preliminarily the applicability of the CMOS-SPAD-system on fluorescent drug's solutions as well as find out the requirements related to quantitative assessments using Raman spectroscopy. In drug research there is also constant need for reliable in vitro cell assays. The assessments made in this study may prove useful to the future applications e.g. measurements with living cells. An effective fluorescence suppression was achieved to strong fluorescent backgrounds using the novel time-resolved CMOS-SPAD-detection system coupled with the pulsed picosecond 532 nm laser. The setup is potentially a convenient tool to overcome many fluorescence-related limitations of Raman spectroscopy for laboratory and process analytical technology (PAT) use in the pharmaceutical setting. The results achieved encourage to consider that with careful calibration and method validation there is potential for quantitative analysis, biopharmaceutical and biological applications e.g. in vitro cell studies where most Raman techniques suffer from strong fluorescence backgrounds. Other potential fields for future applications can be also considered.
  • Salmi, Pauliina; Eskelinen, Matti A.; Leppänen, Matti T.; Pölönen, Ilkka (MDPI AG, 2021)
    Plants 2021, 10(2), 341
    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 < r < 0.96, p < 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 < r < 0.98, p < 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 < 0.001). Consequently, we described a simple imaging setup and information extraction based on vegetation indices that could be used to monitor algae cultures.
  • Mattlar, Viivi (Helsingin yliopisto, 2018)
    The literature review introduces the rapeseed chemical composition, the rapeseed protein isolate as a novel food ingredient, and protein oxidation. Rapeseed is an economically important oilseed crop since it is one of the largest sources of vegetable oil in the world. Rapeseed expeller is a protein-rich by-product of canola oil extraction. The main use of this protein-rich by-product is animal feed. However, it could be potentially utilized in the food industry, for example, as a source of protein in plant protein products, as a texture-improving ingredient in bakery products or as an alternative for animal proteins. We need more protein for human nutrition, and thus it is important to find new plants that can be used as protein sources. This way we can reduce environmental stress. Because the production process of rapeseed protein expeller already exists, it is a good new potential protein source. Protein and lipid oxidation are significant factors when food and nutrition quality are examined. The objective of this study was to optimize the protein extraction method and to examine the oxidation of rapeseed proteins and the lipid oxidation in two different rapeseed expellers. A few parameters, including extraction solvent (0.1 M and 1 M NaCl), pH (8 and 10), extraction time (4 h and overnight) and the removal of oil, were tested and the parameters that gave the biggest acquisition of soluble proteins were chosen for the experiment. The oxidation of rapeseed protein expeller was measured based on the loss of tryptophan fluorescence and the formation of carbonyls and dityrosine by using fluorescence spectrometry and based on the formation of hexanal by using the headspace gas chromatography method. Protein oxidation was measured in two different ways: in the rapeseed protein expeller during three months and in the extracted protein solution during seven days. The chosen extraction parameters were pH 8, 0.1 M NaCl solution and overnight extraction. The soluble protein amounts of the two rapeseed expeller samples were different after extraction, but this could be explained by the batches’ slightly different chemical compositions, especially their different cruciferin and napin ratios. During both the 7-day and 3-month oxidations, the tryptophan fluorescence decreased. During the 3-month oxidation, the formation of carbonyls increased and no hexanal was detected in any of the rapeseed expeller samples which were measured with headspace gas chromatography. The temperature and preservation time had a considerable effect on the protein oxidation in both the 7-day and 3-month oxidation tests, when only the loss of tryptophan was considered as an oxidation marker. The results revealed that fluorescence spectroscopy is a potential method for investigating the protein oxidation in the rapeseed protein expeller by using the loss of tryptophan and the formation of carbonyls as oxidation markers.