Studying fast dynamics in biological complexes : from photosynthesis in vivo to single DNA molecules in vitro
Farooq, Shazia - \ 2017
Wageningen University. Promotor(en): Herbert van Amerongen, co-promotor(en): Johannes Hohlbein. - Wageningen : Wageningen University - ISBN 9789463431002 - 149
biology - dna - proteins - interactions - probability analysis - förster resonance energy transfer - fluorescence - spectroscopy - photosynthesis - biologie - dna - eiwitten - interacties - waarschijnlijkheidsanalyse - förster resonantie-energieoverdracht - fluorescentie - spectroscopie - fotosynthese
During the last decades, fluorescence spectroscopy has emerged as a powerful tool in the fields of biophysics, biotechnology, biochemistry, cellular biology and the medical sciences. These techniques are highly sensitive, and allow us to study the structure and dynamics of (bio)molecular systems (Valeur 2001). A significant advantage of fluorescence techniques is that they can often be non-invasive and measurements can be performed in real time. In this thesis different advanced fluorescence methods will be used to study two important biological processes: (1) DNA dynamics and (2) plant photosynthesis. The first part aims at improving the smFRET technique for the analysis of DNA dynamics and other fast conformational changes. This improvement is made by combining and developing instrumentation and data evaluation tools. The second part is the continuous development of time-resolved fluorescence spectroscopy methods, as well their application in the field of photosynthesis to study ultrafast processes in thylakoid membranes and leaves. The two fluorescence techniques are technically and conceptually very different, but they are both designed for analysis of biomolecular systems. In this thesis, the techniques are applied to study energy transfer and dynamical changes in DNAs, thylakoid membranes and leaves.
REFERENCE: VALEUR B 2001. Molecular Fluorescence: Principles and Applications. 1 ed: Wiley-VCH.
Plantmonitoring op basis van fotosynthese sensoren : ontwikkelen en testen van sensoren
Dieleman, Anja ; Bontsema, Jan ; Jalink, Henk ; Snel, Jan ; Kempkes, Frank ; Voogt, Jan ; Pot, Sander ; Elings, Anne ; Jalink, Vincent ; Meinen, Esther - \ 2016
Bleiswijk : Wageningen UR Glastuinbouw (Rapport GTB 1405) - 86
teelt onder bescherming - glastuinbouw - kastechniek - sensors - fotosynthese - kooldioxide - energie - energiebesparing - verlichting - kunstlicht - kunstmatige verlichting - ventilatie - kunstmatige ventilatie - fluorescentie - tomaten - solanum lycopersicum - protected cultivation - greenhouse horticulture - greenhouse technology - sensors - photosynthesis - carbon dioxide - energy - energy saving - lighting - artificial light - artificial lighting - ventilation - artificial ventilation - fluorescence - tomatoes - solanum lycopersicum
The basic process for crop growth and production is photosynthesis. Measuring crop photosynthesis is therefore important to monitor the status of the crop and whether the greenhouse climate is set to the needs of the crop. In this project, two monitoring systems for crop photosynthesis were developed and tested. (1) The crop photosynthesis monitor is a soft sensor that can calculate the CO2 uptake of an entire crop. The basis for these calculations are the balance between CO2 supply and CO2 loss via ventilation and crop photosynthesis. By measuring the CO2 concentration and humidity inside and outside the greenhouse, the crop photosynthesis can be calculated. (2) The CropObserver is a fluorescence sensor that measures the light use efficiency of photosynthesis of a large crop area (3 x 3 m2). The crop receives light pulses from a laser in the top of the greenhouse, the sensor measures the fluorescence signal of the crop. Both sensors were tested in a tomato crop in 2014 with promising results. The sensors functioned without problems and delivered patterns of daily photosynthesis which matched the reference measurements reasonably well up to well.
Production very closely linked to amount of intercepted light : plant can use a lot of light
Heuvelink, E. ; Dueck, T.A. ; Noort, F.R. van; Kierkels, T. - \ 2015
In Greenhouses : the international magazine for greenhouse growers 4 (2015)4. - ISSN 2215-0633 - p. 34 - 35.
horticulture - greenhouse horticulture - greenhouses - tomatoes - pot plants - photosynthesis - lighting - fluorescence - temperature - light intensity - tuinbouw - glastuinbouw - kassen - tomaten - potplanten - fotosynthese - verlichting - fluorescentie - temperatuur - lichtsterkte
The photosynthetic process can hardly be bettered. But the utilisation of natural or artificial light certainly leaves room for improvement. In recent years our understanding of light has grown considerably and this has major implications on how we deal with light in horticulture.
Oral coatings: a study on the formation, clearance and perception
Camacho, S. - \ 2015
Wageningen University. Promotor(en): Kees de Graaf, co-promotor(en): Markus Stieger; F. van de Velde. - Wageningen : Wageningen University - ISBN 9789462575653 - 223
afdeklagen - eiwitten - orale toediening - tong - mond - smering - emulsies - in vivo experimenten - sensorische evaluatie - perceptie - dynamica - zoetheid - fluorescentie - coatings - proteins - oral administration - tongue - mouth - lubrication - emulsions - in vivo experimentation - sensory evaluation - perception - dynamics - sweetness - fluorescence
Oral coatings are residues of food and beverages that coat the oral mucosa after consumption. Several studies have reported on the lubrication properties in mouth, and the after-feel and after-taste impact of oral coatings. Further, oral coatings have been suggested to influence subsequent taste perception. Although it is well known that oral coatings can influence sensory perception, there was little information available on the chemical composition and physical properties of oral coatings. As such, the aim of this thesis was to understand which factors influence the composition of oral coatings and their sensory perception.
This study started with the development of an appropriate calibration method for an already described methodology to quantify oil oral coatings: in vivo fluorescence. Further, the samples studied were shifted from pure oil (used on previous studies) to a more realistic food beverage: o/w emulsions. Pig´s tongues are known to be a good model of human tongue. As such, Chapter 2 used pig´s tongues on the calibration of the method, to mimic the fluorescence in mouth of oil coatings. On chapter 2, Confocal Scanning Laser Microscopy images showed that stable o/w emulsions (1-20% (w/w)) stabilised by Na-caseinate created individual oil droplets on the surface of the pigs tongue, as such a new descriptor for oil coatings was developed. Oil fraction, i.e. mass of oil per surface area of the tongue, was shown to be higher on the back compared to the front anterior part of the tongue. This is thought to be due to the morphology of the tongue and abrasion of the oil coating owed to the rubbing with the palate. Further, in vivo measurements showed that oil fraction deposited on the tongue increased linearly with oil content of o/w emulsions. Coating clearance from the tongue was a fast process with around 60% of the oil being removed on the first 45s. After-feel perception (Fatty Film and Flavour Intensity) was shown to be semi-logarithmic related to oil fraction on the tongue.
Chapter 3, further investigated different properties of 10% (w/w) o/w emulsions that influence the oil fraction deposited on the tongue, its clearance and after-feel perception. Three different properties were studied: protein type, protein content and viscosity of the o/w emulsions. To study the influence of protein type, two different proteins which behave differently in-mouth were studied: Na-caseinate - creates emulsions which do not flocculate under in mouth conditions, and lysozyme – creates emulsions which flocculate under in mouth conditions. To study the influence of protein content, three concentrations of Na-caseinate and lysozyme were used (0.2, 3, 5.8% (w/w) all in excess to stabilize the water/oil interface). To study the influence of viscosity of o/w emulsions, three o/w emulsions stabilized with 3% (w/w) Na-caseinate were thickened with varying concentrations of xanthan gum (0-0.5%) (w/w).
Generally, the irreversible flocculation of lysozyme stabilized emulsions with saliva did not create a significant difference on oil deposition compared to emulsions stabilized with Na-caseinate, immediately after expectoration of the emulsions. Nevertheless, lysozyme stabilised emulsions caused slower oil clearance from the tongue surface compared to emulsions stabilized with Na-caseinate. Protein content had a negative relation with oil fraction on the tongue for lysozyme stabilized emulsions and no relation for Na-caseinate stabilized emulsions. The presence of thickener decreased deposition of oil on tongue, although viscosity differences (i.e., thickener content) did not affect oil fraction. After-feel perception of creaminess and fatty-film was strongly influenced by the presence of thickener likely due to lubrication in-mouth, i.e., the higher the concentration of thickener in the emulsions the stronger was the perception. Oral coatings perception was further influenced by the protein used in the emulsions, with Na-caseinate stabilised emulsions creating coatings with higher perception on creaminess and fatty-film.
Chapter 2 and chapter 3 provided knowledge on the deposition and clearance of oil coatings, but little was known on the formation of oil coatings. Chapter 4 focused on the formation of oil coatings formed by Na-caseinate stabilised o/w emulsions (1-20% (w/w)). The formation of oil coatings was a rapid process, where the maximum oil deposition was achieved at normal drinking behaviour (~3s). Further, in Chapter 4 we investigated the hypothesis often referred on literature, in which oil coatings form a physical barrier which prevents tastants to reach the taste buds, and thus create a reduction on taste perception. It was concluded that oil coatings formed by emulsions within one sip did not affect subsequent sweetness perception of sucrose solutions. We suggested that the oil droplets deposited on the tongue (as seen on chapter 2) did not form a hydrophobic barrier that is sufficient to reduce the accessibility of sucrose to the taste buds and consequently does not suppress taste perception.
Previous chapters focused on oral coatings formed by liquid o/w emulsions, however studies describing oral coatings formed by semi-solids and solids are scarce. As such, chapter 5 focused on the formation, clearance and sensory perception of fat coatings from emulsion-filled gels. Four emulsion-filled gelatin gels varying in fat content and type of emulsifier (whey protein isolate - created fat droplets bound to matrix; tween 20 - created fat droplets unbound to matrix) were studied. As in for oil coatings formed by liquid o/w emulsions, fat coatings formed by emulsion-filled gels reach their maximum deposition in the first seconds of mastication. This suggests that the first bites are the most relevant for the formation of fat coatings on the tongue. Further, fat fraction deposited on tongue increased when oral processing time of the gels increased. This trend was clearer for gels with higher fat content (15%) compared to gels with lower fat content (5%). Fatty perception increased with increasing mastication time, and decreased after expectoration with increasing clearance time. Fat fraction deposited on tongue and fatty perception are higher in gels with unbound droplets compared to bound droplets, as well as in gels with 15% fat compared to 5% fat.
To elucidate the role of protein on oral coatings, Chapter 6 focused on the development of a method to quantify protein in the oral coatings. Further, Chapter 6 studied the influence of protein content, in-mouth protein behaviour (lysozyme - protein which creates flocs with saliva vs. Na-Caseinate - protein which does not create flocs with saliva) and presence of thickener on the formation of protein oral coatings and sensory perception of protein coatings. Protein coatings were collected from the front and middle part of the anterior tongue using cotton swabs after subjects orally processed protein solutions for different time periods. Protein concentration of the coating (mass protein/mass coating) was quantified with the Lowry method. Similarly to oil/fat coatings, results show protein coatings are formed rapidly, reaching maximum deposition on the first seconds of the samples´ oral processing. Further, different protein in mouth-behaviour (Na-caseinate vs. lysozyme) did not create differences on protein deposition on the tongue. Presence of xanthan-gum in the processed samples decreased protein deposition on the tongue, compared to when samples without xanthan-gum were processed. The perception of protein coatings was strongly influenced by the viscosity and protein used in the samples. Higher viscosity of the samples lead to higher intensity on creaminess and thickness. Lysozyme samples created coatings with high sweetness and astringent intensity, which is related to the molecular structure of the protein.
Changes in the viscosity of beverages can cause changes in thickness perception. The changes in thickness perception can be accompanied by differences in other sensory properties, such as sweetness and creaminess which might be undesirable when reformulating beverages or developing new products. Knowledge on the differences by which viscosity of beverages can be modified to create a difference in sensory perception is currently lacking. Chapter 7 focus on the determination of the Just Noticeable Difference (the minimal difference that can be detected between two stimuli) for thickness perception of beverages. Oral thickness sensitivity (K=0.26) was found to be comparable to literature values for kinesthetic food firmness and spreadability, creaminess, sourness and bitterness perception.
The aim of this thesis was to determine and characterize factors influencing oral coatings and their sensory perception. For this purpose, reliable methods to quantify oil and protein deposited on the tongue had to be developed to later study the macronutrients deposition. Further, the influence of stimulus properties on the formation and clearance dynamics of oral coatings and their impact on sensory perception were investigated.
Probing functional (re)organisation in photosynthesis by time-resolved fluorescence spectroscopy
Ünlü, C. - \ 2015
Wageningen University. Promotor(en): Herbert van Amerongen. - Wageningen : Wageningen University - ISBN 9789462572829 - 118
algen - fotosynthese - light harvesting complexen - fotosysteem ii - fluorescentie - spectroscopie - chlamydomonas reinhardtii - algae - photosynthesis - light harvesting complexes - photosystem ii - fluorescence - spectroscopy - chlamydomonas reinhardtii
The possible mechanisms for reorganisation of outer LHCs of PSII (LHCII) upon state transitions in Chlamydomonas reinhardtii have been discussed for several decades [38, 43-54]. For a long time people adhered to the opinion that upon the transition from state 1 to state 2, 80% of LHCII detaches from PSII and attaches completely to PSI in Chlamydomonas reinhardtii [38, 45]. This thesis provides new insights for the mechanism of state transitions in Chlamydomonas reinhardtii. In the remainder of this thesis, the role of minor light-harvesting complexes in excitation energy transfer to reaction centers of photosystem II are discussed as well as multiexciton dynamics of the alloyed ZnCdTe quantum dots are studied in detail.
In chapter 2, we demonstrate with picosecond-fluorescence spectroscopy on C. reinhardtii cells that although LHCs indeed detach from Photosystem II in state-2 conditions only a fraction attaches to Photosystem I. The detached antenna complexes become protected against photodamage via shortening of the excited-state lifetime. It is discussed how the transition from state 1 to state 2 can protect C. reinhardtii in high-light conditions and how this differs from the situation in plants.
In chapter 3, we study the picosecond fluorescence properties of Chlamydomonas reinhardtti over a broad range of wavelengths at 77K. It is observed that upon going from state 1 (relatively high 680nm/720nm fluorescence ratio) to state 2 (low ratio), a large part of the fluorescence of LHC/PSII becomes substantially quenched, probably because of LHC detachment from PSII, whereas the fluorescence of PSI hardly changes. These results are in agreement with the proposal in chapter 2 that the amount of LHC moving from PSII to PSI upon going from state 1 to state 2 is very limited.
In chapter 4, we used picosecond-fluorescence spectroscopy to study excitation-energy transfer (EET) in thylakoids membranes isolated from A. thaliana wild-type plants and knockout lines depleted of either two (koCP26/24 and koCP29/24) or all minor Lhcs (NoM). In the absence of all minor Lhcs, the functional connection of LHCII to the PSII cores appears to be seriously impaired whereas the “disconnected” LHCII is substantially quenched. For both double knock-out mutants, excitation trapping in PSII is faster than in NoM thylakoids but slower than in WT thylakoids. In NoM thylakoids, the loss of all minor Lhcs is accompanied by an over-accumulation of LHCII, suggesting a compensating response to the reduced trapping efficiency in limiting light, which leads to a photosynthetic phenotype resembling that of low-light-acclimated plants. Finally, fluorescence kinetics and biochemical results show that the missing minor complexes are not replaced by other Lhcs, implying that they are unique among the antenna subunits and crucial for the functioning and macro-organization of PSII.
In chapter 5, we have performed picosecond fluorescence measurements on ZnCdTe ternary quantum dots at room temperature by using a streak-camera setup in order to investigate in detail the fluorescence kinetics for ZnCdTe quantum dots with different size and structure by using different excitation laser intensities. Our data show that the changes in fluorescence kinetics are mostly related to the changes in structure and size. In heterogeneous structured ZnCdTe quantum dots, the fluorescence kinetics become faster as compared to homogeneous structured ZnCdTe quantum dots. Also, in both homogeneous and heterogeneous ZnCdTe quantum dots, a new peak is observed in the high-energy region of the emission spectrum when using high excitation intensities, which shows that the radiative processes that occur from higher energy states become more favoured as the excitation intensity increases.
CF camera on platform (side crop viewer)
Gorbe Sanchez, E. - \ 2015
tuinbouw - glastuinbouw - kasproeven - tomaten - botrytis - stengels - fluorescentie - fotosynthese - opnameapparatuur - conferenties - horticulture - greenhouse horticulture - greenhouse experiments - tomatoes - botrytis - stems - fluorescence - photosynthesis - recording instruments - conferences
Experiment on how early the CF camera can detect botrytis infection in tomato stems inoculated with botrytis spores. Poster van PlantgezondheidEvent 12 maart 2015.
LUMINEX®: fast fluorescent detection : multiplex detection for the agricultural and food industries
Bergervoet, J.H.W. ; Currie, H.T. - \ 2014
precisielandbouw - teeltsystemen - gewasbescherming - voedselgewassen - detectie - enzymimmunoassay - elisa - immunoassay - voedselveiligheid - fluorescentie - precision agriculture - cropping systems - plant protection - food crops - detection - enzyme immunoassay - elisa - immunoassay - food safety - fluorescence
Luminex®: Detection of mycotoxins, pathogenic fungi, proteins, DNA/RNA
|Antibioticagebruik achterhaald via bot
Rijke, E. de; Raamsdonk, L.W.D. van; Nielen, M.W.F. - \ 2013
Voedingsmiddelentechnologie 23 (2013). - ISSN 0042-7934 - p. 30 - 31.
antibiotica - pluimveehouderij - vleeskuikens - fluorescentie - tests - voedselveiligheid - pluimveevlees - antibiotics - poultry farming - broilers - fluorescence - food safety - poultry meat
Om het ontstaan van antibioticaresistente bacteriën tegen te gaan, mogen pluimveehouders antibiotica alleen nog therapeutisch gebruiken. RIKILT ontwikkelde een fluorescentietest waarmee relatief eenvoudig en snel kan worden achterhaald of kuikens antibiotica hebben gekregen en ook of de antibiotica tegen ziekte of meer als groeibevorderaar zijn ingezet.
Kasklimaatregeling op basis van fotosynthese-metingen: wat zijn de mogelijkheden? : verslag van het eerste werkpakket van het project " Energie besparen door sturing van licht en CO2 op basis van gewasbehoefte"
Dieleman, J.A. ; Pot, S. ; Snel, J.F.H. ; Kromdijk, J. ; Jalink, H. ; Bontsema, J. - \ 2013
Wageningen : Wageningen UR Glastuinbouw (Rapporten GTB 1270) - 26
glastuinbouw - klimaatregeling - fotosynthese - gasuitwisseling - fluorescentie - meting - gewasmonitoring - behoeftenbepaling - lichtregiem - kooldioxide - greenhouse horticulture - air conditioning - photosynthesis - gas exchange - fluorescence - measurement - crop monitoring - needs assessment - light regime - carbon dioxide
Het klimaat in een kas wordt ingesteld om een optimale gewasfotosynthese, assimilatenverdeling en plantvorm te realiseren. Om momentaan het kasklimaat aan te kunnen passen aan de behoeftes van de plant is het van groot belang inzicht te hebben in de directe gevolgen van aanpassingen in het klimaat op de plant prestaties, in het bijzonder op de fotosynthese. Dit is te doen met de volgende methodes: 1. Gasuitwisseling van bladeren: nauwkeurige metingen van de fotosynthese van een stukje blad, met draagbare meetapparatuur. 2. Plantivity: een commercieel verkrijgbare meter die de fluorescentie van een stukje blad meet. 3. Kas-in-kas: een niet-geklimatiseerde meetkamer waarin CO 2 opname van een aantal planten gemeten kan worden. 4. Fluorescentie-imaging: fluorescentie metingen op afstand aan een groter oppervlakte gewas. 5. Fotosynthese-monitor: soft-sensor waarmee de CO 2 opname van een kas berekend wordt op basis van ventilatievoud en metingen van de CO 2 concentratie binnen en buiten de kas. Uit twee workshops met telers bleek dat zij fotosynthese als een belangrijk proces beschouwen in de teelt van hun gewas, en dat zij de fotosynthese van hun gewas graag momentaan online zouden willen meten. Het is daarom wenselijk door te gaan met de ontwikkeling van een robuust en betrouwbaar meetsysteem voor de gewasfotosynthese
Light harvesting and photoprotection in Cyanobacteria
Tian, L. - \ 2013
Wageningen University. Promotor(en): Herbert van Amerongen. - [S.l.] : s.n. - ISBN 9789461735294 - 167
cyanobacteriën - fotosynthese - light harvesting complexen - fluorescentie - lichtverdeling - cyanobacteria - photosynthesis - light harvesting complexes - fluorescence - light distribution
The process of photosynthesis has been studied for centuries, but despite a large amount of progress, there are still many aspects that are not fully understood. An important part of the progress is the fact that many structures of photosynthetic complexes have been resolved 1,2and these complexes have been studied separately in great detail, amongst other with ultrafast spectroscopic techniques. These studies allow to monitor excitation-energy transfer (EET) and charge separation (CS), the first crucial processes after the absorption of a photon. Many picosecond studies have also been performed in vivo in the past before the crystal structures were known, but due to an additional lack of knowledge about the organization and composition of the thylakoid membrane where most of the EET and CS processes take place, the obtained results were difficult to interpret. More recently, new interest has arisen in in vivo studies on photosynthetic organisms because a lot of molecular and organizational information has been obtained but also because the spectroscopic techniques have improved and mutants have become available that allow to study the effect of specific modifications in the organisms. This thesis focuses on the study of the light energy harvesting processes of photosynthetic complexes in cyanobacteria in general by using time-resolved fluorescence techniques, and with particular emphasis on the study of the in vivo protective process of non-photochemical quenching (NPQ) that is induced in the presence of high intensities of blue-green light.
Molecular mechanism of active photoprotein complex formation
Eremeeva, E.V. - \ 2013
Wageningen University. Promotor(en): Willem van Berkel; Ton Visser, co-promotor(en): E.S. Vyotski. - S.l. : s.n. - ISBN 9789461734587 - 194
photo-eiwitten - obeline - moleculaire structuur - bioluminescentie - fluorescentie - photoproteins - obelin - molecular conformation - bioluminescence - fluorescence
|Ziekzoeker in pootaardappelen : spectrale en chlorofylfluorescentie beeldanalyse van virus- en bacteriezieke pootaardappelen
Kamp, J.A.L.M. ; Blok, P.M. ; Polder, G. - \ 2012
Kennisakker.nl 2012 (2012)30 mei.
aardappelen - verwijderen van ongewenste planten - beeldverwerking - spectraalanalyse - fluorescentie - plantenziektebestrijding - pootaardappelen - akkerbouw - potatoes - roguing - image processing - spectral analysis - fluorescence - plant disease control - seed potatoes - arable farming
Dit onderzoek was gericht op het vinden van de meest geschikte opnametechniek voor het ziekzoeken, waarmee een eenduidig onderscheid te maken is tussen zieke en niet-zieke planten. Een belangrijk accent lag op het vroegtijdig herkennen van zieke planten. De nieuwe technologieën hebben vooral meerwaarde als het herkennen mogelijk wordt voordat de symptomen door het menselijk oog waarneembaar zijn. Op basis van de meetresultaten en discussies in de klankbordgroep kan geconcludeerd worden dat de technieken nog niet rijp zijn om naar de praktijk te brengen. Nader onderzoek is nodig.
Proefresultaten Ziekzoeker 2011 : spectrale en chlorofylfluorescentie beeldanalyse virus- en bacteriezieke pootaardappelen
Blok, P.M. ; Polder, G. ; Schoor, R. van der; Jalink, H. ; Kamp, J.A.L.M. - \ 2012
Lelystad [etc.] : Praktijkonderzoek Plant & Omgeving, Business Unit Akkerbouw, Groene Ruimte en Vollegrondsgroenten - 39
aardappelen - pootaardappelen - verwijderen van ongewenste planten - plantenziektebestrijding - beeldverwerking - spectraalanalyse - fluorescentie - akkerbouw - potatoes - seed potatoes - roguing - plant disease control - image processing - spectral analysis - fluorescence - arable farming
Een vroege detectie van zieke planten met moderne vision technieken kan de kosten voor selectie in de pootaardappelteelt flink drukken. De nadruk ligt hierbij op de detectie van Erwinia, gezien de grote financiële schade. In 2010 zijn een eerste serie metingen in pootaardappelen uitgevoerd met een bestaande opstelling voor detectie van viruszieke tulpenbollen. De resultaten hiervan waren niet goed genoeg. Daarom is dit onderzoek met name gericht op het vinden van de meest geschikte opnametechniek voor het ziekzoeken, waarmee een eenduidig onderscheid te maken is tussen zieke en niet-zieke planten. Een belangrijk accent ligt op het vroegtijdig herkennen van zieke planten. De nieuwe technologieën hebben vooral meerwaarde als het herkennen mogelijk wordt voordat de symptomen door het menselijk oog waarneembaar zijn.
Functional silicon nanoparticles
Ruizendaal, M.H. - \ 2011
Wageningen University. Promotor(en): Han Zuilhof; Ernst Sudhölter, co-promotor(en): Jos Paulusse. - [s.l.] : S.n. - ISBN 9789461730213 - 122
deeltjesgrootteverdeling - nanotechnologie - silicium - fluorescentie - synthese - particle size distribution - nanotechnology - silicon - fluorescence - synthesis
Diagnostics in biological systems are continuously searching for novel materials and systems for more specific detection with lower detection limits. Fluorescent probes are often used in the (selective) labeling of tissues and cellular components, since they give a high signal-to-noise ratio. The disadvantage of commonly used organic dyes is severe photobleaching, which makes prolonged studies impractical. Fluorescent quantum dots (QDs) are not prone to photobleaching, moreover, their fluorescence emission wavelength is size-tunable, making them the ideal candidates for bioimaging purposes.
Silicon nanoparticles (Si NPs) in particular have the additional advantage that the core consists of the non-toxic silicon, in contrast to the toxic elements (Cd, Se) typically employed in QDs. The development of a robust synthetic approach towards Si NPs, as well as a versatile functionalization strategy are therefore essential in enabling application in biological systems.
In Chapter 1, a general introduction on quantum dots (QDs) and in particular silicon nanoparticles is given; the origin of fluorescence is explained, and several synthetic methods are discussed. Chapter 2 describes the synthesis of Si NPs via the oxidation of magnesium silicide with bromine, yielding bromine-terminated Si NPs. Subsequent reaction with n-butyl lithium, and purification via column chromatography, resulted in butyl-terminated Si NPs. NMR analysis revealed that the major side-product (multiply brominated octane) was also in part attached to the Si NPs. Detailed characterization by IR and NMR confirmed the attachment of butyl-chains as well as a minor oxidation of the Si core. TEM measurements revealed a Si core size of 2.6 ± 0.7 nm. UV-Vis measurements showed a gradual increase in absorption with decreasing wavelengths, and a fluorescence emission maximum was observed at 390 nm (lexc= 340 nm). The Si NPs were fractionated using size exclusion chromatography, which yielded four fractions containing Si NPs of different sizes. Fluorescence anisotropy measurements, XPS and DOSY NMR spectroscopy confirmed the size-differences between the fractionated samples.The slope of the UV spectrum increases upon smaller Si NP size, whereas a shift in fluorescence emission maxima was observed from 383 to 445 nm (lexc= 340 nm), for respectively the smallest and largest Si NPs. Fluorescence quantum yields did not differ significantly between the different fractions, and the highest QY measured at lexc= 496 nm is 5.2 %. Fluorescence emission lifetimes did not reveal distinct difference in the differently sized Si NPs, most likely due to the relatively small size-differences between the fractions.
In Chapter 3 the synthesis of alkene-terminated Si NPs is described. To this purpose, the bromide-terminated Si NPs were reacted with 3-butenylmagnesium bromide. The resulting Si NPs were purified using SEC, and yielded Si NPs with a core size of 2.4 ± 0.5 nm as measured by TEM. Only minimal oxidation of the silicon core had occurred as observed by IR, while NMR spectroscopy confirmed successful attachment of the terminal alkenes onto the Si NPs. This also allowed for quantification of the amount of bromoalkanes attached to the Si NPs (butene : octane = 1 : 0.36). UV-Vis absorption of the Si NPs did not change significantly as compared to butyl-terminated Si NPs. The extinction coefficient was determined to be 0.14 (mg mL-1)-1 at 300 nm and 0.035 (mg mL-1)-1 at 350 nm. A fluorescence emission maximum was observed at 525 nm (lexc= 430 nm), while a QY of 7.1 ± 1.2 % was measured (lexc= 496 nm).
Modification of the alkene-terminated Si NPs using thiol-ene chemistry is described in Chapter 3. This reaction involves the radical-initiated coupling of a thiol to an alkene. The Si NPs were modified with thiolacetic acid, mercaptoethanol, thiolated triethyleneglycol monomethylether, and a thiolated polyethylene glycol 5000 monomethylether. The thiol-ene modification step did not significantly alter the photophysical properties of the Si NPs. Furthermore, IR and XPS showed that the functionalization step did not oxidize the silicon core. NMR and XPS results confirmed successful attachment of the functional thiols. In Chapter 4, carboxylic acid terminated Si NPs were synthesized by thiol-ene chemistry with 3 different spacer lengths; i.e. no spacer, a tetraethyleneglycol spacer and a PEG3000 spacer. The Si NPs were further functionalized by coupling an NH2-terminated single stranded DNA molecule via EDC/NHS chemistry. Coupling and subsequent hybridization with the complementary strand was confirmed by gel electrophoresis, UV-Vis and fluorescence spectroscopy. This revealed that 2 to 3 DNA strands were attached to the Si NPs. Finally, Chapter 5 describes the initial investigations in the toxicity of Si NPs. To this purpose, Si NPs were synthesized via reduction of silicon tetrachloride, followed by hydrosilylation with functional alkenes. The Si NPs were capped with –C3H6NH2, –C11H22N3and –C3H6COOH groups, yielding positively and negatively charged, as well as neutral Si NPs. Two types of tests were preformed: the MTT test for mitochondrial activity, and the BrdU test for cell proliferation, both in the presence and absence of FCS. Upon exposure of human colonic Caco2 cells, the negatively charged Si NPs showed no observable cytotoxic effects, whereas the N3-terminated Si NPs display a moderate toxicity with an IC50 of 500 mg/L in the presence of FCS in the MTT test, and the NH2-terminated Si NPs display strong cytotoxic effects on the cells with an IC50 of 20 mg/L in the presence of FCS in the MTT assay. The described synthesis, followed by the versatile functionalization and bioconjugation, in combination with the low inherent cytotoxicity, shows that the Si NPs are readily suitable for applications in biological systems.
|Meten of de plant zich lekker voelt: Fotosynthesemeter laat de plant spreken (interview met Henk Jalink)
Sleegers, J. ; Jalink, H. - \ 2011
Vakblad voor de Bloemisterij 66 (2011)12. - ISSN 0042-2223 - p. 32 - 33.
fotosynthese - meting - plantenfysiologie - fluorescentie - glastuinbouw - photosynthesis - measurement - plant physiology - fluorescence - greenhouse horticulture
WUR glastuinbouw werkt aan een meter die van planten continu de fotosynthese kan bepalen. Daarmee is direct vast te stellen hoe een gewas reageert op bijvoorbeeld een giet-beurt. In de toekomst is het wellicht mogelijk het kasklimaat te sturen op de plant.
Monitoren van plantkwaliteit via werking fotosynthese
Jalink, H. - \ 2010
sierteelt - gewaskwaliteit - fotosynthese - beeldanalyse - kwaliteitscontroles - monitoring - opnameapparatuur - fluorescentie - ornamental horticulture - crop quality - photosynthesis - image analysis - quality controls - monitoring - recording instruments - fluorescence
Door met de gepulste MIPS-LED camera opnamen te maken kan van hele planten objectief worden bepaald of de fotosynthese optimaal functioneert. Uit het beeld kan worden berekend of er schade op gaat treden of dat er al schade is ontstaan.
Chlorophyll Fluorescence of seeds A non-destructive marker foor seed maturity and seed quality
Jalink, H. - \ 2010
fluorescentie - chlorofyl - zaden - methodologie - kwaliteitszorg - rijpheid - fluorescence - chlorophyll - seeds - methodology - quality management - maturity
A method has been developed for the assessment of maturity and quality of seeds. The method is based on a non-destructive measurement of chlorop¬hyll-a in individual seeds.
Beoordeling van Zaadkwaliteit met behulp van Chlorofyl Fluorescentie Beelden
Jalink, H. - \ 2010
zaadkieming - chlorofyl - embryo's - fluorescentie - zaden - technieken - capsicum - glastuinbouw - seed germination - chlorophyll - embryos - fluorescence - seeds - techniques - capsicum - greenhouse horticulture
Tijdens het kiemingsproces van zaden wordt o.a. chlorofyl gevormd. Dit chlorofyl wordt aangemaakt door het embryo. Het is gebleken dat de toename van chlorofyl een maat is voor het verloop van het kiemingsproces. Dit chlorofyl kan gevoelig worden gemeten met een fluorescentietechniek.
Verkennend onderzoek naar blauwalgengroei in de woonomgeving : blauwalgen in stadswater
Lürling, M.F.L.L.W. ; Oosterhout, J.F.X. ; Beekman-Lukassen, W.D. ; Dam, H. van - \ 2010
Amersfoort : Stowa (Rapport / STOWA 2010 20) - ISBN 9789057734830 - 57
oppervlaktewater - plassen - stedelijke gebieden - aquatisch milieu - cyanobacteriën - monitoring - inventarisaties - kwantitatieve analyse - fluorescentie - oppervlaktewaterkwaliteit - noord-brabant - gelderland - surface water - ponds - urban areas - aquatic environment - cyanobacteria - monitoring - inventories - quantitative analysis - fluorescence - surface water quality - noord-brabant - gelderland
De leerstoelgroep Aquatische Ecologie en Waterkwaliteitsbeheer van Wageningen University is in 2006 begonnen met een inventarisatie van cyanobacteriënbloei in stedelijk water. De hoeveelheid cyanobacteriën, de soortensamenstelling, het voorkomen van drijflagen, de hoeveelheid gifstoffen en een aantal milieuvariabelen werden in kaart gebracht. Om een eerste indruk te verkrijgen van de cyanobacteriënbloei in oppervlaktewater in de woonomgeving, is in de zomer van 2006 (juli, augustus) een kleine selectie van 50 verschillende stadswateren in Noord-Brabant en Gelderland bemonsterd. Twee vijvers zijn gedurende 2006 intensiever bemonsterd om een indruk te verkrijgen van het verloop van de cyanobacteriënbloei in deze vijvers. De cyanobacteriën werden gekwantificeerd en onderscheiden van eukaryote algen met behulp van in vivo chlorofyl-a fluorescentie.
The development of FISH tools for genetic, phylogenetic and breeding studies in tomato (Solanum lycopersicum)
Szinay, D. - \ 2010
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Hans de Jong; Yuling Bai. - [S.l. : S.n. - ISBN 9789085856351 - 147
solanum lycopersicum - plantenveredeling - fylogenetica - fluorescentie - dna - chromosoomanalyse - methodologie - genexpressieanalyse - in situ hybridisatie - solanum lycopersicum - plant breeding - phylogenetics - fluorescence - dna - chromosome analysis - methodology - genomics - in situ hybridization
In this thesis various fluorescence in situ hybridization (FISH) technologies are described to support genome projects, plant breeding and phylogenetic analysis on tomato (Solanum lycopersicum, 2n=24). Its genome is 980 Mb and only 30 % are single copy sequences, which are mostly found in the euchromatin regions. These regions in all 12 chromosomes were therefore focus of the International Solanaceae Genome Sequencing Project. Based on the F2.2000 linkage map bacterial artificial chromosomes (BACs) were selected from three libraries for validating their physical locations by Fluorescence in situ Hybridization (FISH). In Chapter 2 I describe a five-color high-resolution BAC FISH approach and results of the mapping of 75 seed BACs on pachytene complements of chromosome 6. We found differences between the cytogenetic map and the linkage map. Most of the discrepancies occurred in the pericentromeric heterochromatin where recombination is highly suppressed. For establishing the BAC coverage of chromosome 6 a pooled BAC FISH method was used to hybridize all seed BACs simultaneously. A few larger gaps were discovered mostly on the long arm, where our ‘BAC-by-BAC’ sequencing approach could not manage to close the gaps by extending contigs. Afterwards new candidate BACs were tested by pooled-BAC FISH. Finally we demonstrated the heterochromatin / euchromatin distribution focusing on its borders by mapping pooled repetitive sequences (Cot 100) together with border BACs. In Chapter 3 the repeat content of chromosome 7 was analyzed by combining BAC and extended fiber FISH mapping with bioinformatics of 169 BACs. Repeats are important due to their challenging interpretations in genome sequencing. Tandem arrays of Tomato Genome Repeat I (TGRI) were found in BACs close to the distal end of chromosome 7 as well as on the long arm interstitial knobs. Phylogenetic analysis by neighbor-joining approach showed clustering of the TGRI blocks that suggested their independent origin. TGRI is likely to be transposed by extrachromosomal circular DNA molecules during anaphase. The dispersed TGR repeats (TGRII, TGRIII, TGRIV) all belong to the Ty3-Gypsy LTR class of retrotransposons. All of them cover the pericentromeric heterochromatin but overlap only partly as shown by FISH and BAC sequencing. TGRII hybridized through the whole pericentromere, TGRIII overlapped with TGRII except for the distal regions of the heterochromatin on the long arm, whereas TGRIV showed coverage in the most proximal parts of the short arm heterochromatin. BAC sequences corresponded well to the FISH data except that there were solo LTRs of TGRII found in the euchromatin. In the pericentromere heterochromatin truncated and solo LTRs were present of both TGRII and TGRIII. The TGRIV repeat could not be further investigated due to too high repeat content. Further this chapter offers some clues why TGR repeats are distributed in a certain way in the pericentromere.
In Chapter 4 a comparative mapping study was carried out between tomato and potato (Solanum tuberosum) chromosome 6 using BACs from both species. The BACs were hybridized on both species by FISH. Due to some repeat-rich BACs Cot 100 blocking was necessary as well as lowered stringent washing to achieve unique and clear signals. We detected a novel paracentric inversion on the short arm of chromosome 6. The two break points are close to the distal heterochromatin end and to the eu- heterochromatin border. The BAC order revealed colinearity on the long arm. The two investigated tomato cultivars- Heinz 1706 and Cherry VFNT- were colinear for all of the used BACs. One (RH98-856-18) out of six potato clones differed by a small rearrangement in the middle of the inversion. This study gave a first idea for evolutionary investigative studies in the Solanum genus using chromosomal rearrangements as detected by FISH and which are elaborated in Chapter 5. It is known that chromosomal rearrangements happen frequently, but rarely get fixed during evolution. The reason is that chromosomal rearrangements have often a negative influence on fertility and on the progeny. In Solanum mostly inversions were previously reported. We selected repeat poor and evenly distributed tomato and potato BACs and after labeling those by fluorescence dyes we hybridized them across related wild species, tomato breeding lines, potato, eggplant and pepper (which is a close relative outside of the genus). We could reveal synteny between these species. In this way we discovered five undescribed inversions and found discrepancies with previous literature claiming chromosomal rearrangements. Our results correspond well to published phylogeny on Solanum, suggesting that our approach would be suitable for studying unknown genomes and resolving relationships on a higher level, such as sections.
Finally this thesis discusses the crucial points of FISH technology; such as spatial resolution, detection sensitivity and applicability. It highlights the strength, weaknesses, opportunities and threads of FISH. In conclusion FISH is an indispensible technique for sequencing large genomes and defining repeat content with support of bioinformatics. Moreover, hidden chromosomal rearrangements can be visualized in regions where recombination is suppressed, which is important for plant breeding and definitely for phylogenetic studies.