Talk Abstracts
Please note that the talk by Professor Weihong Tan, from University of Florida, USA has been cancelled. Dr. Seth Olsen from University of Queensland, Australia will be giving a talk instead.
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Plasmon Enhanced Fluorescence: Ultrasensitive Detection and Biomedical Applications.
Zygmunt Gryczynski, Julian Borejdo, Evegenia Matveeva, Nils Calander *, and Ignacy Gryczynski
Health Science Center, University of North Texas,
Fort Worth, TX. * Department of Physics, Chalmers University of Technology, Göteborg, Sweden.
Fluorescence technology is the foundation of numerous analyses in sensing, microscopy, medical diagnostics, biotechnology, and gene expression. Recently we demonstrated incredible capability for modifying radiative rates of fluorophores in close proximity to metallic nanostructures. The useful changes of spectral properties of fluorophore in proximity to conducting metallic surfaces/ nanostructures include increased rates of excitation, increased quantum yields, decreased fluorescence lifetime with increased photostability, directional rather isotropic eprojects and drastically increased multi-photon excitation. These effects are result of strong interactions between light electromagnetic field/molecular orbital excitation and free oscillating electrons in metallic structures (plasmons). Also our theoretical predictions and experimental observations indicate potential for significant detection volume reduction to a level of 1-2 attoL.
Such plasmon enhanced fluorescence (PEF) presents incredible potential for use in microscopy, biological assays, immunoassays, and for studying biophysical properties of macromolecules on a nanoscale level. These open new capabilities for single molecule detection and makes feasible development of fluorescence assays based on single molecule system. In particular we applied plasmonic technology to immunoassays in dens physiological media (plasma and blood) and detection of single cross bridges in the muscle.
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Femtosecond Laser Based Biophotonics
Min Gu
Centre for Micro-Photonics, Faculty of Engineering and Industrial Sciences,
Swinburne University of Technology, Melbourne, Australia
Since 1960, the invention of lasers has revolutionised optical sciences. Lasers have opened up numerous opportunities for photonics, information technology and biomedical sciences. Laser scanning microscopy is a basic approach for optical bioimaging, which allows multi-dimensional image and applications to a broad range of live biological species. The power of modern microscopy lies in its ability to collect images in optical sections with enhanced spectral accessibility from biological specimens. Focused laser beams can be also used as laser tweezers designed to trap and manipulate biological specimens.
This presentation will be focused on the research activities currently carried out at Centre for Micro-Photonics, with an emphasis on the application of femtosecond laser beams for cellular imaging, manipulation and engineering. Discussion will include nonlinear optical microscopy and nonlinear fiber-optic endoscopy for early cancer detection. Basic principles of femtosecond laser trapping in far field and near-field regions will be explained and demonstrated. Micro-fabrication based on femtosecond lasers facilitates the three-dimensional micro/nano-struactures which can be used as novel micro-fluidic devices and bioreactors in stem cell engineering.
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Application of magnetic/phosphorescent nanoparticles in immunoassays.
D. Dosev [a], M. Nichkova [b], R. Dumas [c], K. Liu [c], I. M. Kennedy [a]
[a] Department of Mechanical and Aeronautical Engineering,
[b] Department of Entomology,
[c] Department of Physics, University of California Davis, CA 95616
Many types of fluorescent nanoparticles have been investigated as alternatives to conventional organic dyes in biochemistry. In addition, magnetic beads also have a long history of biological applications. In this work we apply flame spray pyrolysis in order to engineer a novel type of nanoparticle that has both phosphorescent and magnetic properties. The particles have magnetic cores of Nd/Fe oxide and phosphorescent shells of Eu – doped gadolinium oxide (Eu:Gd 2O 3). Measurements on a Vibrating Sample Magnetometer showed an overall paramagnetic response of these composite particles. Fluorescence spectroscopy showed spectra typical of the Eu ion in a Gd 2O 3 host – a narrow eprojects peak centered near 615 nm. Our synthesis method offers low-cost, high-rate synthesis allowing a wide range of biological applications of magnetic/fluorescent core/shell particles. We have demonstrated an immunoassay using the magnetic and phosphorescent properties of the particles for separation and detection purposes. Three different antibodies were attached in controlled concentrations to the Eu doped nanoparticles. A sandwich immunoassay was used to detect the presence of captured antigen with secondary antibodies that were labeled with three different fluorophores. Each fluorophore provided a measure of the amount of antigen present in the magnetically separated material, while the Eu signal provided a measure of the total amount of antibodies that were present. This allowed us to normalize the fluorophore signals with an internal standard, yielding accurate and reliable standard curves for the assays.
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Establish biophotonics core in a biomedical research community- from enabling technology to critical applications
Chi-Hung Lin
Biophotonics Interdisciplinary Research Center, Genome Research Center,
Instrumentation Resource Center, National Yang-Ming University, Taipei, Taiwan
To address basic cell biology in molecular terms, conventional biochemical and molecular biology experiments are very powerful in identifying molecules and their interacting partners involving in certain biological activities of interest. These techniques, however, typically can only study "average" behaviors of biomolecules in a cell, and study limited structure information (by X-ray, NMR, cryoEM) from molecules “isolated’ from the cells or made in vitro. In order to understand the biological functions of biomolecules, it is "NECESSARY" that they are studied in the living cell environment with spatial 3-D and temporal dynamic information. The applications of photonics to biology can meet these needs from many aspects. Photonic techniques allow one 1) to study the biomolecules in action within the context of living cells, 2) to detect beyond the resolution limit of light, or at very high temporal resolutions, and 3) to investigate the biophysical characteristics of the cell in details, as well as intermolecular forces, fluctuation, transport mechanisms, rhythm etc. In light of this enormous potential of biophotonics on biological and medical researches, we in National Yang-Ming University have put efforts in the past three years to construct in Yang-Ming University a biophotonics core, including instrumentation, human resources and logistics, to promote and support the related researches and development.
I will share our experiences that are being gathered along this process, from establishing technological platforms to identifying and launching biomedical investigation projects. Some recent results generated by selected researches will be presented and discussed.
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Optical Spectroscopy for Probing the Structure-Property-Function Relationships of Eumelanin - a Key Functional Bio-macromolecule
1 P. Meredith, 1J. Riesz, 1S. Nighswander-Rempel, 2Ben J. Powell, 2J. Gilmore & 3Mark R. Pederson
1 Soft Condensed Matter Physics Group & Centre for Biophotonics & Laser Science, School of Physical Sciences, University of Queensland, Brisbane, QLD 4072, Australia
2 Theory of Condensed Matter Physics Group, School of Physical Sciences, University of Queensland, Brisbane, QLD 4072, Australia
3 Center for Computational Materials Science, Naval Research Laboratory, Washington, D.C. 20375, USA
The melanins are a class of functional bio-macromolecules responsible for photoprotection and pigmentation in humans and many other species. They possess a unique collection of physico-chemical properties including condensed phase electrical conductivity and photoconductivity, strong broad band UV and visible absorption, and very strong non-radiative conversion of photo-excited states. Despite many decades of research activity in the melanin field, significant fundamental questions concerning their basic structure-property-function relationships still exist. One of the most important issues yet to resolve is that of the structure at the primary and secondary level. We have been studying eumelanin (the predominant form of the macromolecule in humans) for several years with a view to understanding how the molecular and supramolecular structures relate to observable macroscopic properties. Key tools in this effort have been steady state and time resolved optical spectroscopies coupled with state-of-the-art quantum chemical calculations and predictions. In my talk I will review the current state of knowledge in the field, and describe our recent spectroscopic and molecular findings. I will show how a combined experiment-theory approach can shed light on this difficult problem – a classic case of “molecular biophotonics”.
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New Light Induced Molecular Switch allows Sterically Oriented Micrometer
Sized Immobilization of Biomolecules
Steffen B. Petersen, Teresa Neves Petersen
Aalborg University, Denmark
Fundamental knowledge on protein structures and the effect of ultraviolet light on these structures has paved the way for the development of a unique light-based immobilization technology that allows oriented protein immobilization onto micrometer sized spots. The methodology is considered to be a strong alternative to the conventional procedures which often include the use of harsh conditions such as strong chemicals and elevated temperatures. The technology behind this immobilization technique – here termed "light assisted immobilization" - is based on the fact that disulphide bridges that are naturally present within the protein structure can be broken as a result of UV-illumination. The free thiol groups (-SH) created upon disruption of a disulphide bridge are very reactive and can be used as linkers for covalent attachment to a surface. The surface can for example be gold or thiol-derivatized silicon, making this technology extremely useful for a large range of application areas, including biosensors
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Biophotonics, Nanophotonics, Nanochemistry and Nanomedicine
Paras N. Prasad
Institute for Lasers, Photonics and Biophotonics
Departments of Chemistry, Physics, Electrical Engineering and Medicine
University at Buffalo The State University of New York, New York 14260
www.biophotonics.buffalo.edu
Biophotonics deals with interaction of light with biological or biologically relevant matter [1]. It provides novel optical probes for interactions and dynamics of cellular and tissue processes. The applications range from optical diagnostics, bioimaging and biosensing, to light-guided and light–activated therapy. New opportunities exist for the application of new fluorescence-based as well as nonlinear optical techniques for biophotonics.
Nanophotonics, defined as nanoscale optical Science and Technology, is another new frontier [2]. A major thrust area of nanophotonics is photonic nanomaterials in which excitation dynamics, energy transfer and nonlinear optical interactions can be controlled and photon localization can be manifested. Nanophotonics enables us to tailor the optical properties of the nanostructures for biomedical applications. Nanochemistry provides opportunities to produce, in a highly controlled way, nanoprobes and nanostructures for nanophotonics.
Nanomedicine provides prospects for revolutionary diagnostic and therapeutic approaches for the 21 st century health care. It is an emerging field which utilizes nanostructures to probe cellular processes, to perform bioimaging, to effect biosensing, and to provide a more effective, targeted therapy. Nanomedicine also provides opportunity for real time monitoring of a therapy. Nanochemistry provides exciting prospect to produce multifunctional nanomaterials with combined magnetic, optical, plasmonic and biologic functionalities, necessary for the advancement of nanomedicine. Examples of these multifunctional nanomaterials and their in-vitro and in-vivo studies will be presented from the on-going research at our Institute. The use of these nanoparticles for bioimaging, photodynamic therapy and optically trackable gene delivery will also be presented.
The talk will conclude with a subjective view of opportunities for both research and commercializable technology development in these fields.
- P.N. Prasad, “Nanophotonics”, John Wiley & Sons, New York (2004)
- P. N. Prasad, “Introduction to Biophotonics”, John Wiley & Sons (2003)
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The chromophore of all-protein chromophores
Mark Prescott , Pascal Wilmann, Jion Battad, Jan Petersen, Rod Devenish, Kristina Turcis, Ashley Buckle, Sophie Dove, Michael Ling, Anne Pettikiriarachchi, Jamie Rossjohn
Monash University, Melbourne, Australia
All-protein chromophores (APCs) such as the green fluorescent protein are indispensable tools for studying a wide range of key events in the living cell. Intense interest and research activity has resulted in the isolation and development of a range of APCs that have intriguing, novel and biotechnologically useful properties such as photoconvertability. The diversity is such that hundreds of different cDNAs have been isolated from marine organism that can be categorised into roughly 30 different groups based on their properties.
We have used protein X-ray crystallography to investigate the structural basis for some of these intriguing optical properties. It is apparent the chemical nature and conformation of the chromophore in these proteins is considerably more subtle and complex than one might have suspected. The chromophore in particular proteins adopts alternative conformations within the b-barrel structure of the protein. The non-fluorescent but intensely coloured chromoproteins, Rtms5 and AsCP, contain a trans non-coplanar chromophore whereas the highly red-fluorescent protein, EqFP611, contains a trans coplanar chromophore. In other highly fluorescent proteins such as GFP and DsRed the chromophore is cis coplanar. More than one configuration can exist in particular proteins. It has been established these different configurations underly the reversible photoswitch (fluorescent/non-fluorescent) of particular chromoproteins. In addition to alterations in configuration of the chromophore, distinct differences in the post-translational chemistry of the chromophore have been identified. For example, the main polypeptide backbone can be cleaved within the native protein resulting in the formation of new chromophores.
An overview of these structures in relationship to their optical properties will be given together with some disussion on how some of their novel properties maybe applied.
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Employing high-resolution liquid crystal displays for novel methods in biomedical optics
A. Jesacher, S. Fürhapter, S. Gspan, A. Meyer, C. Meusburger, C. Heinrich, S. Bernet, and M. Ritsch-Marte,
Division of Biomedical Physics, Innsbruck Medical University, Muellerstr.44, A-6020 Innsbruck, Austria
The new generation of spatial light modulators (SLM), i.e. high-resolution LCDs, opens up new possibilities to generate pre-designed optical field distributions that may e.g. be employed for contact-free optical micromanipulation of transparent particles under the microscope. Designing a special Fresnel-type holographic set-up, we have successfully generated arrays of individually addressable optical traps of selectable shape: apart from simple focus points, annular, elliptic and rectilinear traps have been demonstrated. This may for instance be utilized for size-dependent trapping of particles. One may even use static light intensity distributions for producing a continuous flow of micro-particles. Such an all-optical micro-pump makes use of the fact that light can carry orbital angular momentum that can be transferred from the laser light to the particles.
A totally different application of SLMs is to implement an all-optical method for generating and steering ultrasonic beams: By using the optoacoustic effect in a layer attached to the surface of a water tank which absorbs time-modulated laser pulses, ultrasonic beams in the kHz to MHz regime can be generated. By sending a pattern corresponding to a computer-generated phase-hologram to the SLM one creates an acoustic analogue to "diffractive optical elements" in laser optics, which leads to the eprojects of ultrasonic waves in a predetermined way. Several ultrasound patterns have been created using optoacoustic holography, including ultrasonic "doughnut" modes, Bessel beams and field distributions corresponding to annular beams which are very difficult to generate by means of piezo-transducers. Optoacoustic ultrasound generation with holographic spatio-temporal beam steering has the potential to become a flexible method promising new applications in medical and technical ultrasound diagnostics. Finally, a new type of functional microscopy based on nonlinear optics will also be discussed briefly.
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Multispectral Cytometry: Studying Cells at the speed of light
J. Paul Robinson,
Professor of Immunopharmacology & Biomedical Engineering
Purdue University, West Lafayette, IN USA
Flow cytometry is a technology that can analyze and physically sort a single cell from mixtures of tens of thousands in seconds. It is a technology that is embeded within the medical diagnostics field. We have developed a flow cytometer with the capability of collecting 32 channels of fluorescence spectra enabling a quantum leap in flow cytometry systems. This instrument enables us to transform the entire spectrum of a particle, bead, or cell into a spectral parameter and opens up the opportunity of creating a spectral fingerprint of every cell at speeds of several thousand cells per second.
This is a paradigm shift in the design of cytometers. The new paradigm establishes spectral fingerprint analysis as a core method of screening and classification, in contrast to current intensity-based techniques. These current well-established screening methods used in diagnostic instruments rely on organic fluorochromes & eprojects band-detection systems. They are powerful, but by nature are probably restricted to a dozen or so simultaneous fluorescent probes, have complicated and numerous optics as well as restricted spectral bandwidths, & must also deal with the very complex issues such as compensation. This method of signal acquisition is not well suited for spectral fingerprint detection, where sensitivity and versatility may be sacrificed for the benefit of higher spectral resolution. Next-generation biophotonics systems capable of handling this level of data collection are demanding.
There are many issues that must be successfully resolved in order for such a technology to be commercially successful. First, design of an electronic system of detection with a reasonable signal strength to allow adequate data collection via new optical designs. Second, adaptation of staining technologies to become useful for biologically compatible spectral coding. Lastly, the design of quite advanced integrated software package for rapid analysis of spectral data. We will demonstrate the powerful nature of the above approach using biological and non-biological examples.
For flow cytometry to meet the 21st century demands of biotechnology and cell biology, minor advances in instrumentation will be insufficient. This presentation demonstrates the effectiveness of a truly next-generation approach to flow cytometry collection and analysis that has the promise of being fundamental to future diagnostic instruments.
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From cells to organs with lasers
M. J. Friese 1,2 G. Knoener 2, T. Nieminen 2, S. Parkin 2, J. Ross 2, W. Singer 2, C. Talbot 1,2, N.R. Heckenberg 2 , and Halina Rubinsztein-Dunlop 2,
1 Centre for Magnetic Resonance
2 Centre for Biophotonics and Laser Science, School of Physical Sciences,
The University of Queensland, Brisbane, 4072 Queenslan, Australia.
Biophotonics is one of the major pioneering fields at the interface of physical, biological and medical sciences of the twenty first century. It is the science of generating and harnessing light to image, detect and manipulate biological materials and is an enabling science, able to spawn new technologies. Biophotonics helps to discover, develop and apply light- and laser-based science and technology to advance biology, biomedicine and health.
This talk will outline three examples of “biophotonics at work” These are: (1) Production of hyperpolarised gases for use in nuclear magnetic resonance imaging of whole organs. This technique enables imaging of organs such as lungs and brain which otherwise are very difficult to image; (2) Measurement of light-activated processes, such as fluorophore transport. The usefulness of this technique is derived from its capacity to decouple the imaging and activation processes, allowing fluorescent imaging of fluorophore transport at a convenient activation wavelength; (3) Measurement of viscoelastic properties of biological liquids using rotating optical tweezers. This method enables analysis of femtoliter volumes of liquid and gives information of the properties of liquid which are otherwise very difficult to obtain.
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Tissue imaging with optical coherence tomography
D. D. Sampson, S. G. Adie, S. A. Alexandrov, J. J. Armstrong, T. R. Hillman, M. S.
Leigh
Optical+Biomedical Engineering Laboratory, School of Electrical, Electronic & Computer Engineering,
The University of Western Australia, Perth, Australia
Optical coherence tomography (OCT) [1] is an optical sectioning microscopy that employs temporal coherence as its axial sectioning mechanism. Despite its early success as a medical imaging modality in ophthalmology, much still remains to be done at both the fundamental and the applied levels. There is an imperfect understanding of the OCT image formation process and the limits set in imaging biological tissue by such phenomena as absorption, dispersion, and multiple scattering. There is significant scope for improvements in the technology; in particular, to make higher resolution imaging more widely available, and there are many new biological and clinical applications that have yet to make the transition from the research phase to practice. In this talk, we shall briefly review the basic principles of OCT imaging and present a selection of the more prominent application areas. We shall consider several topical issues: firstly, the limits on resolution set by absorption and dispersion in biological tissue. We shall describe what is required to achieve ultra-high resolution (around 1 micron) at depths of a few hundred microns and beyond [2]. We shall show that for the common OCT wavelength of 1300 nm water absorption sets strong constraints and that in the 800 nm window the demands on dispersion compensation are high. Speckle is a ubiquitous feature of coherent imaging systems that can destroy the fidelity of the image, and speckle statistics are often sample independent. Our recent work shows under what conditions OCT speckle contrast is correlated with optical sample properties – such measurements may provide useful diagnostic information on samples or on the OCT image itself [3]. A currently active area in biomedical optics is the exploitation of the angular and spectral properties of elastically scattered light. We shall discuss applying
another coherent imaging technique, Fourier holography, to this problem [4]. Finally, we shall describe the evolution of OCT into a medical imaging modality suitable for clinical application [5].
References
1. D. D. Sampson, T. R. Hillman, Optical Coherence Tomography, in Lasers and Current OpticalTechniques in Biology , G. Palumbo, R. Pratesi, Eds, (Royal Society of Chemistry, Cambridge, UK, ISBN 0-85404-321-7), pp. 481-571, 2004.
2. T. R. Hillman, D. D. Sampson, “Effect of water dispersion and absorption on axial resolution in ultrahigh-resolution optical coherence tomography”, Opt. Express , vol. 13, pp. 1861-1874, 2005.
3. T. R. Hillman, S. G. Adie, V. Seemann, J. J. Armstrong, S. L. Jacques, and D. D. Sampson, “Correlation of static speckle with sample properties in optical coherence tomography”, Opt. Lett. , vol. 31, no. 2, 2006, in press.
4. S. A. Alexandrov, T. R. Hillman, D. D. Sampson, “Spatially resolved Fourier holographic light scattering angular spectroscopy”, Opt. Lett. , vol. 30, no. 24, 2005, in press.
5. J. J. Armstrong, M. S. Leigh, D. D. Sampson, J. H. Walsh, D. R. Hillman, P. R. Eastwood, “Quantitative upper airway imaging during sleep and wakefulness with anatomical optical coherence tomography”, Am. J. Respir. Crit. Care Med. , vol. 172, 2005; published ahead of print on October 20, 2005 as doi:10.1164/rccm.200510-1604OE.
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The Bright, Excited State of Fluorescent Protein Chromophores
Seth Olsen and Sean C. Smith
Centre for Computational Molecular Science
The University of Queensland , St. Lucia , QLD 4067, Australia
The green fluorescent protein and its growing family of homologues have become the driving force behind a revolution in biophotonics applications and technology. Their usefulness stems from their ability to autocatalytically form the chromophores which give them their identity. Trends that map chromophore structure and properties to protein photophysics have become apparent, implying that studies of simplified chromophore models can yield valid insights into the relevant photobiology. We will review modeling efforts to date, highlighting our own efforts to understand the family by comparison across a range of different chromophore structures. We will outline the potential and limitations of modeling studies for deriving insights into the photophysics of the proteins.
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Medical Imaging in Thick Tissues Using Diffuse Optics
Bruce J. Tromberg
Beckman Laser Institute and Medical Clinic,
University of California, Irvine 92617-1475
Medical diagnostic techniques based on near infrared (NIR) transillumination were first introduced more than 70 years ago to detect breast cancer. Although NIR light penetrates tissue to depths of several centimeters, early methods were not successful due to the fact that these approaches were qualitative and did not account for distortions from multiple light scattering.
Recent advances in time- and frequency-domain “photon migration” now make it possible to separate light absorption from scattering in thick tissues. Time-domain techniques measure the delay and spread of a picosecond pulse, while frequency-domain methods measure the phase shift and amplitude of MHz - GHz intensity-modulated waves (1). Both approaches are based on comparing measured data with radiative transport models to form images, i.e. “diffuse optical imaging (DOI)”, and acquire spectra, i.e. “diffuse optical spectroscopy (DOS)”.
This talk reviews principles of light propagation in tissue and describes the development of DOI/DOS for non-invasively characterizing tissue structure and biochemical composition. Particular emphasis is placed on the development of “broadband DOS” for quantitative, high-resolution measurements of NIR absorption and scattering spectra between 600-1000 nm (2). These data are used to determine the tissue concentration of deoxygenated hemoglobin, oxygenated hemoglobin, methemoglobin, lipid, and water, as well as the tissue “scatter power”. Clinical study results are shown highlighting the sensitivity of broadband DOS to metabolic changes in breast cancer detection and therapeutic drug monitoring (3,4). These findings will be placed in the context of conventional imaging methods, such as MRI, in order to assess the current and future role of diffuse optics in medical imaging (5).
References
1. Cerussi, A. E. and Tromberg, B. J. "Photon Migration Spectroscopy," in Biomedical Optics Handbook, Tuan Vo-Dinh, Ed., CRC Press (Boca Raton, FL), (2002).
2. Bevilacqua, F., A. J. Berger, A. E. Cerussi, D. Jakubowski, and B. J. Tromberg. Broadband Absorption Spectroscopy in Turbid Media by Combined Frequency-Domain and Steady-State Methods, Applied Optics 34, 6498-507 (2000).
3. Shah, N., Cerussi, A., Eker, C., Espinoza, J., Butler, J., Fishkin, J., Hornung, R., Tromberg, B. Non-Invasive Functional Optical Spectroscopy of Human Breast Tissue. Proceedings of the National Academy of Science 98, 4420-4425, (2001).
4. Jakubowski, D.B.; Cerussi, A.E.; Bevilacqua, F.E.; Shah, N.; Hsiang, D., Butler, J., Tromberg, B.J., Monitoring neoadjuvant chemotherapy in breast cancer using quantitative diffuse optical spectroscopy: a case study, Journal of Biomedical Optics, 9, 230-238, 2004.
5. Merritt S, Bevilacqua F, Durkin AJ, Cuccia DJ, Lanning R, Tromberg BJ, Gulsen G, Yu H, Wang J, Nalcioglu O. Coregistration of diffuse optical spectroscopy and magnetic resonance imaging in a rat tumor model, Applied Optics 42, 2951-2959, (2003).
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Naturally Fluorescent. – Fluorophores from Fungi
Duncan Veal 1,2
1 FLUOROtechnics Pty Ltd (www.fluorotechnics.com)
2 Department of Chemistry and Biomolecular Science, (http://www.chem.mq.edu.au)
Macquarie University, NSW 2109, Australia.
Fluorochromes used in biological analyses are derived from both chemical and biological sources. Biological sources include the photosynthetic pigments of plants and algae (eg Phycoerythrin and PerCP). In addition various fluorescent proteins such as green fluorescent protein (from the jellyfish Aequorea vitoria) have become amongst the most widely used tools for studying gene expression in living organisms.
Fungi are an extremely diverse biological group, and due to their unusually biosynthetic capacity are exploited commercially to produce a plethora of biological molecules (antibiotics, steroids, enzymes, biopharmaceuticals, etc). Although fungi have been used since antiquity as sources of dyes for textiles and foods they have not been explored as a source of fluorophores.
As a result of serendipity we discovered a new class of fluorescent molecule which are secondary metabolites of the fungus Epicoccum nigrum. This family contains a large number of spectrally distinct members. A number of products have been developed and we have built a company around this discovery.
Epicocconone 1 (one member of this new class of fluorophore) is a low molecular weight (410), water soluble, heterocyclic, organic fluorophore. On spontaneous binding to primary amines (such as lysine residues in proteins) epicocconone yields an intensely fluorescent, red product 2 (see figure 1).
This change in the fluorescence properties on conjugation provides a novel approach to the sensitive quantification of proteins across a wide variety of platforms (e.g. in solution, gels, blots, arrays, cell, etc) 3-6.
The covalent binding of epicocconone to proteins is only stable at low pH (pH 2.5) and the fluor can be removed by simply washing at a higher or lower pH 2. This pH dependent, reversible binding renders proteins amenable to subsequent analysis by mass spectrometry, Edman-based sequencing, and to functional assays of non-denatured proteins.
Epicocconone shows a marked increase in quantum yield in the hydrophobic environments around proteins, membranes and lipids, is freely cell permeable, non-toxic and has no effect on the growth of a wide range of cell types (bacteria, yeast, mammalian). These features enable live cells to be brightly stained, without permeablization steps, or the requirement to remove unbound fluorophore through washing. This has led to the use of epicocconone for intracellular fluorescent imaging of live cells 4.
Epicocconone is excitable by common lasers such as Violet (405 nm) argon ion (488 nm) and He-Neon (543 nm), enabling analysis by standard fluorescence instrumentation (fluorescence and confocal laser scanning microscopy, flow cytometry, etc).
The large Stokes’ shift of epicocconone when bound to proteins (ca 200 nm) enables simple multiplexing with a wide range of short Stokes’ shift fluorophores (fluorescein, DAPI, Hoechst 33342, and STYO family dyes) using a single light source 4.
- Bell, P.J.L. and Karuso, P. (2003) Epicocconone, a novel fluorescent compound from the fungus Epicoccum nigrum. Journal of American Chemical Society. 125, 9304.
- Coghlan, D. R., Mackintosh, J. & Karuso, P. (2005). Mechanism of reversible fluorescent staining of protein with Epicocconone. Organic Letters. 7, 2401-240
- Mackintosh, J.A., Veal, D.A. and Karuso, P. (2005) FluoroProfile, a fluorescence based assay for rapid and sensitive quantification of proteins in solution. Proteomics, 5, 4673-4677.
- Choi, H.-Y., Veal, D.A. & Karuso, P. (2005) Epicocconone, A New Cell-Permeable Long Stokes’ Shift Fluorescent Stain for Live Cell Imaging and Multiplexing. Journal of Fluorescence. In press.
- Malmport, E., Mackintosh, J., Ji, H., Veal, D. & Karuso, P. (2005). Visualization of proteins electro-transferred on Hybond ECL and Hybond-P using Deep Purple Total Protein Stain. GE Healthcare Life Science News. 19, 12-13.
- Mackintosh, J.A., Choi, H.-Y., Bae, S.-H., Veal, D.A., Bell, P.J., Ferrari, B.C., van Dyk, D., Verrills, N.M., Paik, Y.-K. & Karuso, P. (2003). A fluorescent natural product for ultra sensitive detection of proteins in 1-D and 2-D gel electrophoresis. Proteomics. 3, 2273-2288.
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Studies of Protein Dynamics with Fluorescence Correlation Spectroscopy:
From Molecules to Cells
Ton Visser
MicroSpectroscopy Centre, Laboratory of Biochemistry
Wageningen University, Dreijenlaan 3
6703 HA Wageningen, The Netherlands
Understanding interactions, structure and dynamics of biomolecules and of properties of biomolecular networks in living cells is of central importance in life sciences. Fluorescence correlation spectroscopy (FCS) is an example of a single-molecule fluorescence detection technique that can provide detailed information on biological systems. FCS is a sensitive technique to study diffusion properties and conformational dynamics of proteins on the microsecond timescale and longer. We will present several examples of FCS applications in vitro and in vivo.
To understand how a linear chain of amino acids folds into a functional protein molecule with complex three-dimensional structure is one of the major challenges in structural biology. For this study apoflavodoxin is covalently labeled with Alexa 488 maleimide to the only accessible Cys and is unfolded using guanidine hydrochloride. FCS has been used to characterize the denaturant induced conformational changes in the protein. The results indicate that the unfolded protein has a 30% larger hydrodynamic radius than the native protein molecule, which is in agreement with an expanded coil-like structure.
FCS can also provide a detailed picture of the conformational dynamics of single flavoenzymes in action. We illustrate this approach using the flavoenzyme p-hydroxybenzoate hydroxylase (PHBH) that carries out the hydroxylation of the aromatic substrate p-hydroxybenzoate (PHB). We have labeled the only accessible Cys of PHBH with Alexa 488 maleimide. This label shows overlap between its fluorescence spectrum and the absorption spectrum of the flavin prosthetic group. In this way a single-pair FRET system is designed that is only operational (spFRET active) in the resting (or oxidized) state of the enzyme and is not operational (spFRET inactive), when the enzyme is reduced during catalysis. The FCS autocorrelation curves of the labeled enzyme show an extra relaxation process - related with the modulation of the flavin redox state – under enzymatic turnover conditions using both substrates PHB and NADPH.
Dictyostelium discoideum , a soil amoeba, uses the process of chemotaxis to develop from a unicellular to a multicellular form. A crucial step in chemotaxis is the receptor-mediated activation - by the chemoattractant cAMP - of heterotrimeric GTP-binding proteins (G-proteins). The G-proteins, composed of G a , G b and G g subunits, dissociate upon activation into a and bg subunits, which in turn activate other proteins in the cell. Using FCS we have studied the diffusion of GFP tagged G b and G g subunits in Dictyostelium cells. The results indicate that the G b and G g subunits are associated with each other in a tight complex. In the cytoplasm of chemotaxing cells the G bg complex shows increased mobility as compared to vegetative cells. The diffusion of the G bg complex in the front of the cell is significantly faster than in the mid or back of the cell. We propose that this variable diffusion contributes to the amplification of the chemotactic response.
By using the concept of Fluorescence Cross-Correlation Spectroscopy (FCCS) it is possible to determine the interaction between two proteins each tagged with a different fluorescent protein in a very specific way. We report on the application of FCCS to study transmembrane receptors in plant cells. The receptor is fused with both CFP and YFP and expressed in plant protoplasts. With FCCS the distribution, diffusion and degree of interaction of the receptors can be determined.
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Fluorescence and photochemical reactions: lessons from teeth and tissues
Laurence J. Walsh
The University of Queensland, Brisbane, Australia
This lecture will provide an outline of two main topics, laser-induced fluorescence for the diagnosis of dental decay and dental calculus (and its use in feedback control of laser ablation), and laser-induced photochemical reactions for tooth whitening and killing bacteria in sites difficult to treat with conventional methods. The presence of bacterial biofilms and their interaction with organic substrates leads to novel uses for fluorescence signals from bacterial porphyrins. The characteristic patterns of fluorescence emitted beyond 700 nm when irradiated with pulsed 655nm laser light can be exploited to detect dental decay present within the structure of teeth as well as deposits of dental calculus within pockets around the teeth. Novel sapphire prism designs exploiting reflection can allow clinicians to investigate these areas, with the fluorescence data contributing to decisions regarding invasive treatments. Using thresholds for fluorescence, destructive laser energy can be applied in a controlled manner to achieve selective removal of bacteria and their products. This provides for minimally invasive clinical patient care.
The use of photoreactive materials applied onto or into teeth is a second major emerging area of interest. Materials such as tolonium chloride and rhodamine B at high pH conditions irradiated under 635nm and 532 nm laser excitation respectively can yield reactive oxygen species for killing bacteria or triggering other chemical reactions – such as destroying organic chromogens and thus whitening teeth. Examples of these methods and their effectiveness in clinical trials will be presented, and placed in the context of the challenges with traditional approaches.
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Application of real-time imaging for the analysis of NF-κB signalling dynamics
Michael RH White
School of Biological Sciences
Liverpool University, Crown St, Liverpool, L69 7ZB
Multiple signalling pathways control cell fate through regulation of transcription. The dynamic configuration of signalling networks enables cells to respond appropriately to biological signals & stresses. Genome sequencing has identified the global set of human genes, but a problem lies in elucidation of gene function and how gene functions are integrated to control cellular processes in time and space. The number of central signalling nodes that regulate key processes in the mammalian cell remains relatively small. Loss or abnormal functioning of key proteins (e.g. p53 or NF- κ B ) can result in aberrant cell function leading to diseases such as inflammation and cancer. The efficacy of the cellular information processing steps that filter multiple signal information is critical for the organism to avoid disease.
Some of the most important tools for the elucidation of intracellular signalling in living cells have been proteins from luminescent organisms. We pioneered the use of low light level imaging for the imaging of firefly luciferase activity as a reporter for transcription in single cells. The discovery of naturally fluorescent proteins from jelly fish and corals that could form their fluorescent chromophores without the need for exogenous substrates in different cell types was particularly significant. These proteins have now been mutagenised to form a wide range of colours and to make brighter derivatives. In addition they have been used as sensors for other processes such as measurement of calcium, proteolysis and protein interactions. These tools have led to a revolution in quantitative non-invasive measurement of cellular processes.
We have developed multi-parameter imaging approaches to study signalling, gene expression and cell fate in single living mammalian cells. Signalling by the transcription factor Nuclear Factor kappa B (NF- k B) involves its release from Inhibitor kappa B (I k B) in the cytosol, followed by translocation into the nucleus. NF- k B regulation of I k B a transcription represents a delayed negative feedback loop that drives oscillations in NF- k B translocation. Single cell time-lapse imaging and computational modeling of NF- k B (RelA) localization showed asynchronous oscillations following cell stimulation that decreased in frequency with increased I k B a transcription (1). Transcription of target genes depended on oscillation persistence, involving cycles of RelA phosphorylation and dephosphorylation. The functional consequences of NF- k B signalling may depend on number, period and amplitude of oscillations and may depend on the kinetics of formation of different NF-kB complexes in the nucleus (2).
The discovery of these complex dynamic characteristics of the important NF-κB signalling pathway underlines the importance of timelapse imaging in single cells. The discovery that the other important cellular stress pathway, p53, is also oscillatory, raises the possibility that other signalling networks may oscillate with different frequencies, which may be important in signal pathway cross-talk. Through a DTI-funded Beacon project with Manchester University we are developing transfected cell arrays to investigate these processes with a higher throughput. This involves integrated development of genomics, imaging, automated image analysis and database technology.
1. Nelson, D.E., Ihekwaba, A.E.C., Elliott, M., Johnson, J.R., Gibney, C.A., Foreman, B.E., Nelson, G., See, V., Horton, C.A., Spiller, D.G., Edwards, S.W., McDowell, H.P., Unitt, J.F., Sullivan, E., Grimley, R., Benson, N., Broomhead, D., Kell, D.B. & White,. M.R.H. (2004) Science 306: 704-8.
2. Nelson, D.E., Sée, V., Nelson, G. and White M.R.H. Oscillations in transcription factor dynamics: a new way to control gene expression.(2004) Biochem. Soc Trans. 30: 1090-192.
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Photo-Oncology: The Application of Photonic Sciences and Technologies to Cancer Treatment, Response Monitoring and Diagnosis
Brian C Wilson
Ontario Cancer institute/University of Toronto, Canada
Advances in photonics are helping drive the development of a range of new techniques applied to early cancer detection and cancer treatment. The emerging field of photo-oncology is illustrated by diverse examples where photonics provide critical enabling capabilities. The applications presented include: photodynamic therapy of solid tumors using light-activated drugs (both single and 2-photon) and early cancer detection using fluorescence imaging/spectroscopy, Raman spectroscopy and optical coherence tomography. The future enhancement of these capabilities by emerging nanotechnologies is also discussed and illustrated. The current limitations and possible new applications of photonics in cancer treatment, diagnosis and research are highlighted.
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New Advances in Optical Imaging of Living Cells and Tissues
Sunney Xie
Harvard University
Department of Chemistry and Chemical Biology
By integrating new biochemical probes with advanced microscopy, our group is able to monitor individual turnovers of a single enzyme molecule and to detect protein molecules generated one at a time in living cells.
Recent developments of coherent anti-Stokes Raman scattering (CARS) microscopy have allowed noninvasive vibrational imaging of live cells and tissues with high sensitivity. Compelling applications of CARS microscopy to biology and medicine are beginning to emerge.
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