Conference Speakers

The list of confirmed speakers for the Light in Life Sciences (LILS) Conference 2009 will continue to grow. Please check this list weekly for updated speaker confirmations.

Invited speakers confirmed to present at the LILS 2009 Conference are as follows:

 

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Speaker
Institution
Dr. Arjen Bader Universiteit Utrecht,Netherlands
Prof. Wolfgang Becker Becker & Hickl GmbH, Germany
Prof. David Birch University of Strathclyde, UK
Dr. Christoph Biskup Jena University, Germany
A/Prof. Filip Braet The University of Sydney, Australia
A/Prof. Robert Campbell University of Alberta, Canada
Dr. Andrew Clayton Ludwig Institute for Cancer Research, Australia
Dr. Martin de Jonge Australian Synchrotron, Australia
Dr. Katharina Gaus University of New South Wales, Australia
A/Prof Michelle Gee University of Melbourne, Australia
Dr. Vladimir Ghukasyan National Yang-Ming University, Taiwan
Dr. Michal M. Godlewski Macquarie University, Sydney
A/Prof. Liz Harry University of Technology, Sydney
Dr. Rainer Heinztmann King's College London, UK
Dr. Will Hughes Garvan Institute,Australia
Dr. Stefan Jakobs Max Planck Institute, Germany
Prof. Anita Jones University of Edinburgh, Scotland
Prof. Aaron Lewis Nanonics Imaging Ltd., Israel
Prof. Don McNaughton Monash University,Australia
Prof. David Millar The Scripps Institute, USA
Prof. Paul Mulvaney University of Melbourne, Australia
Prof. Mutsuo Nuriya Keio University, Japan
Dr. Mike Roberts University of Queensland, Australia
Prof. Paul Robinson Purdue University, USA
Prof. Sarah Russell Swinburne University of Technology, Australia
Prof. Markus Sauer University of Bielefeld, Germany
Prof. Herbert Schneckenburger Institut fur Angewandte Forschung, Germany
Prof. Jenny Stow University of Queensland, Australia
Dr. Klaus Suhling Kings College London, UK
Prof. Leann Tilley La Trobe University, Australia
Prof. Matt Trau University of Queensland, Australia
A/Prof. Vladislav Verkhusha Einstein College of Medicine, USA.
Dr. Joerg Wiedenmann University of Southampton, UK
Prof. Tony Wilson Oxford University, UK
Prof. Paul Wiseman McGill University, Canada

 

Talk Abstracts

 

 

Dr. Arjen Bader

IN-VIVO NON-LINEAR SPECTRAL MICROSCOPY OF SKIN


In Multi-photon excitation, fluorescent molecules having absorption bands in the visible or UV are excited by the simultaneous absorption of two or more Infra Red (IR) or Near Infra Red (NIR) photons. The non-linear dependency of the two-photon absorption probability on the excitation intensity results in the confinement of the excitation volume. This behavior can be employed for 3-D imaging without a pinhole in multi-photon excitation microscopy. TPE lifts some of the problems related to conventional autofluorescence spectroscopy and microscopy. Importantly, it affords excitation of autofluorescing molecules with one photon absorption bands in the UV without the complications related to the use of UV light. Moreover, Multi-photon excitation microscopy enables high resolution imaging deep inside tissues.
In this work a dedicated multi-photon spectral imaging was employed to study the intrinsic emission of tissues. The purpose of the research was to investigate the potential of non-linear excitation methods to derive "optical signatures" from the intrinsic emission from tissue for characterizing skin and eventually diagnose diseases.
We investigated both mouse and human skin. Different layers of skin could be clearly distinguished based on both their spectral signature and morphology. Auto fluorescence images were recorded that contain signals from cellular and extra cellular structures. The fluorescence arises from native chromophores such as NADPH, elastin, collagen and flavins. In addition a Second Harmonic signal generated by collagen is observed. The signals can be separated using linear unmixing techniques. Besides morphology, the technique provides biochemical information about, for instance, metabolism (NADPH, FAD). Moreover, the technique offers potential for the diagnoses of diseases such as skin cancers. The skin of UV exposed mice revealed spectral changes as well as morphological changes that could be associated with tumor formation.

Address:

Dr. Arjen Bader
Universiteit Utrecht,Netherlands
E-mail: A.N.Bader@uu.nl

Reference:
1.J.A. Palero, H.S. de Bruijn; A. van der Ploeg van den Heuvel, H.J.C.M. Sterenborg , H. van Weelden and H.C. Gerritsen, Photochemical & Photobiological Sciences;7(11),1422-5 (2008).
2.J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg and H. C. Gerritsen, Biophys J, 93, 992-1007 (2007).
3.J. A. Palero, H. S. de Bruijn, A. van der Ploeg-van den Heuvel, H. J. C. M. Sterenborg, H. C. Gerritsen, Optics Express 14, 4395 (2006).

 

Dr. Wolfgang Becker

Probing Molecular Interactions by Fluorescence Lifetime Imaging

 

The fluorescence lifetime is a direct indicator of the interactions of a fluorophore with its molecular environment. A Fluorescence Lifetime Imaging (FLIM) technique for biological microscopy has to combine high lifetime accuracy, resolution of multi-exponential decay profiles, and simultaneous recording in several wavelength intervals with minimum exposure of the sample and optical sectioning capability. We will show that the combination of multi-dimensional time-correlated single photon counting (TCSPC) with laser scanning meets these requirements almost ideally. Multi-dimensional time-correlated single photon counting (TCSPC) is based on the excitation of the sample by a high-repetition rate laser and the detection of single photons of the fluorescence signal in several detection channels. Each photon is characterised by its time in the laser period, its wavelength, and the coordinates in the scanning area. Combined with a confocal or two-photon laser scanning microscope, multi-dimensional TCSPC makes a fluorescence lifetime imaging (FLIM) technique with multi-wavelength capability, near-ideal counting efficiency, picosecond time resolution, and the capability to resolve multi-exponential decay profiles. The technique is particularly useful for fluorescence resonance energy transfer (FRET) experiments and autofluorescence imaging. Moreover, it can be used to obtain combined fluorescence lifetime and fluorescence correlation data in diffraction-limited spots of a sample. We will demonstrate the capabilities of the technique for a number of typical applications.

Address:
Dr. Wolfgang Becker
Becker & Hickl GmbH,
Nahmitzer Damm 30, 12277
Berlin, Germany
E-mail: becker@becker-hickl.com

 

Prof. David J S Birch

The Fluorescence of Fibrils

When native soluble protein is destabilized, it tends to aggregate, forming fibrils and β-sheet rich structures. Such behaviour is characteristic of disease associated with protein disfunction such as cystic fibrosis and many forms of cancer and that associated with aggregate cytotoxicity such as the neurodegenerative Alzheimer's, Parkinson's and Huntington's disease. The fragment peptide beta-amyloid (Aβ) is the main constituent of plaques found in the brain in the case of Alzheimer's disease. To date fluorescence studies of fibril formation and aggregation of Aβ have focused on the use of extrinsic probes. However, in this talk I will show how intrinsic fluorescence brings several advantages to bear in the study of fibrils, not only in the case of Aβ, but in metabolic fibrils in general and illustrate this further by describing the unusual fibrils which can be formed in the synthesis of melanin.

Address:
Prof. David J S Birch
Photophysics Group,
Centre for Molecular Nanometrology,
Department of Physics, SUPA,
University of Strathclyde,
Glasgow, Scotland

 

Prof. Christoph Biskup

Think globally! – The benefits of adding new dimensions to fluorescence microscopy

 

The fluorescence light emitted by fluorophores can provide a wealth of information about the hidden life of cells: Fluorescence labelling techniques can be used to reveal and follow up the spatial distribution of proteins. Fluorescence sensors can be used to monitor non-invasively the concentration of ions and bio¬molecules, and fluorescent analogues of ligands provide the possibility to study the binding of ligands to their receptor. Moreover, physical phenomena such as Förster energy transfer (FRET) can be exploited to extend the resolution of a light microscope to a nanometer scale and to test if molecules are in close vicinity to each other.
Fluorescence images do not only provide qualitative information about the sample. They can also be evaluated in a quantitative way to get a deeper insight into the highly regulated networks of biological systems. In conventional fluorescence microscopy only the information conferred by the fluorescence intensity is exploited. But also other intrinsic properties of a fluorophore such as the absorption and emission spectra, the fluorescence lifetime or the fluorescence anisotropy can be used to retrieve additional information from the sample. This information can help to build more precise models of the biological system and to reduce the error of the recovered parameters.
Here, we report on different strategies that can be pursued to improve the results derived from fluorescence measurements. We point out the benefits of acquiring fluorescence data in a temporally and spectrally resolved manner and show how these data sets can be used to evaluate FRET measurements.
Fluorescence measurements can be also combined with other methods such as the patch-clamp technique. This combination allows to record simultaneously fluorescence signals and electrical currents of ion channels in membrane patches. Global analysis of the data can yield valuable information about the processes underlying channel activation. We used this approach to study the activation of homotetrameric CNGA2 and HCN2 channels in inside-out membrane patches. By using fluorescent analogues of cyclic nucleotides as ligands we were able to simultaneously determine ligand binding and channel activation.

Address:
Prof. Christoph Biskup
Biomolecular photonics group
University Hospital Jena
Teichgraben 8, D 07740 Jena, Germany
E-mail: christoph.biskup@mti.uni-jena.de

 

A/Prof. Filip Braet

Contribution of combined and correlative imaging techniques in the study of colorectal (liver) cancer

 

Gone are the days when the typical morphologist sought to understand the complex interior of the cell with his “magnifying glass” while his colleague, possibly even in the same institution, pursued the same quest with advanced molecular biology techniques. Instead, structural biology and molecular biology are closely integrated in the modern research environment thanks to the development of novel imaging biomolecular microscopy platforms, which have arisen from new, powerful computers, from fast high-resolution cameras, from improved image processing software, and from advances in various molecular probes specifically designed for labelling fixed and/or living cells. The current boom in the application of different microscope beam-lines to study a particular research quest illustrates the point: combined, correlative, and integrated microscopy is central to the modern biosciences and is helping to integrate subfields—in this case, structural biology and molecular biology. In this presentation, examples of the application of different correlative multidimensional im¬aging and modelling techniques to study colorectal-mediated subcellular events over length scales will be presented. By applying this approach our team endeavours to gather novel spatial and temporal molecular information involved in the onset of colorectal liver metastasis, which should ultimately contribute to the development of new immunotherapeutic strategies.

Address:
A/Prof. Filip Braet
Australian Key Centre for Microscopy & Microanalysis,
The University of Sydney
E-mail: f.braet@usyd.edu.au


A/Prof. Robert Cambell

Moulding fluorescent proteins into new biotools: engineering of protein surfaces, topology, and chromophores

 

At the heart of the Campbell groups research program lies the extraordinarily useful family of fluorescent proteins (FPs) which includes the green FP (GFP) from Aequorea jellyfish and its numerous homologues of various color from coral. All FPs share the ability to autonomously generate a visible wavelength fluorophore from a sequence of 3 amino acids located in the center of their barrel-like structures. FPs are credited with sparking the explosive and continuing growth in the popularity of live cell fluorescence microscopy; culminating in the 2008 Nobel Prize in Chemistry being awarded to 3 pioneers in this area; Shimomura, Chalfie, and Tsien.

Since its inception in 2003, the Campbell research group has worked to mould (that is, engineer) FPs into improved tools for addressing fundamental questions in life science. For example, we have developed several improved alternatives to some of the traditionally preferred hues of FPs [1,2,3] and exploited their improved properties in new applications. Some of our novel applications include the use of FPs for investigating peptide structure in vivo [4], dual-FRET imaging of caspase activation during apoptosis [5], and a FP-based assay of nucleoside transporter activity (with Joe Casey) [6].

In this seminar I will discuss some of our most recent efforts to create FP variants that have physical or spectral properties that are either improved relative to existing variants or are completely novel and have not been previously reported. As the title of this seminar indicates, this discussion will focus on 3 aspects of FPs; chain topology, surface properties, and chromophore chemistry. Specifically, I will present our most recent progress in the following areas: creation of circularly permuted red FPs through manipulation of topology; engineering of monomeric and heterodimeric FPs through manipulation of surface properties; and engineering of photostable and photconvertable FPs by manipulation of chromophore chemistry and environment.

Address:
A/Prof. Robert Campbell
Department of Chemistry
University of Alberta
Edmonton, Alberta
T6G 2G2 CANADA
E-mail: robert.e.campbell@ualberta.ca

References:
1.Ai,H., Henderson,J.N.,Remington,S.J.&zCampbell,R. E. Biochem. J. 400,531-540(2006).
2.Ai, H.,Shaner,N. C.,Cheng,Z.,Tsien,R. Y. & Campbell,R. E. Biochemistry 46,5904-5910(2007).
3.Ai, H.,Olenych,S. G.,Wong,P.,Davidson,M. W. & Campbell,R. E. BMC Biology 6,13 (2008).
4.Cheng,Z.,Miskolzie,M. & Campbell,R. E. ChemBioChem 8,880-883 (2007).
5.Ai, H.,Hazelwood,K. L.,Davidson,M. W. & Campbell,R. E. Nat. Methods 5,401-403 (2008).
6.Johnson, D. E.,Ai, H. W.,Wong, P.,Young,J. D.,et al. J Biol Chem 284, 20499-20511 (2009).

 

Dr.Andrew H.A. Clayton

EGF receptor in the lime light.


Cell surface EGF receptor exists in monomeric, dimeric and higher order forms. Understanding which form(s) are active or inactive is an important aspect of the regulation of signal transduction and antagonist design. For many years it has been believed that receptor activation occurs via a monomer:dimer transition which is associated with a conformational change which activates the receptor kinase. An alternative model, the rotational twist model, has been proposed based on the observation that a significant fraction of receptors are in a ligand-free dimerized state. However, little is known about the quaternary state of the EGF receptor at normal levels of expression (<105 receptors/cell), in the absence of other EGFR family members and in the absence of ligands. We have employed multi-dimensional microscopy techniques to gain insight into the state of association of the human EGFR, in the absence and presence of ligand, on the surface of intact BaF/3 cells (50,000 receptors/cell). Image correlation microscopy of an EGFR-enhanced Green Fluorescent Protein (eGFP) chimera was used to establish that the average degree of aggregation on the sub-micron scale is 2.2 receptors/cluster. In the presence of ligand the degree of aggregation increase to 3.7 receptors/cluster. Energy transfer between mixtures of FITC-EGF and Alexa555-EGF on the surface of EGFR-Baf/3 cells can be detected using fluorescence lifetime imaging microscopy (FLIM). By varying the donor:acceptor labeling ratio it is possible to gain insight into the spatial disposition of EGFR ligand binding sites on the nanometre scale. This microscopy data can be related to the 3D structure of the ligated EGFR extracellular domain. In the context of a monomer-dimer-oligomer model, our biophysical measurements are consistent with a population of ligated EGFR tetramers comprising two dimers juxtaposed in a side-by-side arrangement. We have explored the relationship between association state and EGF receptor kinase phosphorylation (activation) using a novel hybrid microscopic method. Contrary to the prevailing view that the EGFR dimer is the predominant, active form, our data determine that higher-order EGFR oligomers are the dominant species associated with the ligand activated EGFR tyrosine kinase.

Address:
Dr. Andrew H.A. Clayton,
Melbourne Branch, Ludwig Institute for Cancer Research,
Australia 3050.
E-mail: Andrew.Clayton@ludwig.edu.au

References:
1. Yarden Y., Schlessinger J. (1987). Biochemistry 26(5), 1434-42.
2. Gadella TW Jr, Jovin TM. (1995 J Cell Biol. 129(6), 1543-58.
3. Clayton, A.H., Walker, F., Orchard, S.G., Henderson, C., Fuchs, D., Rothacker, J., Nice, E.C., Burgess, A.W. (2005 J Biol Chem. 280(34), 30392-9.
4. Clayton, A.H., Orchard, S.G., Nice, E.C., Posner, R.G., Burgess, A.W. (2008).Growth Factors 26(6), 316-24.

 

Dr. Martin de Jonge

X-ray fluorescence Microspectroscopy at the Australian Synchrotron; capabilities, present and future

 


The hard x-ray microprobe is ideally suited to the mapping of trace elements situated in a biological context, combining a variety of powerful experimental techniques. Fluorescence mapping can be used to detect sub-ppm quantities in specimens that have undergone minimal preparation, and X-ray Absorption Near-Edge Spectroscopy (XANES) can be used to obtain an in situ chemical fingerprint for a range of metals of interest.
At the Microspectroscopy beamline of the Australian Synchrotron we have recently commissioned two hard x-ray fluorescence microprobes. These microprobes and their ancillary detectors enable a wide range of studies – but necessarily preclude others. In this presentation we discuss the current capabilities of the Microspectroscopy beamline with reference to applications.
We are developing two techniques that will be particularly useful for imaging in the life sciences; a quantitative differential phase contrast imaging scheme that will enable ultrastructural imaging in tandem with elemental mapping, and a fast fluorescence tomography technique. We have developed a prototype of this tomography methodology at the Advanced Photon Source, and will present first reconstructions of a whole cell with a resolution of about 200nm. A much improved version of the technique will be deployed at the Australian Synchrotron over the next 12 months.

Adderss:

Dr. Martin de Jonge
Microspectroscopy Australian Synchrotron
800 Blackburn Road, Clayton 3168,
Australia
E-mail: Martin.deJonge@synchrotron.org.au

 

A/Prof Katharina Gaus

Membrane organization at T cell activation sites: fluorescence super-resolution microscope for cell signalling

 

We employ single molecule imaging techniques to understand how activation of the T cell receptor on the cell surface leads to an intracellular signalling response. To function in an immune response, T cells become activates when a highly specific pathogen-derived peptide binds to the T cell receptor (TCR). However, the concomitant signaling events are not specific to the TCR raising the question how T cells recognize specific signals for activation. Researchers were excited by the idea that lipid domains could organize signalling cascades in time and space and thus establish hierarchies and, ultimately, control signalling outcomes that determine cell function in health and disease. Our previous data revealed that membrane lipids and proteins co-operate to form stable membrane domains and protein clusters that are necessary for full T cell activation. To understand the underlying principles of domain formation, we aim to image and quantify protein localization, diffusion and interaction on a molecular scale employing new imaging approaches including time-correlated single photon counting (TCSPC) and photo-activation localization microscopy (PALM).

Address:
A/Prof Katharina Gaus
Cell Membrane Biology Group
Centre for Vascular Research
University of New South Wales,
2052, Sydney, NSW
Australia
E-mail: k.gaus@unsw.edu.au

 

 

A/Prof. Michelle Gee

Time-Resolved Surface Specific Fluorescence Spectroscopies for Probing Interactions of Proteins and Peptides at Surfaces

 

Gaining a clear understanding of the interactions of proteins and peptides at surfaces has far reaching implications: In medicine, it is thought that biofouling is initiated by the adsorption of proteins to materials used in the manufacture of devices such as catheters or artificial implants. A key component of cell signalling and immune response is the change in conformation and oligomerisation of proteins at cell membranes. Perturbations to protein-protein interactions can cause abnormalities in signalling networks and can lead to disease states such as cancer. To combat this, there is much activity looking at peptides as therapeutics targeted towards diseased cells through their interactions at cell membranes.

Recently, we have shown that novel surface specific fluorescence spectroscopies can yield new insights into the in situ molecular conformation and dynamics proteins and peptides at an interface. In these Variable-Angle Time-Resolved Evanescent Wave-Induced Fluorescence Spectroscopic (VA-TREWIFS) techniques, an evanescent field, created through total internal reflection of a femtosecond laser pulse, excites a fluorphore associated with a protein or peptide at an interface. The resulting fluorescence decay kinetics is measured using single photon counting methods and reports on protein conformation or peptide location. By coupling VA-TREWIFS with Time-Resolved fluorescence Anisotropy Measurements (EW-TRAMS), the mobility of the protein or peptide can be probed.

Here, results of two studies will be presented in overview to highlight these surface specific techniques. The first study will illustrate the conformational change that a protein undergoes when interacting with an inorganic surface. The second study will show the interaction of a peptide with a membrane mimic.

Address:
A/Prof. Michelle Gee
School of Chemistry, University of Melbourne,
Parkville, Australia.
E-mail: mlgee@unimelb.edu.au

 

Dr. Vladimir Ghukasyan

Metabolic mapping of cell culture growth by NADH fluorescence lifetime imaging

 

Fluorescence lifetime imaging microscopy (FLIM) has been demonstrated as advantageous at discrimination between free and protein-bound forms of the NADH coenzyme, providing not only with the lifetimes of the both states (shorter τ1and longer τ2),but also with the ratio of their relative concentrations (fractions a1and a2correspondingly). In this aspect, FLIM has been applied in a number of studies, revealing a potential in a number of diagnostic and sensing techniques, such as early discrimination of apoptosis from necrosis, pre-cancer biochemical changes, glucose sensing, etc. However, for the discrimination of pathological states, a proper characterization of NADH dynamics at physiological conditions has to be conducted.
We have conducted metabolic mapping with NADH FLIM during cell culture growth. The factor is also important since the density of the cells increases as the tumor development progresses. The fluorescence lifetime measurement results appeared to be dependent on the cell culture growth stage. Thus, during the first three days after plating as the cells attach, spread on the cover slip and recover from stress and spread on the cover slip, an increase of the a1/a2ratio has been observed. As the cells proceed to the exponential growth, the a1/a2decreases gradually from 4.0±0.23 on the 3rd to 3.3±0.21 on the 6th day. As the cell culture growth reaches the confluency state (slowing down and entering plateau on the 7th and 8th days), the ratio increases slightly to the value of 3.6 and stays there consecutively for 2 last days. To our best knowledge, this is the first demonstration of such correlation between the NADH fluorescence lifetime parameters and cell culture growth dynamics.

Address:
Dr. Vladimir Ghukasyan,
Institute of Biophotonics, National Yang-Ming University
155, Li-Nong St., Sec. 2, Taipei 112, Taiwan
Tel.: (886 2) 2826 7000 ext. 5709
E-mail:v.gukassyan@gmail.com

 

Dr. Michal M. Godlewski

Exploring the early events on the secretory pathway in filamentous fungus Trichoderma reesei with quantitative and qualitative cytometry

 

Trichoderma reesei is an industrially exploited producer of biomass degrading enzymes. It has been extensively modified by random mutagenesis and genetic engineering to improve production yields of its native cellulolytic enzymes. As a eukaryote, T. reesei has potential to become an efficient expression host for a variety of heterologous proteins originating from plants and mammals. However, attempts to express heterologus proteins in the high secreting T. reesei mutants have been only partially successful and the yields have remained low. Although the desired proteins have been expressed, they have been caught in unknown bottlenecks in the secretory pathway. Until recently, improvement strategies for the production of both native and heterologous proteins have not considered various alterations in the physiology of high producing mutants. Little is known of the secretory pathway of filamentous fungi, its evolution and the progress of proteins through the hyphae. In this study the wild-type strain Trichoderma reesei QM6a, its high secreting mutant RutC30, modified to produce high amounts of the native cellobiohydrolase I (CBHI) and a transformant strain x45 producing a heterologous bacterial xylanase B (XYNB) were investigated.
Expression of the native CBHI followed a previously discovered pulsing pattern during the first 36 h of hyphal growth. This pattern was consistent from the wild type to the high-secreting mutant RutC30. In the transformant producing heterologus XYNB, the enzyme slowly accumulated in the hyphae. The morphology of RutC30 and x45 showed typical signs of cell stress. Based on these findings, ER, the major organelle involved in the protein production, quality control and stress sensing was investigated. Organelle labelling revealed a massive recycling of membranes between ER and Golgi occurring in all analysed strains within the first 24 h of growth. This corresponded to the alterations in the organisation of actin filaments. Genetic labelling of CBHI and XYNB revealed that in the RutC30 mutant, CBHI was quickly secreted outside the hyphae, whereas in the x45 transformant the XYNB excited ER but was retained within the hyphae. Furthermore, x45 strain showed extensive disruptions in the organisation of actin filaments which was subsequently linked with the microautophagy processes.
In conclusion, this study shows how dramatically the physiology of RutC30 has been altered to cope with the demands of massive enzyme production. It explains aspects of fungal secretory pathway that have not been previously reported and the cellular consequences of expression of a heterologous protein. Therefore, it is elementary to understand the physiological effects of high-level expression and secretion when developing fungi as efficient cell factories.

 

Address:
Dr. Michal M. Godlewski
CBMS, Faculty of Science, Macquarie University,
Sydney, 2109 NSW, Australia
Department of Physiological Sciences,
Faculty of Veterinary Medicine, Warsaw University of Life Sciences,
Nowoursynowska 159, 02-776 Warsaw, Poland
E-mail: mickgodl@hotmail.com

 

 

A/Prof. Liz Harry

The commitment to bacterial cytokinesis: Z ring assembly

 

The idea of a bacterial cytoskeleton arose just ten years ago with the identification of the cell division protein, FtsZ, as a tubulin homolog. FtsZ plays a pivotal role in bacterial division. It is present in virtually all prokaryotes as well as in some eukaryotic organelles, and is hotly pursued by industry as a target for the development of novel antibiotics.
The first commitment to cell division in bacteria is the self-assembly of FtsZ into a ring structure known as the Z ring, which subsequently constricts the cell membrane during cytokinesis. The Z ring marks the future division site, and its formation is under complex temporal and spatial control. We recently showed that in addition to forming a ring, FtsZ also assembles into dynamic helical structures along the cell, that we propose act as precursors to the Z ring via a cell cycle-mediated FtsZ polymer remodelling. The fine structure of the FtsZ helix and ring structures are completely unknown but crucial for identifying the molecular details of Z ring assembly and its regulation by various known factors. Two of these factors are the Min system and nucleoid occlusion. The new model for Z ring formation challenges previous models for the function of these factors in bacteria. We will present this data and also present our observations of FtsZ in B. subtilis using super-resolution microscopy which reveal a highly irregular and discontinuous helix of FtsZ, very different to the smooth cable-like appearance observed by conventional fluorescence optics. This type of microscopy also identified a novel FtsZ helical structure of smaller pitch that is invisible to standard optical methods, identifying a possible third intermediate in the finely controlled pathway to Z ring assembly, which commits the bacterial cell to divide.

Address:
A/Prof. Liz Harry
Institute for the Biotechnology of Infectious Diseases,
University of Technology,
Sydney (UTS), NSW,
E-mail: Elizabeth.Harry@uts.edu.au

Co-Author:
Phoebe C Peters1, Leigh G Monahan1, Michael P. Strauss1, Guy C Cox2 and Elizabeth J Harry1

1. Institute for the Biotechnology of Infectious Diseases,
University of Technology,
Sydney (UTS), NSW,

2.Australian Key Centre for Microscopy & Microanalysis,
University of Sydney, NSW

 

 

Dr.Rainer Heintzmann

Structured Illumination and Image Inversion Interferometry

 


An overview of recent advances in high resolution fluorescence microscopy will be given.
In structured illumination the sample is illuminated with a number of different patterns of light. In our case this is a series of sinusoidal grids at different grid positions and orientations generated by a programmable spatial light modulator or a physical phase grating. Experimental datasets acquired under these conditions and reconstructed results from these data, demonstrating a resolution improvement of up to a factor of two over standard widefield microscopy are presented.

The non-linear approach of saturating optical transitions (for structured illumination as well as beam-scanning approaches) has a great potential especially in combination with photo-switchable dyes such as the recently described IrisFP protein from Ulrich Nienhaus’ group or the Cy3-Alexa647 system used in Xiaowei Zhuang’s group. An interesting approach is to push molecules into dark states in a patterned way shortly before imaging and exploiting the saturation of this transition.

Finally a method will be presented in which the emitted fluorescence of a confocal microscope passes through two separate paths. These paths are interferometrically recombined in such a way that the images undergo a mutual rotation of 180 degrees. The self-interference of the fluorescent light is only constructive, if it originated from the optical axis of the scanning laser beam, thus leading to an efficient detection of a high resolution fluorescence images.

 

Author Details:
Dr. Rainer Heintzmann
King’s College London, Randall Division, Guy’s Campus,
London SE1 1UL, United Kingdom
Email: heintzmann@googlemail.com

Co-Author:
Kai Wicker, Marie Walde, Enno R. Oldewurtel, Liisa Hirvonen,
Ondrej Mandula, Simon Sindbert,

1.King’s College London, Randall Division, Guy’s Campus,
London SE1 1UL, United Kingdom
Email: heintzmann@googlemail.com

 

 

Dr. Will Hughes

How the cytoskeleton can control vesicle trafficking – studies using TIRFM

 

The insulin-stimulated trafficking of GLUT4 to the plasma membrane in muscle and fat tissue constitutes a central process in blood glucose homeostasis. The tethering, docking and fusion of GLUT4 vesicles with the plasma membrane represent the most distal steps in this pathway and have been recently shown to be key targets of insulin action. However, it remains unclear how insulin influences these processes to promote the insertion of the glucose transporter into the plasma membrane. In this study we have identified a previously uncharacterised role for cortical actin in the distal trafficking of GLUT4. Using high frequency total internal reflection fluorescence microscopy (TIRFM) imaging we show that insulin increases actin polymerisation near the plasma membrane. Disruption of this process inhibited GLUT4 exocytosis. Using TIRFM in combination with probes that could distinguish between vesicle transport and fusion we found that defective actin remodelling was accompanied by normal insulin-regulated accumulation of GLUT4 vesicles close to the PM but the final exocytotic fusion step was impaired. These data clearly resolve multiple steps of the final stages of GLUT4 trafficking, demonstrating a crucial role for actin in the final stage of this process.


Address:

Dr. Will Hughes
The Garvan Institute of Medical Research
384 Victoria Street, Darlinghurst
Sydney NSW 2010 Australia
E-mail:w.hughes@garvan.org.au

 

 

Dr. Stefan Jakobs

Focusing on mitochondria with STED microscopy

 

Practically, all that we know about the inner architecture of mitochondria is from electron microscopy, which in the 1960s and 70s revealed that these organelles assume a tubular shape of 200-500 nm diameter with a smooth outer and a highly folded inner membrane. Optical microscopy has failed to visualize inner mitochondrial structures because discerning details smaller than half the wavelength of light (400-800 nm) has been precluded by diffraction. This is unfortunate because light microscopy would offer a number of striking potential applications, arguably, most notably the visualization of the distribution, assembly and spatial dynamics of inner mitochondrial protein complexes.
In the recent past, various physical concepts emerged for overcoming the diffraction resolution barrier in lens-based fluorescence microscopy. Providing three-dimensional (3D) resolution on the nanoscale, these fluorescence nanoscopy methods, including stimulated emission depletion (STED) microscopy, have all been applied to imaging mitochondria. The latest advances in the imaging of sub-mitochondrial protein distributions using diffraction-unlimited STED microscopy will be reported.

Address:
Dr. Stefan Jakobs
Mitochondrial Structure and Dynamics Group
Dept. of NanoBiophotonics
Max Planck Institute for Biophysical Chemistry
Am Fassberg 11
37077 Goettingen
Germany
E-mail: sjakobs@gwdg.de

 

 

Prof. Anita C. Jones

Probing DNA Conformation and DNA-Enzyme Interaction by Time-Resolved Fluorescence


The dynamic behaviour of the DNA bases plays an important role in processes that are critical to the maintenance and function of the duplex, including electron transport and many fundamental DNA-enzyme interactions. The conformational properties of DNA can be probed using the fluorescent adenine analogue, 2-aminopurine (2AP). 2AP forms Watson-Crick base pairs with thymine and, therefore, does not disrupt the DNA double helical structure. The absorption maximum of 2AP (~305 nm) is red-shifted relative to the natural bases, allowing selective excitation, and its fluorescence properties are sensitive to the local molecular environment.
We use time-resolved fluorescence measurements of 2AP-labelled DNA, in solution, single crystals and frozen matrices at 77K, to investigate the influence of base dynamics on the populations and properties of the conformational states of the duplex.1 Recently we have found that 2AP in DNA exhibits long-wavelength fluorescence, in addition to the familiar short-wavelength spectrum, as a result of formation of an electronically coupled ground-state dimer with an adjacent natural base.2 The observation of this dual emission from 2AP in a variety of oligodeoxynucleotide duplexes and single strands demonstrates the generality of this phenomenon. Electronic coupling between the bases in DNA is of great interest because of its relevance to charge transport and the possible involvement of long-lived excited dimer states in the formation of UV-induced photolesions.
The DNA duplex undergoes conformational change in response to interaction with agents such as enzymes and drugs. A particularly remarkable example of localised conformational distortion is the phenomenon of nucleotide flipping, induced by enzymes, such as methyltransferases, that need to access a base to perform chemistry. Nucleotide flipping involves 180o rotation of the target nucleotide around the phosphate backbone, out of the DNA helix and into the reactive site of the enzyme.

Time-resolved fluorescence measurements 2AP-labelled DNA duplexes complexed with methyltransferase enzymes, in single crystals and in solution, allow us to explore in detail the nature of the interaction between enzyme and duplex and the conformational properties of the nucleotide-flipped complex.3-5. Recently we have used this approach to investigate the newly discovered use of nucleotide flipping by restriction enzymes (which do not chemically modify DNA) as part of their DNA recognition mechanism.6

Address:
Prof. Anita C. Jones
School of Chemistry and Collaborative Optical Spectroscopy, Micromanipulation and Imaging Centre (COSMIC), University of Edinburgh, Edinburgh EH9 3JJ, UK
E-mail: a.c.jones@ed.ac.uk

References:
1. R.K. Neely and A.C. Jones, J. Am. Chem. Soc., 2006, 128, 15952-3
2. E.Y.M. Bonnist and A.C. Jones, ChemPhysChem, 2008, 9, 1121-9.
3. R.K. Neely, D. Daujotyte, S. Grazulis, S.W. Magennis, D.T.F. Dryden, S. Klimasauskas and A.C. Jones, Nucleic Acids Res, 2005, 33, 6953-6960.
4. T. Lenz, E.Y.M. Bonnist, G. Pljevaljčić, R.K. Neely, D.T.F. Dryden, A.J. Scheidig, A.C. Jones and E. Weinhold, J. Am Chem. Soc., 2007, 129, 6240-6248.
5. B. Youngblood, E.Y.M. Bonnist, D.T.F. Dryden, A.C. Jones and N.O. Reich, Nucleic Acids Res. 2008, 36, 2917-2925.
6. R.K. Neely, G. Tamulaitis, K. Chen, M. Kubala, V. Siksnys and A. C. Jones, Nucleic Acids Res (in press).

 

Prof. Aaron Lewis

New Directions in AFM & NSOM Bio-Imaging in Liquids


Atomic force microscopy (AFM) with tuning fork feedback is the best method of AFM imaging known today. We now report the operation of this feedback mechanism in liquid. This allows for liquid cell AFM and NSOM operation in physiological media without any optical or mechanical constraints or interference. The extension of this feedback mechanism to liquids allows for scanned probe microscopy (SPM) liquid cell imaging fully integrated with any optical microscope including upright, 4 pi or standard Raman microprobes. It also will be shown that water immersion objectives can now be used with SPM and that these new directions allow for live cell bioimaging with NSOM for the first time. The advances reported in this paper, along with additional innovations in probe and scanner developments, allow for the dream of multiprobe NSOM/SPM to be implemented in physiological media. The results of these efforts portend important advances in the application of SPM in structural and functional bioimaging.

Address:
Prof. Aaron Lewis
Nanonics Imaging Ltd., Israel
Manhat Technology Park, Malcha
Jerusalem 91487, Israel
E-mail: aaron@Nanonics.co.il

 

 

Prof. Don McNaughton

Infrared and Raman micro-spectroscopy and imaging of fixed and live cells.

 

Infrared and Raman micro-spectroscopy techniques provide methodologies that can be adapted for obtaining direct molecular signatures in fixed and live cells and so provide ways of directly monitoring molecular process in cells. Synchrotron infrared microspectroscopy, whilst still limited by diffraction, provides an efficient way of imaging whole cells and monitoring macromolecular change and the results of experiments aimed at defining oocyte maturation markers will be outlined. Raman microscopy, through enhancement techniques, can provide single molecule spectroscopy and using near field techniques spatial resolution down to 10-20nm can be achieved. This promises to provide methodologies capable of monitoring even drug interaction in live cells and the results of preliminary experiments on sectioned red blood cells using Tip Enhanced Raman Spectroscopy (TERS) will be presented.

Address:
Prof. Don McNaughton
Professor of Molecular Sciences and Australian Professorial Fellow
Director, Centre for Biospectroscopy
School of Chemistry
Monash University
Clayton, Victoria 3800
Australia
E-mail: Don.McNaughton@sci.monash.edu.au

 

Prof. David Millar

Biomolecular Folding and Assembly at the Single-Molecule Level

 

Single-molecule fluorescence spectroscopy is a powerful tool for detailed mechanistic studies of the assembly and conformational dynamics of macromolecular complexes involved in a wide variety of essential cellular functions, such as DNA replication, RNA transport and protein translation and targeting. Measurements at the single-molecule level are able to resolve multiple folding and assembly pathways and provide kinetic information in situations where it is impossible to synchronize a population of molecules. To illustrate these capabilities, I will describe three systems currently under study in my laboratory. (1) DNA polymerases replicate DNA substrates with extraordinarily high fidelity because of their ability to discriminate between cognate and non-cognate nucleotide substrates during each cycle of nucleotide incorporation. Correct nucleotide selection is thought to arise from an induced-fit mechanism, whereby a correct incoming nucleotide induces a large conformational change of the polymerase. In addition, many polymerases have the ability to excise incorrectly incorporated nucleotides using an exonuclease activity located in a separate enzyme domain. Two complementary FRET systems have been developed to monitor conformational changes of individual DNA polymerase molecules during nucleotide incorporation and exonucleolytic proofreading processes. (2) The HIV-1 protein Rev mediates the nuclear export of unspliced (genomic RNA) and partially spliced mRNAs encoding viral structural proteins. This is a complex process during which multiple Rev monomers must assemble on the Rev Response Element, a highly conserved element within the viral mRNA, and several cellular proteins are subsequently recruited to the ribonucleoprotein (RNP) particle in order to promote nuclear export of the unspliced and partially spliced mRNAs. TIRF microscopy is being used to visualize oligomerization of Rev on individual RRE molecules with single monomer resolution, revealing the mechanism of assembly and the influence of selected cellular cofactors on RNP complex assembly. (3) The signal recognition particle (SRP) mediates the cotranslational targeting of proteins destined for the secretory pathway. In humans, this essential RNP is composed of a 300 nt RNA and six proteins. Single-pair FRET methods are being used to monitor RNA folding events during the early assembly of the SRP, revealing the role of specific SRP proteins and the mechanism of protein-mediated RNA folding.

Address:
Prof. David Millar,
Department of Molecular Biology,
The Scripps Research Institute,
La Jolla, CA, USA.
E-mail: millar@scripps.edu

 

 

Prof. Paul Mulvaney

Colloidally Stable, Water Soluble, Biocompatible, Semiconductor Nanocrystals with a Small Hydrodynamic Diameter

 

Analytical Ultracentrifiugation (AUC) provides a useful tool for characterizing quantum dots and bioconjugated nanocrystals. We describe our work on preparing CdSe based quantum dots for work in biolabelling, and the effects of ligands and coatings on their behaviour in AUC. In principle, AUC can be used to determine the stoichiometry of QD bioconjugates.

We also report a simple, economical method for generating water soluble, biocompatible nanocrystals that are colloidally robust and have a small hydrodynamic diameter. The nanocrystal phase transfer technique utilizes a low molecular weight amphiphilic polymer that is formed via maleic anhydride coupling of poly(styrene-co-maleic anhydride) with either ethanolamine or Jeffamine M-1000 polyetheramine. The polymer encapsulated water soluble nanocrystals exhibit the same optical spectra as those formed initially in organic solvents, preserve photo-luminescence intensities, are colloidally stable over a wide pH range (pH 3-13), have a small hydrodynamic diameter and exhibit low levels of non-specific binding to cells.

Address:
Prof. Paul Mulvaney
ARC Federation Fellow
School of Chemistry & Bio21 Institute
Level 2 North ,30 Flemington Road
University of Melbourne
Parkville, VIC, 3010
Australia
E-mail: pcmulvaney@gmail.com

References:
1. E. E. Lees, T.-L. Nguyen, A. H. A. Clayton and P. Mulvaney_ACS Nano 3,1121-28 (2009).
2. Emma E. Lees, et al. Nanoletters 8, 2883-90 (2008).

 

 

Prof. Mutsuo Nuriya

Imaging membrane potential dynamics in fine structures using the second harmonic generation imaging.


In neurons and other excitable cells, fast information processing is mainly performed electrically. Therefore, quantitative information on membrane potential dynamics is the key to the understanding of physiology of these cells. In neurons, however, this is challenged by their extremely elaborate and fine structures including axons and dendrites, often below a micrometer in diameter. To gain quantitative information on membrane potential dynamics in these structures, second harmonic generation (SHG) imaging was applied to neurons. Point-scan SHG imaging from dissociated cultured hippocampal neurons allowed fast measurement of electrical signals from sub-micron structures. Furthermore, as was predicted from theoretical studies, SHG signals showed a linear response to membrane potential changes, enabling the quantitative imaging of membrane potential changes in any visible places in neurons. In this presentation, application of this technique to membrane potential imaging of fine neurites that were hardly accessible with other techniques will be introduced. In addition, potentials of SHG imaging for other applications will be discussed.

Address:
Prof. Mutsuo Nuriya
Department of Pharmacology
School of Medicine, Keio University
35 Shinanomachi, Shinjuku, Tokyo, 160-8582, Japan
E-mail: mnuriya@sc.itc.keio.ac.jp

 

 

Dr. M.S. Roberts

Use of fluorescence lifetime imaging (FLIM) to measure skin biology and solute/ nanoparticle transport in the viable epidermis

 

Cosmeceutical solutes and nanoparticles are routinely used in cosmetic products topically applied onto human skin. The transport of these solutes and nanoparticles beyond the outer layer of skin, the stratum corneum, is a critical determinant in their efficacy and in the potential toxicity of these solutes and nanoparticles. However, defining what occurs in the epidermis noninvasively is difficult. Multiphoton tomography (MPT) and FLIM with the application of image quality techniques are used to directly image the viable epidermis. Here, we use these techniques to show the skin viability changes with time after excision and to demonstrate the relative penetration of different types of solutes and nanoparticle into normal and diseased skin.

Address:

Dr. M.S. Roberts
Therapeutics Research Unit,School of Medicine,
University of Queensland
Princess Alexandra Hospital,Ipswich Rd
Buranda, Qld 4102,Australia
E-mail: email m.roberts@uq.edu.au

Co-Author:
W. Sanchez, Jnr, W. Sanchez, J. Grice, M. Wu and T. Prow
Universities of Queensland and South Australia

 

 

Professor J. Paul Robinson

Next generation cellular analysis

 

The field of cytomics broadly defined as the systematic study of biological organization and behavior at the cellular level has begun to mature and establish itself as an integral component in cell biology. The necessary tools for integration of cytomics into the fundamental nature of cell systems analysis are maturing but new tools are demanded to achieve our goals. While there is a long way to go before we have tools that can perform true cytomics analysis, cytometry is a subset of tolls that is tremendously powerful and from which we can extract a significant subset of information about many biological systems.

It is important therefore to realize that for major advances to be realized new technologies must be developed for cytomics to become a reality. For example there will be a need for essential development of new sensor technologies that provide both sensitivity and selection in the visible and near IR spectrum. Secondly, a better integration between different measurement and detection tools will be needed. We simply cannot make independent measurements and hope to integrate these tools easily. Thirdly, in order to analyze the complex data sets resulting from new technology integration a major advance is needed to accommodate analysis of these data sets. Fourthly, chemistries must advance to permit greater selectivity of tracking tools. These will most likely expand beyond fluorescence to accommodate enhanced scatter analysis as well as chemical composition. This enhances the opportunities for evaluation of the size, shape and texture of cells, features that enrich automated analysis significantly.

Together, these advances place the cytomic opportunity into a new dimension for understanding metabolic responses in single cells and ultimately defining new functional populations of cells. The result will be new research tools as well as a toolset for clinical and diagnostic utility.

Address:
Prof. J. Paul Robinson
SVM Professor of Cytomics
Purdue University Cytometry Laboratories
Bindley Bioscience Center
1203 West State Street
Discovery Park, Purdue University
West Lafayette, IN 47907-2057
E-mail: jpr@flowcyt.cyto.purdue.edu

 

Prof. Sarah Russell


The role of asymmetric sorting of proteins in determining T cell fate


We have previously shown that T cells utilize an evolutionarily conserved network of polarity proteins to orchestrate cell shape and polarity, and that these proteins are required for migration and immunological synapse formation in T cells. We now have in vitro evidence using the OT-1 model system that T cells utilize this polarity network to coordinate asymmetric cell division. We demonstrate that naive T cells remain attached to antigen presenting cells (dendritic cells pulsed with ovalbumin peptide) throughout cell division, and utilize this attachment to orient their axis of cell division. We have developed microfabricated grids with which to contain T cells for long term imaging, and have written software with which to assess protein polarity in rapidly moving cells. Using these tools, we find that certain proteins are distributed asymmetrically both before and during cell division, resulting in two daughters with different compositions of proteins. Molecular disruption of polarity or of the orientation of the mitotic spindle alters the distribution of cell fate determinants, with subsequent effects on T cell differentiation. By maintaining the asymmetry originally associated with immunological synapse formation, the daughters of the T cell division inherit different molecular characteristics, which provide the capacity to dictate different subsequent fates.

Address:
Prof. Sarah M Russell
Peter MacCallum Cancer Centre and Centre for Microphotonics,
Swinburne University of Technology, Australia.
E-mail: srussell@swin.edu.au

Co-Author:
Jane Oliaro, Daniel Day, Ze’ev Bomzon, Vanessa van Ham, Kerrie-Ann McMahon, Mandy Ludford-Menting, Min Gu

 

Dr. Markus Sauer

DSTORM: Super-resolution imaging with small organic fluorophores

 

We introduce a general approach for multicolor super-resolution fluorescence imaging based on photoswitching of standard small organic fluorophores. Photoswitching of organic rhodamine and oxazine fluorophores, i.e. the reversible transition from a fluorescent to a non-fluorescent state in aqueous buffers exploits the formation of long-lived triplet radical anions through reaction with thiol compounds and repopulation of the singlet ground state by reaction with molecular oxygen. We unravel the underlying switching mechanism and demonstrate super-resolution imaging with different commercially available organic fluorophores. Furthermore, we provide evidence that the method can be advantageously used for live cell imaging with ~ 20 nm optical resolution.

Address:
Dr. Markus Sauer
Applied Laser Physics and Laser Spectroscopy
and Bielefeld Institute for Biophysics and Nanoscience,
Bielefeld University,
Universitätsstrasse 25,33615 Bielefeld,
Germany.
E-mail: sauer@urz.uni-heidelberg.de

 

Dr. Herbert Schneckenburger

Multi-dimensional Fluorescence Microscopy in Biomedicine

 

An overview on recent applications of fluorescence microscopy with high spatial, spectral and temporal resolution is given. Spectral imaging is used to characterize membrane stiffness as a function of temperature and cholesterol content. Fluorescence lifetime seems to be an appropriate parameter of malignancy in tumour diagnostics, but also can be used to probe intermolecular interactions, e.g. in pathogenesis of Alzheimer’s disease or in sensing of apoptosis. Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) as well as methods of structured illumination are used to obtain high axial resolution, whereas polarization microscopy is applied for measuring cell and membrane dynamics. Finally microscopic techniques are modified for applications of fluorescence reader technology in various fields of diagnostics.

Address:
Dr. Herbert Schneckenburger,
Hochschule Aalen, Biophotonics Group,
Anton-Huber-Str.21,
73430 Aalen,
Germany.
E-mail:herbert.schneckenburger@htw-aalen.de

 

Prof. Jennifer L Stow

Imaging Trafficking Pathways and Organelles Inside Macrophages

 

Newly synthesized, endocytosed and recycling proteins are constantly transported through intracellular exocytic and endocytic pathways in mammalian cells. In immune cells like macrophages these pathways allow cells to perform immune functions such as engulfing microbes and secreting soluble mediators. We have used live cell fluorescence imaging to map pathways in macrophages and to characterize organelles such as recycling endosomes that act as a hub for the trafficking of divergent cargo. Fluorescent labelling of multiple cargo proteins and of cellular machinery proteins has provided insights into the handling and deployment of proteins destined for secretion or for specific membrane domains. The coexpression of multiple fluorescently tagged cytokines, for instance, has revealed cargo separation into subcompartments within recycling endosomes that may contribute to protein sorting. Definition of these subcompartments has been aided by development of image analysis methodologies to automatically segment and quantify protein colocalization in individual organelles within a three-dimensional (3D) multichannel image. Insights obtained using these programs are reshaping our understanding of endosome function and of protein deployment during cellular immune responses.

Address:
Prof. Jennifer L Stow and Nick Hamilton
Institute for Molecular Bioscience,
The University of Queensland, Brisbane,
Australia, 4072
E-mail:j.stow@imb.uq.edu.au

 

Dr. Klaus Suhling

Fluorescence Lifetime Imaging of molecular rotors to map microviscosity in cells

 

Fluorescence lifetime imaging (FLIM) can report on photophysical events that are difficult or impossible to observe by fluorescence intensity imaging, because FLIM allows the separation of fluorophore concentration and quenching effects.1 We report on FLIM of modified hydrophobic BODIPY dyes that act as molecular rotors, and show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.2,3 Incubating cells with these dyes, we find a punctate and continuous distribution of the dye in cells. The viscosity value obtained in vesicles in live cells is around 100 times higher than that of water and of cellular cytoplasm.2,3 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Porphyrin-based molecular rotors4,5 also yield microviscosity values considerably higher than those of water and of cellular cytoplasm. In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of BODIPY molecular rotors.

Other Authors:
Marina Kuimova*, James Levitt, Pei-Hua Chung, Gokhan Yahioglu,

Address:
Dr. Klaus Suhling
Department of Physics, King’s College London, Strand, London WC2R 2LS, UK.
Chemistry Department, Imperial College London, Exhibition Road, London SW7 2AZ, UK.
PhotoBiotics Ltd, 21 Wilson Street, London EC2M 2TD, UK
E-mail: klaus.suhling@kcl.ac.uk

References
1. F. Festy, S.M. Ameer-Beg, T. Ng and K. Suhling. Molecular BioSystems 3(6), 381-391, 2007.
2. M.K. Kuimova, G. Yahioglu, J.A. Levitt and K. Suhling.J. Am. Chem. Soc., 130(21), 6672–6673, 2008
3. J.A. Levitt, M.K. Kuimova, G. Yahioglu, P.-H. Chung, K. Suhling, and D. Phillips.J Phys Chem C, in press DOI: 10.1021/jp9013493
4. M.K. Kuimova, H. Collins, M. Balaz, E. Dahlstedt, J. Levitt, N. Sergent, K. Suhling, M. Drobizhev, N. Makarov, and A. Rebane, Organic and Biomolecular Chemistry 7, 889-896, 2009.
5. M.K. Kuimova, S.W. Botchway, A.W. Parker, M. Balaz, H.A. Collins, H.L. Anderson, K. Suhling, and P.R. Ogilby, Nature Chemistry 1, 69-73, 2009.

 

Prof. Leann Tilley

Imaging malaria parasite-infected erythrocytes using environment-sensitive probes and high resolution modalities

 

The malaria parasite, Plasmodium falciparum, develops within human red blood cells (RBCs), consuming host hemoglobin in order to support its own growth. The digestive vacuole of the malaria parasite is the site of hemoglobin digestion and heme detoxification and, as a consequence, the site of action of chloroquine and other antimalarials. However the mechanism for genesis of the digestive vacuole and the precise pH of the vacuole and the endocytic vesicles that feed it are still debated. We have monitored the uptake of a pH-sensitive fluorescent dextran from the RBC cytoplasm and estimated the pH of the parasite's endocytic compartments. In early ring stage parasites we show that the host cytoplasm is internalized via cytostome-derived vesicles and concentrated into several acidified peripheral structures and that hemoglobin digestion and hemozoin formation are initiated. As the parasite matures the hemozoin-containing compartments coalesce to form a large acidic vacuole that is fed by hemoglobin-containing vesicles.

Reactive oxygen species (superoxide and hydrogen peroxide) are by-products of hemoglobin digestion and are believed to exert significant oxidative stress on the parasite. The chromophore, BODIPY 581/591, has an extended conjugated system that reacts with oxygen centered-radicals leading to changes in its spectral characteristics. We have used of a phosphotidylcholine derivative of BODIPY 581/591 (BODIPY-PC) to monitor oxidative stress in individual cells and in different compartments within cells in P. falciparum-infected RBCs We have also characterised a cell permeant, far red fluorescent nucleic acid-binding dye, SYTO 61, that can be used to distinguish between uninfected and infected RBCs in a flow cytometric format. We have used SYTO 61 in combination with the fluorescent reactive oxygen species reporter 5-(and-6)-chloromethyl-2',7'-dichlorodihydro-fluorescein diacetate acetyl ester to probe oxidative stress in the cytoplasmic compartment in different stages of live P. falciparum.

The resolution of conventional optical microscopy techniques is limited to about half the wavelength of the illuminating photons. Transmission x-ray cryo-tomography can achieve a higher resolution than optical microscopy due to the shorter wavelength of x-rays and can exploit the natural contrast between organic matter and water in the "water window" of photon energies. X-ray imaging can be readily performed on whole cells. Three-dimensional structured illumination microscopy is a visible light microscopy modality that circumvents the diffraction limit of light and permits analysis of whole cell samples that are specifically labelled with fluorescent probes. We have examined these techniques to examine the cellular architecture of P. falciparum-infected RBCs.

Address:
Prof. Leann Tilley
Department of Biochemistry and
Centre of Excellence for Coherent X-ray Science,
La Trobe University, Victoria,
Australia,
E-mail: l.tilley@latrobe.edu.au

 

Prof. Matt Trau

Shedding Light on Biomarkers: Nanoscaled Biosensors for applications in Early Disease Detection, Personalized Medicine and Diagnostics


The development of low cost, point of care, molecular-based diagnostics that can detect diseases while they are still curable, represents one of the most promising approaches to reducing the growing disease burden of mankind. Diagnostics that detect diseases such as cancer at an early stage, when the disease is most responsive to contemporary therapies, can provide enormous social and economic benefits to society1. This has been demonstrated in early disease detection programs such as the PAP smear cervical cancer test and colonoscopy for colon cancer. Numerous other applications of molecular diagnostics require the stratification (personalization) of diseases into molecular types. Unfortunately, current diagnostic protocols typically depend on a complicated variety of tests based on a wide range of different, and often expensive, technological platforms. Each different platform requires significant investment in single-use equipment and training. Despite this investment, results can be ambiguous and require multiple, different tests to produce a confirmed result for a single pathogen. Nanoscaled biosensors offer the promise of miniaturized, inexpensive, flexible and robust “plug-and-play” molecular readout systems which can be effectively deployed in the field. In this talk we will present several platforms which our Centre is currently developing for such applications2-9.

Address:
Prof. Matt Trau
ARC Federation Fellow & Professor of Chemistry
Director, Centre for Biomarker Research and Development
Australian Institute for Bioengineering and Nanotechnology (AIBN)
The University of Queensland, QLD 4072, AUSTRALIA
E-mail: m.trau@uq.edu.au

 

A/Prof. Vladislav V. Verkhusha

Fluorescent proteins that change color after illumination or with time.

 

Kinetic pathway for the red chromophore formation, in which an anionic red chromophore is formed from the neutral blue chromophore form, provides a basis to develop several types of new fluorescent proteins (FPs) on the basis of red FPs (RFPs). The first type is bright blue fluorescent proteins (BFPs) with a tyrosine residue in the chromophore. The second type is fluorescent timers (FTs), which change their fluorescence spectrum from blue to red over time. The third type is photoactivatable from dark to fluorescent state red fluorescent proteins (PA-RFPs). To develop BFPs, a rational design strategy was applied to five RFPs of the different genetic background such as TagRFP, mCherry, HcRed1, M355NA and mKeima, which all were converted into blue probes. Further improvement of the TagRFP’s blue variant resulted in an enhanced monomeric protein, called mTagBFP, with the excitation/emission maxima at 399/456 nm. The mTagBFP is characterized by the 1.8-fold higher brightness, faster chromophore maturation, and better pH stability than currently available BFPs with a histidine-based chromophore. To develop FTs, the rational design followed by random mutagenesis was applied to mCherry. The resulting monomeric FTs exhibit distinctive fast, medium and slow blue-to-red chromophore maturation rates that depend on the temperature. At 370C the maxima of the blue fluorescence were observed at 0.25, 1.2 and 9.8 hours for the Fast-FT, Medium-FT and Slow-FT, respectively. The half-maxima of the red fluorescence were reached at 7.1, 3.9 and 28 hours, respectively. In the case of PA-RFPs, we have developed three photoactivatable variants of mCherry. The best variant, called PAmCherry1, is originally dark but when is irradiated with a violet light, such as 405 nm laser, it becomes a red fluorescent with excitation/emission maxima at 564/595 nm. The photoactivation contrast of PAmCherry1 is 4,000-fold in purified protein samples and more than 100-fold in mammalian cells. The resulting brightness is a half of that of mCherry. As compared to other monomeric or tandem-dimeric PA-RFPs, PAmCherry1 has the higher pH stability with an apparent pKa of 6.5, higher photostability, and similar number of photons per molecule. It makes PAmCherry1 an excellent probe for both conventional diffraction-limited microscopy and super-resolution imaging techniques. Lastly, the blue-to-red FTs provide a reliable way to analyze spatial-temporal history of the FT-fused proteins, to determine their age, and to study activation and repression of their synthesis and degradation.

Address:
A/Prof.Vladislav Verkhusha, Ph.D.
Albert Einstein College of Medicine
Department of Anatomy and Structural Biology
and Gruss-Lipper Biophotonics Center
1300 Morris Park Avenue, Bronx,
NY 10461,USA.
E-mail:vverkhus@aecom.yu.edu

 

Dr. Joerg Wiedenman

Engineering of photoswitchable proteins

 

The discovery of GFP-like proteins in marine invertebrates gave access to a breathtaking variety of potentially useful marker proteins. Organisms such as jellyfish, corals and sea anemones, copepods and even basic chordate animals yielded a broad spectrum of fluorescent proteins (FPs) with emission colors ranging from cyan to red. Moreover, proteins were isolated from natural sources that allow the manipulation of the fluorescence properties by altering the quantity and quality of the incident light. The emission of these proteins can be irreversibly converted from one color to another or reversibly switched on and off. The possibility to control the fluorescence with light pulses allowed exciting applications in cell biology such as regional optical marking and fluorescence nanoscopy. However, most natural FPs require further engineering to fully unfold their potential as cellular markers. This talk will focus on recent progress in the development of photoswitchable proteins.

Address:
Dr. Joerg Wiedenman
National Oceanography Centre, Southampton,
University of Southampton Waterfront Campus,
European Way, Southampton SO14 3ZH,
E-mail: joerg.wiedenmann@noc.soton.ac.uk


Prof. Tony Wilson

Fluorescence microscopy advances- filterless imaging and fast focus control

 

We will describe fliuorescence microscope that is capable of performing the simultaneous detection of the full spectrum of emission wavelengths using broadband illumination over the same wavelength range. Separation of excitation and emission light is performed not using wavelength filters, but rather through a combination of coherent spatial filtering and polarisation filltering. Spatially coherent, broadband illumination is provided by a photonic crystal fibre-based supercontinuum source. The illumination spectrum covers wavelengths in the range 450-680nm and fluorescence emission is detected over the same range. The microscope is combined with structured illumination microscopy to provide three dimensionally resolved images and improved background rejection.

A common requirement in high-resolution fluorescence microscopy is to obtain a three-dimensional representation of the object under investigation. This is typically achieved by using an optical sectioning technique to obtain a clean in-focus image of a single plane within the sample and then repeating this process a number of times at different focal settings to produce a full three dimensional image stack of the object. In dynamic studies of biological processes, a series of such image stacks are often acquired in quick succession and a computer reconstruction used to observe biological movements in three dimensions. Clearly, the speed of the fastest biological process that can be observed in this way is limited by how quickly each constituent stack in the time series can be acquired.

Developments in scanning techniques now permit practical microscope systems to acquire a single in-focus image of the specimen very quickly and the process of refocusing usually provides the real bottleneck when trying to acquire three dimensional image stacks quickly. For fundamental optical reasons, the only satisfactory method for refocusing high NA microscope systems, up until now, has been to change the distance between the objective and specimen mechanically. This is generally a slow process as it involves moving either the specimen stage or objective lens, which are both relatively heavy. This method also suffers from additional disadvantages, such as specimen agitation, which make the imaging of delicate samples, such as live cell cultures, very diffcult.

We will describe an alternative optical focusing method that does not involve mechanical movements near the specimen. This enables refocusing to be carried out remotely without the introduction of systematic aberrations. We will present a number of practical applications of this method to structured illumination microscopy as well as a two photon microscopy.

Address:
Prof. Tony Wilson
University of Oxford,
Department of Engineering Science,
Parks Road, Oxford,
OX1 3PJ, United Kingdom
E-mail: tony.wilson@eng.ox.ac.uk



Prof. Paul W. Wiseman

Cellular Cartography: Mapping protein transport and interactions in cells using new developments in image correlation spectroscopy

 

Image correlation methods provide a new window of analysis for measurement of protein-protein interactions and macromolecular transport properties from fluorescence images of living cells. These approaches are based on space and time correlation analysis of fluctuations in fluorescence intensity within images recorded as a time series on a laser scanning or TIRF microscope. We recently introduced spatio-temporal image correlation spectroscopy (STICS) which measures vectors of protein flux in cells based on the calculation of a spatial correlation function as a function of time from an image time series. Here we will describe the application of STICS and its two color extension, spatio-temporal image cross-correlation spectroscopy (STICCS), for measuring transport maps of adhesion related macromolecules such as integrin (alpha5, alpha6, alphaL), alpha-actinin, paxillin, talin, and vinculin. within, or associated with the basal membrane in living fibroblast and CHO cells. These measurements have allowed us to propose a model for the molecular clutch that regulates connections between the extracellular matrix, integrins in the membrane and the cytoskeleton during cell protrusion and migration. The seminar will also highlight recent advances we have made with a new form of reciprocal (k-) space ICS, called kICS, that allows us to measure unbiased transport coefficients of fluorescently labeled membrane proteins even if there is complex photophysics (such as nanoparticle emission blinking) of the probe. We will describe kICS measurements of the transport properties of quantum dot labeled receptors in the cell membrane as well as determination of clustering properties of QD labeled receptors based on kICS correlation studies of changes in the nanoparticle blinking.

Address:
Prof. Paul W. Wiseman
Departments of Physics and Chemistry
McGill University
Canada
E-mail: paul.wiseman@mcgill.ca

 


 

Updated Tuesday, 10 th November 2009