Developing Bioimage Informatics – from Microscopy to Software Solutions – with alpha2beta1 Integrin as a Case Study

Biokuvainformatiikan kehittäminen – mikroskopiasta ohjelmistoratkaisuihin – sovellusesimerkkinä α2β1-integriini Kun ihmisen genomi saatiin sekvensoitua vuonna 2003, biotieteiden päätehtäväksi tuli selvittää eri geenien tehtävät, ja erilaisista biokuvantamistekniikoista tuli keskeisiä tutkimusmenetelmiä. Teknologiset kehitysaskeleet johtivat erityisesti fluoresenssipohjaisten valomikroskopiatekniikoiden suosion räjähdysmäiseen kasvuun, mutta mikroskopian tuli muuntua kvalitatiivisesta tieteestä kvantitatiiviseksi. Tämä muutos synnytti uuden tieteenalan, biokuvainformatiikan, jonka on sanottu mahdollisesti mullistavan biotieteet. Tämä väitöskirja esittelee laajan, poikkitieteellisen työkokonaisuuden biokuvainformatiikan alalta. Väitöskirjan ensimmäinen tavoite oli kehittää protokollia elävien solujen neliulotteiseen konfokaalimikroskopiaan, joka oli yksi nopeimmin kasvavista biokuvantamismenetelmistä. Ihmisen kollageenireseptori α2β1-integriini, joka on tärkeä molekyyli monissa fysiologisissa ja patologisissa prosesseissa, oli sovellusesimerkkinä. Työssä saavutettiin selkeitä visualisointeja integriinien liikkeistä, yhteenkeräytymisestä ja solun sisään siirtymisestä, mutta työkaluja kuvainformaation kvantitatiiviseen analysointiin ei ollut. Väitöskirjan toiseksi tavoitteeksi tulikin tällaiseen analysointiin soveltuvan tietokoneohjelmiston kehittäminen. Samaan aikaan syntyi biokuvainformatiikka, ja kipeimmin uudella alalla kaivattiin erikoistuneita tietokoneohjelmistoja. Tämän väitöskirjatyön tärkeimmäksi tulokseksi muodostui näin ollen BioImageXD, uudenlainen avoimen lähdekoodin ohjelmisto moniulotteisten biokuvien visualisointiin, prosessointiin ja analysointiin. BioImageXD kasvoi yhdeksi alansa suurimmista ja monipuolisimmista. Se julkaistiin Nature Methods -lehden biokuvainformatiikkaa käsittelevässä erikoisnumerossa, ja siitä tuli tunnettu ja laajalti käytetty. Väitöskirjan kolmas tavoite oli soveltaa kehitettyjä menetelmiä johonkin käytännönläheisempään. Tehtiin keinotekoisia piidioksidinanopartikkeleita, joissa oli "osoitelappuina" α2β1-integriinin tunnistavia vasta-aineita. BioImageXD:n avulla osoitettiin, että nanopartikkeleilla on potentiaalia lääkkeiden täsmäohjaussovelluksissa. Tämän väitöskirjatyön yksi perimmäinen tavoite oli edistää uutta ja tuntematonta biokuvainformatiikan tieteenalaa, ja tämä tavoite saavutettiin erityisesti BioImageXD:n ja sen lukuisten julkaistujen sovellusten kautta. Väitöskirjatyöllä on merkittävää potentiaalia tulevaisuudessa, mutta biokuvainformatiikalla on vakavia haasteita. Ala on liian monimutkainen keskimääräisen biolääketieteen tutkijan hallittavaksi, ja alan keskeisin elementti, avoimen lähdekoodin ohjelmistokehitystyö, on aliarvostettu. Näihin seikkoihin tarvitaan useita parannuksia,

Determination of macromolecular exchange and PO2 in the microcirculation: a simple system for in vivo fluorescence and phosphorescence videomicroscopy

We have developed a system with two epi-illumination sources, a DC-regulated lamp for transillumination and mechanical switches for rapid shift of illumination and detection of defined areas (250-750 µm²) by fluorescence and phosphorescence videomicroscopy. The system permits investigation of standard microvascular parameters, vascular permeability as well as intra- and extravascular PO2 by phosphorescence quenching of Pd-meso-tetra (4-carboxyphenyl) porphine (PORPH). A Pechan prism was used to position a defined region over the photomultiplier and TV camera. In order to validate the system for in vivo use, in vitro tests were performed with probes at concentrations that can be found in microvascular studies. Extensive in vitro evaluations were performed by filling glass capillaries with solutions of various concentrations of FITC-dextran (diluted in blood and in saline) mixed with different amounts of PORPH. Fluorescence intensity and phosphorescence decay were determined for each mixture. FITC-dextran solutions without PORPH and PORPH solutions without FITC-dextran were used as references. Phosphorescence decay curves were relatively unaffected by the presence of FITC-dextran at all concentrations tested (0.1 µg/ml to 5 mg/ml). Likewise, fluorescence determinations were performed in the presence of PORPH (0.05 to 0.5 mg/ml). The system was successfully used to study macromolecular extravasation and PO2 in the rat mesentery circulation under controlled conditions and during ischemia-reperfusion.

Coupling thin layer electrochemistry with epifluorescence microscopy: an expedient way of investigating electrofluorochromism of organic dyes

The first example of thin layer electrochemistry coupled to epifluorescence microscopy in the total internal reflectance mode is described and applied to the investigation of electrochemically modulated fluorescence of an organic dye (chloromethoxytetrazine) in solution. This technique allows to generate full redox switch of fluorescence when converting reversibly the dye into its anion radical, as well as to record the spectral features of the electrogenerated species. Recording simultaneously fluorescence intensity and lifetime along with coulombic charge as a function of the electrode potential will lead to a deep insight into the redox quenching mechanism.

Two-photon excitation with pico-second fluorescence lifetime imaging to detect nuclear association of flavanols

Two-photon excitation enabled for the first time the observation and measurement of excited state fluorescence lifetimes from three flavanols in solution, which were ∼1.0 ns for catechin and epicatechin, but <45 ps for epigallocatechin gallate (EGCG). The shorter lifetime for EGCG is in line with a lower fluorescence quantum yield of 0.003 compared to catechin (0.015) and epicatechin (0.018). In vivo experiments with onion cells demonstrated that tryptophan and quercetin, which tend to be major contributors of background fluorescence in plant cells, have sufficiently low cross sections for two-photon excitation at 630 nm and therefore do not interfere with detection of externally added or endogenous flavanols in Allium cepa or Taxus baccata cells. Applying two-photon excitation to flavanols enabled 3-D fluorescence lifetime imaging microscopy and showed that added EGCG penetrated the whole nucleus of onion cells. Interestingly, EGCG and catechin showed different lifetime behaviour when bound to the nucleus: EGCG lifetime increased from <45 to 200 ps, whilst catechin lifetime decreased from 1.0 ns to 500 ps. Semi-quantitative measurements revealed that the relative ratios of EGCG concentrations in nucleoli associated vesicles: nucleus: cytoplasm were ca. 100:10:1. Solution experiments with catechin, epicatechin and histone proteins provided preliminary evidence, via the appearance of a second lifetime (τ2 = 1.9–3.1 ns), that both flavanols may be interacting with histone proteins. We conclude that there is significant nuclear absorption of flavanols. This advanced imaging using two-photon excitation and biophysical techniques described here will prove valuable for probing the intracellular trafficking and functions of flavanols, such as EGCG, which is the major flavanol of green tea.

Subdiffraction fluorescence imaging of biomolecular structure and distributions with quantum dots

We introduce semiconductor quantum dot-based fluorescence imaging with approximately 2-fold increased optical resolution in three dimensions as a method that allows both studying cellular structures and spatial organization of biomolecules in membranes and subcellular organelles. Target biomolecules are labelled with quantum dots via immunocytochemistry. The resolution enhancement is achieved by three-photon absorption of quantum dots and subsequent fluorescence emission from a higher-order excitonic state. Different from conventional multiphoton microscopy, this approach can be realized on any confocal microscope without the need for pulsed excitation light. We demonstrate quantum dot triexciton imaging (QDTI) of the microtubule network of U373 cells, 3D imaging of TNF receptor 2 on the plasma membrane of HeLa cells, and multicolor 3D imaging of mitochondrial cytochrome c oxidase and actin in COS-7 cells.

Effect of bleaching on sound enamel and with early artificial caries lesions using confocal laser microscopy

The aim of this study was to evaluate effect of bleaching agents on sound enamel (SE) and enamel with early artificial caries lesions (CL) using confocal laser scanning microscopy (CLSM). Eighty blocks (4 × 5 × 5 mm) of bovine enamel were used and half of them were submitted to a pH cycling model to induce CL. Eight experimental groups were obtained from the treatments and mineralization level of the enamel (SE or CL) (n=10). SE groups: G1 - unbleached (control); G2 - 4% hydrogen peroxide (4 HP); G3 - 4 HP containing 0.05% Ca (Ca); G4 - 7.5% hydrogen peroxide (7.5 HP) containing amorphous calcium phosphate (ACP). CL groups: G5 - unbleached; G6 - 4 HP; G7 - 4 HP containing Ca; G8 - 7.5 HP ACP. G2, G3, G6, G7 were treated with the bleaching agents for 8 h/day during 14 days, while G4 and G8 were exposed to the bleaching agents for 30 min twice a day during 14 days. The enamel blocks were stained with 0.1 mM rhodamine B solution and the demineralization was quantified using fluorescence intensity detected by CLSM. Data were analyzed using ANOVA and Fisher's tests (α=0.05). For the SE groups, the bleaching treatments increased significantly the demineralization area when compared with the unbleached group. In the CL groups, no statistically significant difference was observed (p>0.05). The addition of ACP or Ca in the composition of the whitening products did not overcome the effects caused by bleaching treatments on SE and neither was able to promote remineralization of CL.

Bothropstoxin-I reduces evoked acetylcholine release from rat motor nerve terminals: Radiochemical and real-time video-microscopy studies

Understanding the biological activity profile of the snake venom components is fundamental for improving the treatment of snakebite envenomings and may also contribute for the development of new potential therapeutic agents. In this work, we tested the effects of BthTX-I, a Lys49 PLA2 homologue from the Bothrops jararacussu snake venom. While this toxin induces conspicuous myonecrosis by a catalytically independent mechanism, a series of in vitro studies support the hypothesis that BthTX-I might also exert a neuromuscular blocking activity due to its ability to alter the integrity of muscle cell membranes. To gain insight into the mechanisms of this inhibitory neuromuscular effect, for the first time, the influence of BthTX-I on nerve-evoked ACh release was directly quantified by radiochemical and real-time video-microscopy methods. Our results show that the neuromuscular blockade produced by in vitro exposure to BthTX-I (1 μM) results from the summation of both pre- and postsynaptic effects. Modifications affecting the presynaptic apparatus were revealed by the significant reduction of nerve-evoked [3H]-ACh release; real-time measurements of transmitter exocytosis using the FM4-64 fluorescent dye fully supported radiochemical data. The postsynaptic effect of BthTX-I was characterized by typical histological alterations in the architecture of skeletal muscle fibers, increase in the outflow of the intracellular lactate dehydrogenase enzyme and progressive depolarization of the muscle resting membrane potential. In conclusion, these findings suggest that the neuromuscular blockade produced by BthTX-I results from transient depolarization of skeletal muscle fibers, consequent to its general membrane-destabilizing effect, and subsequent decrease of evoked ACh release from motor nerve terminals. © 2012 Elsevier Ltd.

Grating coupled surface plasmon enhanced fluorescence spectroscopy

Over the last three decades, sensors based on the phenomenon of surface plasmon resonance have proven particularly suitable for real time thin film characterization, gas detection, biomolecular interaction examination and to supplement electrochemical methods. Systems based on prism coupling have been combined with fluorescence detection under the name of surface plasmon fluorescence spectroscopy to increase sensitivity even further. Alternatively, metal gratings can be employed to match photons for plasmon resonance. The real time monitoring of binding reactions not yet been reported in the combination of fluorescence detection and grating coupling. Grating-based systems promise more competitive products, because of reduced operating costs, and offer benefits for device engineering. This thesis is comprised of a comprehensive study of the suitability of grating coupling for fluorescence based analyte detection. Fundamental properties of grating coupled surface plasmon fluorescence spectroscopy are described, as well as issues related to the commercial realization of the method. Several new experimental techniques are introduced and demonstrated in order to optimize performance in certain areas and improve upon capabilities in respect to prism-based systems. Holographically fabricated gratings are characterized by atomic force microscopy and optical methods, aided by simulations and profile parameters responsible for efficient coupling are analyzed. The directional emission of fluorophores immobilized on a grating surface is studied in detail, including the magnitude and geometry of the fluorescence emission pattern for different grating constants and polarizations. Additionally, the separation between the minimum of the reflected intensity and the maximum fluorescence excitation position is examined. One of the key requirements for the commercial feasibility of grating coupling is the cheap and faithful mass production of disposable samples from a given master grating. The replication of gratings is demonstrated by a simple hot embossing method with good reproducibility to address this matter. The in-situ fluorescence detection of analyte immobilization and affinity measurements using grating coupling are described for the first time. The physical factors related to the sensitivity of the technique are assessed and the lower limit of detection of the technique is determined for an exemplary assay. Particular attention is paid to the contribution of bulk fluorophores to the total signal in terms of magnitude and polarization of incident and emitted light. Emission from the bulk can be a limiting factor for experiments with certain assay formats. For that reason, a novel optical method, based on the modulation of both polarization and intensity of the incident beam, is introduced and demonstrated to be capable of eliminating this contribution.

Atomic Force Microscopy Studies of the Amyloidogenic Processes of Intrinsically Unstructured Proteins related to Neurodegenerative Diseases

Protein aggregation and formation of insoluble aggregates in central nervous system is the main cause of neurodegenerative disease. Parkinson’s disease is associated with the appearance of spherical masses of aggregated proteins inside nerve cells called Lewy bodies. α-Synuclein is the main component of Lewy bodies. In addition to α-synuclein, there are more than a hundred of other proteins co-localized in Lewy bodies: 14-3-3η protein is one of them. In order to increase our understanding on the aggregation mechanism of α-synuclein and to study the effect of 14-3-3η on it, I addressed the following questions. (i) How α-synuclein monomers pack each other during aggregation? (ii) Which is the role of 14-3-3η on α-synuclein packing during its aggregation? (iii) Which is the role of 14-3-3η on an aggregation of α-synuclein “seeded” by fragments of its fibrils? In order to answer these questions, I used different biophysical techniques (e.g., Atomic force microscope (AFM), Nuclear magnetic resonance (NMR), Surface plasmon resonance (SPR) and Fluorescence spectroscopy (FS)).

Fluorescence-encoded gold nanoparticles: library design and modulation of cellular uptake into dendritic cells.

In order to harness the unique properties of nanoparticles for novel clinical applications and to modulate their uptake into specific immune cells we designed a new library of homo- and hetero-functional fluorescence-encoded gold nanoparticles (Au-NPs) using different poly(vinyl alcohol) and poly(ethylene glycol)-based polymers for particle coating and stabilization. The encoded particles were fully characterized by UV-Vis and fluorescence spectroscopy, zeta potential and dynamic light scattering. The uptake by human monocyte derived dendritic cells in vitro was studied by confocal laser scanning microscopy and quantified by fluorescence-activated cell sorting and inductively coupled plasma atomic emission spectroscopy. We show how the chemical modification of particle surfaces, for instance by attaching fluorescent dyes, can conceal fundamental particle properties and modulate cellular uptake. In order to mask the influence of fluorescent dyes on cellular uptake while still exploiting its fluorescence for detection, we have created hetero-functionalized Au-NPs, which again show typical particle dependent cellular interactions. Our study clearly prove that the thorough characterization of nanoparticles at each modification step in the engineering process is absolutely essential and that it can be necessary to make substantial adjustments of the particles in order to obtain reliable cellular uptake data, which truly reflects particle properties.

Total phytoplankton abundance and biomass in the surface and fluorescence maximum layers and contributions of dominant species to their values in the Werstern Arctic in July-August 2003

Phytoplankton community was studied in the Bering Strait and over the shelf, continental slope, and deep-water zones of the Chukchi and Beaufort Seas in the middle of the vegetative season (July-August 2003). Its structure was analyzed in relation to ice conditions and seasonal patterns of water warming, stratification, and nutrient concentrations. Overall variations in phytoplankton abundance from 200 to 6000000 cells/l and biomass from 0.1 to 444.1 µg C/l.were estimated. The bulk of phytoplankton cells concentrated in the seasonal picnocline at depths 10-25 m. The highest values of cell abundance and biomass were recorded in regions influenced by inflow of Bering Sea waters or characterized by intense hydrodynamics, such as the Bering Strait, Barrow Canyon, and the outer shelf and slope of the Chukchi Sea. In the middle of the vegetative season, phytoplankton in the study region of the Western Arctic proved to comprise three successional (seasonal) assemblages: early spring, late spring, and summer assemblages. Their spatial distribution was dependent mainly on local features of hydrological and nutrient regimes rather than on general latitudinal trends of seasonal succession characteristic of arctic ecosystems.

Contiunous surface water uncorrected and quench-corrected active fluorescence and derived chlorophyll a concentration using calibration by HPLC pigments during POLARSTERN cruise ANT-XXV/1

The relationship between phytoplankton assemblages and the associated optical properties of the water body is important for the further development of algorithms for large-scale remote sensing of phytoplankton biomass and the identification of phytoplankton functional types (PFTs), which are often representative for different biogeochemical export scenarios. Optical in-situ measurements aid in the identification of phytoplankton groups with differing pigment compositions and are widely used to validate remote sensing data. In this study we present results from an interdisciplinary cruise aboard the RV Polarstern along a north-to-south transect in the eastern Atlantic Ocean in November 2008. Phytoplankton community composition was identified using a broad set of in-situ measurements. Water samples from the surface and the depth of maximum chlorophyll concentration were analyzed by high performance liquid chromatography (HPLC), flow cytometry, spectrophotometry and microscopy. Simultaneously, the above- and underwater light field was measured by a set of high spectral resolution (hyperspectral) radiometers. An unsupervised cluster algorithm applied to the measured parameters allowed us to define bio-optical provinces, which we compared to ecological provinces proposed elsewhere in the literature. As could be expected, picophytoplankton was responsible for most of the variability of PFTs in the eastern Atlantic Ocean. Our bio-optical clusters agreed well with established provinces and thus can be used to classify areas of similar biogeography. This method has the potential to become an automated approach where satellite data could be used to identify shifting boundaries of established ecological provinces or to track exceptions from the rule to improve our understanding of the biogeochemical cycles in the ocean.

Fluorescence-intensity distribution analysis and its application in biomolecular detection technology

A methodology, fluorescence-intensity distribution analysis, has been developed for confocal microscopy studies in which the fluorescence intensity of a sample with a heterogeneous brightness profile is monitored. An adjustable formula, modeling the spatial brightness distribution, and the technique of generating functions for calculation of theoretical photon count number distributions serve as the two cornerstones of the methodology. The method permits the simultaneous determination of concentrations and specific brightness values of a number of individual fluorescent species in solution. Accordingly, we present an extremely sensitive tool to monitor the interaction of fluorescently labeled molecules or other microparticles with their respective biological counterparts that should find a wide application in life sciences, medicine, and drug discovery. Its potential is demonstrated by studying the hybridization of 5′-(6-carboxytetramethylrhodamine)-labeled and nonlabeled complementary oligonucleotides and the subsequent cleavage of the DNA hybrids by restriction enzymes.

Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor.

We extend the sensitivity of fluorescence resonance energy transfer (FRET) to the single molecule level by measuring energy transfer between a single donor fluorophore and a single acceptor fluorophore. Near-field scanning optical microscopy (NSOM) is used to obtain simultaneous dual color images and emission spectra from donor and acceptor fluorophores linked by a short DNA molecule. Photodestruction dynamics of the donor or acceptor are used to determine the presence and efficiency of energy transfer. The classical equations used to measure energy transfer on ensembles of fluorophores are modified for single-molecule measurements. In contrast to ensemble measurements, dynamic events on a molecular scale are observable in single pair FRET measurements because they are not canceled out by random averaging. Monitoring conformational changes, such as rotations and distance changes on a nanometer scale, within single biological macromolecules, may be possible with single pair FRET.