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The application of fluorescence in drug discovery, high-throughout screenings in genomics and proteomics is and will be evidently successful. The increased use of fluorescence techniques is greatly enhanced by the improved instrumentation pioneered by inventive scientists and now made available commercially by several high-tech companies. Moreover, the design and development of many new molecular probes with higher selectivity for specific microenvironmental properties has stimulated many new researchers to employ fluorescence techniques for solving their problems. Probably the most significant breakthrough in fluorescence is its use in detection of single molecules and even of their real-time dynamics. Also, probing inside living cells has become a hot topic in the life sciences. This topic book reflects the updates of scientific progress presented by frontline researchers.



Fluorescence Spectroscopy: New Approaches and Probes


Chapter 1. Advanced Luminescent Labels, Probes and Beads and their Application to Luminescence Bioassay and Imaging

The design of fluorescent probes (and labels) is as challenging as it ever was. Such probes enable studies on the molecular dimensions and dynamics of even complex (bio)matter, but also bioanalytical and screening assays whose sensitivity can reach the single molecule level. The design of advanced labels for bioassays is paralleled by developments in (laser) fluorescence spectroscopy, opto-electronics and data processing. Light-emitting diodes (LEDs) and diode lasers (DLs) are particularly attractive light sources and we therefore have focused our research (a) on labels that are LED- or DL-compatible, and (b) on applications of such labels to various analytical formats.
O. S. Wolfbeis, M. Böhmer, A. Dürkop, J. Enderlein, M. Gruber, I. Klimant, C. Krause, J. Kürner, G. Liebsch, Z. Lin, B. Oswald, M. Wu

Chapter 2. Fluorescence Spectral Engineering — Biophysical and Biomedical Applications

Fluorescence spectroscopy is a central research tool in biology and has also become the dominant method enabling the revolution in biotechnology. At present, almost all fluorescence experiments are performed in a free space at condition, in which the oscillating dipole can radiate isotropically into space. We now describe a new opportunity, fluorescence spectral engineering (FSE), in which metallic surfaces or particles are used to modify the emission rates and spatial distribution of the emission. In this article we first summarize results from the physics literature which demonstrate the effects of metallic surfaces, colloids or islands to increase or decrease emissive rates, increase the quantum yields of low quantum yield chromophores, decrease the lifetimes, increase the distances for resonance energy transfer, and to direct the typically isotropic emission in specific directions. This latter effect is not the result of reflection, but rather as the result of the metal modifying the photon mode density surrounding the fluorophores and the fluorophore dipole interacting with free electrons in the metal. We then show experimental results, which demonstrate increased quantum yields, decreased lifetimes, increased photostability, and increased energy transfer distances near metal particles. Fluorescence spectral engineering provides opportunities for new types of fluorescence experiments and assays.
J. R. Lakowicz, I. Gryczynski, Y. Shen, J. Malicka, S. D’Auria, Z. Gryczynski

Chapter 3. Fluorescence Nanometrology in Sol-Gels

We describe recent fluorescence studies of the formation dynamics and structure of sol-gel glasses from nanometre particles composed of silica clusters in sols to nanometre pores in silica gels. The “kinetic life-history” of silica produced under both acidic and alkaline conditions from sodium silicate in a hydrogel and from an alkoxide in an alcogel is now starting to be revealed by fluorescence techniques and the influence of key parameters such as pH and silica concentration quantified at the molecular level. Through careful choice of fluoro-probe, anisotropy decay has been shown to provide particle size as well as viscosity information and offer advantages over traditional techniques for silica particle sizing based on small angle neutron, X-ray or light scattering. Fluorescence resonance energy transfer (FRET) can now be used to determine the donor-acceptor spatial distribution function without making any a-priori assumptions as to its form. This in turn promises to make FRET a better means of monitoring pore morphology in the wet gel during drying and ageing, offering distinct advantages over dry gel techniques such as mercury porosimetry and nitrogen adsorption. The insight into sol-gel processes provided by these new interpretations of fluorescence decay data promises to have implications for both our fundamental understanding and the production of sol-gel systems.
D. J. S. Birch, C. D. Geddes, J. Karolin, R. Leishman, O. J. Rolinski

Chapter 4. Integrated Supramolecular Systems: From Sensors to Switches

Integrated supramolecular systems with a receptor built in a photo- or electroactive unit have been reviewed with the focus on their particular electronic properties and different photochemical and electrochemical processes which make them suitable for cation sensing or switching. The fluoroionophores with an electron donating ionophore have been the most investigated and their initial weakness related to cation decoordination in the excited state. The small blue-shift of the fluorescence spectrum and the slight change of the emission quantum yield upon cation complexation, have now been overcome by a careful combination of several donor and acceptor units, which provide new low-lying excited states decoupled from the complexed ionophore and by using TICT probes where the electronic coupling between the D and A parts is too small to induce decoordination of the cation during the excited state lifetime. On the contrary the switching action requires that the binding ability of the ionophore be lowered or increased on a larger time scale. This has been done by electrochemical oxidation and by insertion of the ionophore into a photochromic system. Differences in binding ability of three to four orders of magnitude have been obtained and it is our belief that integrated supramolecular systems combining an ionophore and a photochromic moiety (photoionochromics) will be for cation switching as successfull as integrated fluoroionophores have been for sensing cations.
J.-P. Malval, I. Gosse, J.-P. Morand, R. Lapouyade

Chapter 5. Ratiometric Probes: Design and Applications

With the aim of substantial improvement of solvatochromic and electrochromic fluorescence probes by coupling their response with an excitedstate reaction, a series of 3-hydroxychromone derivatives have been synthesized. They demonstrate two well-separated emission bands that originate from normal and phototautomer forms due to excited-state intramolecular proton transfer (ESIPT) reaction. In the studies of solvent polarity effect, electrochromism (internal Stark effect) and red edge effect a strong amplification of probe response is achieved by recording of relative intensity ratios of these fluorescence bands instead of their spectral shifts. This result is an illustration of a new principle in design of fluorescence probes, which may allow the achievement of almost perfect on-off switching behavior.
A. P. Demchenko, A. S. Klymchenko, V. G. Pivovarenko, S. Ercelen

Chapter 6. Binding of Ethidium to Yeast tRNAPhe: A New Perspective on an Old Bromide

We have reinvestigated the binding of ethidium bromide (EB) to yeast tRNAphe using frequency domain fluorometry and Global Analysis. Previous fluorescence investigations of EB — tRNA interactions, carried out for more than 30 years, have indicated a “strong” binding site with a lifetime near 26 ns and one or more “weak, non-specific” binding sites with reduced lifetimes. In our study, under specific conditions in which only one EB is bound, a fluorescence lifetime of 27 ns was obtained. However, as the EB/ tRNA ratio increased, shorter lifetime components appeared. Global Analysis of the lifetime data was consistent with a model in which the second EB molecule bound has a lifetime of only 5.4 ns. Global Analysis also indicated that this second binding event leads to a reduction in the lifetime of the first EB bound, namely from 27 ns to 17.7 ns. The lifetime decrease associated with the “strong” binding site could be due to a quenching process arising either from energy transfer between EB molecules or from alterations in the conformation of the tRNA, or both. These results are considered in light of recent NMR observations on an EB/tRNA system. We also investigated the effect of ionic strength on the lifetime and relative affinities of these two binding components and found that NaCl levels up to 900 mM did not significantly affect the results.
M. Tramier, O. Holub, J. C. Croney, T. Ishi, S. E. Seifried, D. M. Jameson

Chapter 7. Experimental Aspects of DNA Computing by Blocking: Use of Fluorescence Techniques for Detection

The suitability of fluorescence techniques for the experimental implementation of a DNA based algorithm was studied. Two different assays based on the use of PicoGreen and FRET, respectively, have been used for the detection of hybridisation. The limitations of both assays are discussed.
K. A. Schmidt, C. v. Henkel, G. Rozenberg, H. P. Spaink

Fluorescence Spectroscopy of Single Molecules and Molecular Assemblies


Chapter 8. Multiparametric Detection of Fluorescence Emitted from Individual Multichromophoric Systems

While few years ago the main goal in room temperature single molecule fluorescence spectroscopy (SMS) was to visualize individual molecules, nowadays experiments are designed such that multiparametric observation of different fluorescence characteristics of the investigated molecular systems is allowed. The simultaneous observation of fluorescence characteristics such as spectral peak position, fluorescence decay times, polarization properties or wavelength integrated intensity of fluorescence during the survival time of the investigated molecules leads to a more detailed picture of the molecular states and environment changes experienced by the probed molecules. In this contribution a diffraction-limited scanning stage confocal microscope set-up allowing real time multiparametric observation of fluorescence detected from single molecules immobilized in thin polymer matrix is described. By using pulsed excitation in combination with burst integrated fluorescence lifetime (BIFL) type detection, the simultaneous acquisition of fluorescence spectra, wavelength integrated fluorescence intensity time traces and time-resolved decay curves is demonstrated for synthetic as well as biological systems. By using continuous wave excitation and BIFL type detection, two dimensional fluorescence intensity time traces containing photons resolved in time with an accuracy of 50 ns and carrying polarization information can be recorded from individual immobilized molecules. In combination with specific analysis procedures, the SMS set-up is proved to be a suitable tool for the identification of different emitting species as well as for monitoring dynamical processes at the single molecule level.
M. Cotlet, J. Hofkens, M. Maus, F. C. de Schryver

Chapter 9. Fluorescence Intensity Distribution Analysis (FIDA) and related fluorescence fluctuation techniques: theory and practice

Fluorescence fluctuation methods are a versatile means of probing molecular interactions with single molecule sensitivity. We review a family of such techniques that are centrally concerned with the monitoring of the fluorescence intensity of dilute, heterogeneous samples through histogramming photon counts in consecutive time intervals. The subsequent fitting of a theoretical model to this intensity data resolves and quantifies different molecular species present in the sample. Each of these methods shares a common confocal experimental set-up with fluorescence detection using avalanche photodiodes. Using the “parent” method, fluorescence intensity distribution analysis (FIDA), it is possible to determine both the concentration and specific brightness values of a number of fluorescent species in solution. FIDA may be extended to enable simultaneous monitoring of two different polarization states or spectral bands (2D-FIDA). Further pertinent molecular information can be revealed through measurement of the fluorescence lifetime or the diffusion coefficient of the sample under investigation. It is possible to extend FIDA to incorporate measurement of either of these properties. In the case of fluorescence intensity and lifetime distribution analysis (FILDA) simultaneous determinations of concentration, brightness and fluorescence lifetime are made. Fluorescence intensity multiple distribution analysis (FIMDA) enables simultaneous measurement of the concentration, brightness and diffusion coefficient.
P. Kask, C. Eggeling, K. Palo, Ü. Mets, M. Cole, K. Gall

Chapter 10. Single Molecule Reactions of the Enzyme LDH and of Restriction Endonucleases in the Fluorescence Microscope

Two types of single molecule enzyme reactions can be directly observed in the fluorescence microscope: reactions, which convert nonfluorescing small substrate molecules into fluorescing products (or vice versa) and reactions of enzymes on macromolecules stained by a fluorescence dye or visualized otherwise. As an example of the first type of reaction, the conversion of nonfluorescent NAD+ into fluorescing NADH, or vice versa, by a few molecules of lactate dehydrogenase in femtodroplets is described. The femto-droplet-pipetting method is essentially a subattomol technique with high accuracy. Lineweaver Burk plots are obtained with approximately the kinetic constants of the enzyme known from conventional biochemistry. On the other hand, the femtodroplet-in-substrate method allows the observation of the action of individual enzyme molecules. The second type of single molecule enzyme reactions is the sequence-specific cutting of individual DNA molecules held by optical tweezers. It is shown that such molecules can be characterized by the cutting (restriction) pattern generated by the restriction endonucleases ApaI, SmaI and EcoRI.
B. Nasanshargal, B. Schäfer, K. O. Greulich

Chapter 11. Monitoring γ-Subunit Movement in Reconstituted Single EF°F1 ATP Synthase by Fluorescence Resonance Energy Transfer

The membrane-bound enzymes H+ ATP synthases contain two coupled rotary motors that drive catalysis. We applied a single molecule spectroscopy approach to monitor the internal rotation of the γ-subunit of the F1 part against its static counterpart, the b-subunits of the F0 part. We specifically attached two fluorophores to H+ ATP synthase from E. coli, namely Cy5 at the γ-subunit and tetramethylrhodamine at one b-subunit. After reconstitution into liposomes, these enzymes regained their full catalytic activity as measured by ATP synthesis rates. Fluorescence resonance energy transfer (FRET) was monitored in photon bursts of freely diffusing proteoliposomes using a confocal setup for single molecule detection. Incubation with non-hydrolyzable AMPPNP resulted in stable intensity ratios within a photon burst. This corresponds to a fixed γ-subunit orientation. We detected three different FRET efficiencies, i.e., γ-subunit orientations. After addition of ATP a consecutive order of three distinguishable FRET efficiencies was observed within the bursts, indicating a stepwise unidirectional γ-subunit movement against the b-subunits.
M. Börsch, M. Diez, B. Zimmermann, R. Reuter, P. Gräber

Application of Fluorescence in Biological Membrane and Enzyme Studies


Chapter 12. Application of the Wavelength-selective Fluorescence Approach to Monitor Membrane Organization and Dynamics

Wavelength-selective fluorescence comprises a set of approaches based on the red edge effect in fluorescence spectroscopy, which can be used to monitor directly the environment and dynamics around a fluorophore in a complex biological system. A shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a shift in the excitation wavelength toward the red edge of the absorption band, is termed red edge excitation shift (REES). This effect is mostly observed with polar fluorophores in motionally restricted media such as very viscous solutions or condensed phases where the dipolar relaxation time for the solvent shell around a fluorophore is comparable to or longer than its fluorescence lifetime. REES arises from slow rates of solvent relaxation (reorientation) around an excited state fluorophore, which is a function of the motional restriction imposed on the solvent molecules in the immediate vicinity of the fluorophore. Utilizing this approach, it becomes possible to probe the mobility parameters of the environment itself (which is represented by the relaxing solvent molecules) using the fluorophore merely as a reporter group. Furthermore, since the ubiquitous solvent for biological systems is water, the information obtained in such cases will come from the otherwise “optically silent” water molecules. This makes REES and related techniques extremely useful since hydration plays a crucial modulatory role in a large number of important cellular events including lipid-protein interactions and ion transport. The application of REES and related techniques (wavelength-selective fluorescence approach) as a powerful tool to monitor organization and dynamics of probes and peptides bound to membranes and membrane-mimetic medium such as micelles is discussed.
A. Chattopadhyay

Chapter 13. Fluorescence Approaches for the Characterization of the Peripheral Membrane Binding of Proteins Applied for the Blood Coagulation Protein Prothrombin

Different fluorescence spectroscopic approaches for the characterization of the peripheral membrane binding of proteins are summarized and compared for the blood coagulation protein prothrombin. Basically, the problem of membrane binding can be approached from both the protein side, using either intrinsic tryptophan fluorescence or dye-labeled protein, as typically employed in fluorescence recovery after photobleaching experiments, or from the membrane side, studying solvent relaxation, fluorescence anisotropy or excimer formation of suitable membrane probes. For the case of prothrombin and its fragment 1 the combined use of these fluorescence methods shows a slightly tighter binding of the fragment 1 portion in the case of the entire prothrombin molecule compared to the isolated fragment 1 while no evidence could be obtained for hydrophobic membrane binding sites in the “nonfragment 1” part of prothrombin. Tryptophan fluorescence of fragment 1 is able to detect differences in binding in response to the kind of procoagulant membrane lipid.
R. Hutterer, M. Hof

Chapter 14. Assessment of Membrane Fluidity in Individual Yeast Cells by Laurdan Generalised Polarisation and Multi-photon Scanning Fluorescence Microscopy

Here we describe techniques that we developed for monitoring membrane fluidity of individual yeast cells during environmental adaptation and physiological changes. Multi-photon scanning fluorescence microscopy using laurdan as a membrane probe enables determination whether fluidity changes seen by spectroscopy reflect universal responses or changes only of subpopulations.
R. P. Learmonth, E. Gratton

Chapter 15. Formation of Higher Order Signal Transduction Complexes as Seen by Fluorescence Spectroscopy

Fluorescence spectroscopy is the primary method to view the interactions between membrane-bound proteins in real time. We have been using fluorescence homo- and heterotransfer methods to follow the associations and oligomerization of membrane associated proteins. One system we have focused on is the G-protein-phospholipase Cβ signaling system. This pathway is responsible for transducing extracellular signals that bind to heptahelical surface receptors such as ions, hormones and neurotransmitters. Binding of these agents activate heterotrimeric G proteins which activates phospholipase Cβ (PLCβ), which in turn causes an increase in intracellular calcium and an activation of protein kinase C. Using a combination of fluorescence, biochemical and molecular biology techniques, we have measured the interaction energies of these proteins taking into account their dependence on the surface area of the lipid membrane. We find that activation of PLCβ is mediated by the pleckstrin homology domain, a structural module found in a wide variety of signal transduction proteins. Activation of the catalytic domain by the PH domain plays an analogous role in proteins that are not activated by G proteins. Using energy transfer, we also find that both PLCβ and G proteins have secondary binding sites for each other and for a protein that regulates G protein signaling (RGS). Our data suggest that in cells these proteins form stable signaling complexes capable of mediating high output, rapid and localized signals. We are currently investigating the role that lipid domain or rafts have in stabilizing these protein interactions.
L. Dowal, S. Scarlata

Chapter 16. Mechanisms of the Modulation of Membrane Interfacial Enzyme Catalysis by Non-lamellar Forming Lipids: Comparison with the Behavior of a Fluorescent Probe in Membranes

The activities of several membrane proteins are modulated by the presence of non-lamellar forming lipids. Although several proteins are activated by the presence of these lipids, the molecular mechanism by which this activation occurs is different for different proteins. We have studied two enzymes that are activated by non-lamellar forming lipids in different ways; protein kinase C (PKC) and CTP:phosphocholine cytidylyltransferase (CT). CT is the enzyme that regulates the rate of synthesis of phosphatidylcholine. For many lipid systems, there is a quantitative correlation between the calculated curvature strain of the membrane and the activation of this enzyme. For many lipid systems, including liposomes containing a series of homologous di-18:l phosphatidylethanolamines, there is a quantitative correlation between the extent of activation of CT and the curvature strain in the membrane. In contrast, the order in which this series of di-18:l phosphatidylethanolamines enhances the activity of protein kinase C does not correlate with membrane curvature strain. However, the order in which these lipids affect the quenching of the fluorescent probe 4-[(n-dodecylthio)-methyl]-7-(N,N-dimethylamino)-coumarin by doxyl groups positioned in the acyl chain region of the membrane is well correlated with the extent of activation of protein kinase C. This fluorescent probe is monitoring a property of the membrane that is affected by the presence of non-lamellar forming lipids.
R. M. Epand, R. Cornell, S. M. A. Davies, R. Kraayenhof

Chapter 17. Emission Spectroscopy of Complex Formation between Escherichia coli Purine Nucleoside Phosphorylase (PNP) and Identified Tautomeric Species of Formycin Inhibitors Resolves Ambiguities Found in Crystallographic Studies

The ubiquitous purine nucleoside phosphorylases (PNPs) play a key role in the purine salvage pathway, and are widely considered as major targets in the chemotherapy of immune system diseases, as well as in potentiation of the antitumor and antiviral activities of therapeutically active nucleoside analogues, by preventing them from phosphorolytic cleavage. Therefore, wide attention is devoted to development of more potent and specific inhibitors of the enzyme from various sources, and to studies of the mechanism of enzyme action. This review recalls the results of studies by steadystate and time-resolved emission (fluorescence and phosphorescence) spectroscopy, X-ray crystallography and enzyme kinetics on the interaction of highly purified bacterial (E. coli) purine nucleoside phosphorylase with a specific formycin A inhibitor (antibiotic) and its N-methylated analogues. The red shift of absorption and emission spectra of the ligands versus the enzyme permits selective excitation of ligand in the enzyme-ligand complex, as well as selective detection of enzyme or ligand emission.
B. Kierdaszuk

Microscopic Imaging Techniques and their Application for the Study of Living Cells


Chapter 18. Fluorescence Lifetime Imaging Implemented with Resonant Galvanometer Scanners

Fluorescence Lifetime Imaging (FLIM) typically utilises specialised image intensifiers to obtain a sequence of images at known times relative to a periodic excitation source. Either time-domain or frequency-domain gating characteristics of such devices have been used to derive fluorescence lifetime images on the nanosecond timescale. However, such devices can be problematical in terms of cost, robustness and complexity. This paper explores an alternative method of obtaining lifetime images by using continuously oscillating scanning elements at defined frequencies. Employing a frequency domain approach, sinusoidally modulated laser excitation at frequencies suitable for nanosecond timescale emission is scanned rapidly and symmetrically over a line by using such resonant scanners. By introducing a sampling frequency on the optical data stream critically related to both the excitation modulation frequency and the scanner frequency, it becomes possible to encode the lifetime-related phase delay and demodulation data as a function of position. The sampling achieves the necessary down shifting of the high frequency data in addition to imposing a continuous instrumental phase shifting function. By combining sampled data for a given pixel across repeated passes of the scanner action, formulas are derived for both the steady-state intensity and lifetime-related phase and demodulation data. The overall method is illustrated by simulations and by experiments on model systems.
J. J. Birmingham

Chapter 19. Spectral Imaging of Single CdSe/ZnS Quantum Dots Employing Spectrally- and Time-resolved Confocal Microscopy

A spectrally- and time-resolved study of single CdSe/ZnS quantum dots (QDs) is presented. To this end a versatile, high sensitivy spectrograph is coupled to a confocal laser-scanning microscope. The spectrograph is built in-house and is especially developed for use in fluorescence microscopy. The high sensitivity is achieved by using a prism for the dispersion of light in combination with a state-of-the-art back-illuminated charge-coupled device (CCD) camera. The detection efficiency of the spectrograph, including the CCD camera, amounts to 0.77 ±0.05 at 633 nm. Full emission spectra with a 1–5 nm spectral resolution can be recorded at a maximum rate of 800 spectra per second. The spectrograph can easily be fiber-coupled to any confocal laser-scanning microscope.
W. G. J. H. M. Van Sark, P. L. T. M. Frederix, M. A. H. Asselbergs, D. J. Van den Heuvel, A. Meijerink, H. C. Gerritsen

Chapter 20. Imaging of Oxidative Stress in Plant Cells by Quantitative Fluorescence Microscopy and Spectroscopy

In this chapter the application of two different fluorescent probes is described that are able to interrogate lipid peroxidation in whole plant cells and hydrogen peroxide production in plant cell suspensions.
J. W. Borst, M. A. Uskova, N. V. Visser, A. J. W. G. Visser

Chapter 21. The Biomedical Use of Rescaling Procedures in Optical Biopsy and Optical Molecular Imaging

Using fluorescence imaging in biomedicine, the analysis of laser-induced autofluorescences shows comparable strong intensity distortion for endogenous NADH in the UV and synthesized markers in the NIR range due to tissue optics. Rescaling, taking into account biochemical and biooptical methods, results in the chromophore profile in the observed tissue region. One of the outstanding advantages of near infrared so-called optical molecular imaging (OMI) is the high-detection sensitivity and the capability to brightly modulate the fluorescence injectable markers, e.g., by specific molecular interaction with tumor-specific enzymes. For the in vivo tests of the dyes an experimental NIR imager was used. NIR fluorescence of the entire body of small animals can be imaged. For first experiments an undifferentiated superficial tumor of mouse thigh was used. Corrections due to tissue optics must take care of a stronger scattering of the light in the NIR range in comparison with the UV fluorescence, like in optical biopsy. For example, the diameter of the fluorescent volume is apparently larger for the same reason. Therefore, the established rescaling from the UV adapted to the NIR range is important for the interpretation of fluorescence pictures in biomedicine.
O. Minet, J. Beuthan, K. Licha, C. Mahnke

Chapter 22. Looking into a Living Cell

Microscopic detection of fluorescent dyes is a powerful tool to monitor dynamics of intracellular parameters, in the living cell. In contrast to green fluorescent proteins (GFPs), which require expertise in molecular biology, the ease at which fluorescent dyes can be used makes them appealing for a larger audience of biologists. In this overview we will highlight certain methodologies and considerations when imaging a selection of cellpermeable fluorescent dyes and endogenous fluorescent molecules. We will do this with an emphasis on pH and mitochondrial energetics in cardiomyocytes.
M. Van Borren, N. R. Brady, J. Ravelsloot, H. V. Westerhoff

Chapter 23. Expression of Multicolor Fluorescent Fusion Proteins in Zebrafish Cell Cultures: A Versatile Tool in Cell Biology

A genetically stable zebrafish cell line was transfected with plasmids encoding fluorescent marker proteins of various colors. The markers for human tubulin, actin, ER, golgi and endosome are located at their correct position in zebrafish cells. We discus the benefits of transfected zebrafish cell lines over mammalian cells in cell biological studies.
C. K. D. Breek, F. Van Iren, S. E. Wijting, N. Stuurman, H. P. Spaink


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