Poster Abstracts

 

Poster Session 1 - Wednesday 25th November 2009
11.00 am-12.30am

 

1. Jion. M. Battad

Biochemistry and Molecular Biology,
Monash University, Clayton, VIC, Australia
E-mail: jion.battad@med.monash.edu.au

 

Properties of a chromoprotein containing chromophore amino acid substitutions.

 

Rtms5 is a non-fluorescent (λ_max^Em, 615nm, QY=0.001), highly absorbing (λ_max^Abs, 592nm; ε=80,000 M-1cm-1) deep blue coloured GFP-like protein isolated from the coral Montipora efflorescens. The chromophore tri-peptide sequence Gln⁶⁶Tyr⁶⁷Gly⁶⁸ has a trans, non-coplanar configuration and is extended by the presence of an acylimine. Tyrosine at position 67 is conserved in naturally occurring GFP-like proteins. In order to further investigate chromophore-protein matrix interactions we investigated the structural and optical properties of Rtms5 variants in which Tyr⁶⁷ was substituted by His, Phe or Trp. Rtms5-Y⁶⁷F ((λ_max^Abs, 440nm; ε=1,600 M^-1cm^-1, λ_max^Em, 500nm, QY=0.09) and Rtms5 Rtms5X^Tyr-67X^His (λ_max^Abs 400nm; ε=900 M^-1cm^-1, λ_max^Em, 460nm, QY=0.03) but not Rtms5-Y⁶⁷W form chromophores. In addition, Rtms5-Y⁶⁷H underwent a polypeptide backbone cleavage adjacent to the chromophore. Polypeptide backbone cleavage is suppressed in the doubly substituted variants Rtms5-Tyr⁶⁷His, ^His146X^Ser and Rtms5-^Tyr67^His,^His146^Asnwhich mature to form acylimine-containing chromophores. We observed that Rtms5-^Tyr67^His,^His146^Serundergoes a gradual increase in yellow fluorescence after acylimine formation while Rtms5-^Tyr67^His, ^His146^Asndoes not. The X-ray crystal structure of Rtms5-^Tyr67^His,^His146^Ser was determined and shows the chromophore has adopted a cis, coplanar conformation. We conclude along with other analysis that the chromophore in Rtms5-^Tyr67^His,^His146^Ser undergoes trans-cis isomerization after acylimine formation. The Asn146 side chain of Rtms5-^Tyr67^His,^His146^Asn does not allow this to occur. These results provide a deeper understanding of the chemistry of chromophore formation in GFP-like proteins.

Co-Author:
E. Byres (1,2), D. Traore(1,2), C. DonPaul (1), R. J. Devenish (1), J. Rossjohn (1,2), M. Wilce(1,2), M. Prescott(1)

1 Biochemistry and Molecular Biology,
Monash University, Clayton, VIC, Australia

2 The Protein Crystallography Unit,
Clayton, VIC, Australia

 

 

 

 

2. Toby D M Bell

Steven J Langford,
Monash University, Clayton,
E-mail: Toby.Bell@sci.monash.edu.au
Vic 3800

 

Ensemble and single molecule studies of new water soluble "SANDIs" for fluorescence labelling.

 

There is an on-going need for new fluorescent molecules for use in labelling and imaging applications. This is particularly so for labels which can meet the exacting requirements for study as single molecules. Substituted naphthalene diimides (NDIs) are an almost unexplored class of molecules with many properties making them suitable candidates for single molecule labelling applications. We have previously reported on two novel amino substituted (SANDI) compounds that showed strong visible wavelength emission and excellent photostability as single molecules. (Bell et al., Chem. Asian J. DOI: 10.1002/asia.200900215) We now report on the synthesis and photophysical characterisation of two new water soluble SANDI compounds. The compounds are rendered water soluble by polyether groups attached at the imide nitrogens and to the amino substitutions. The fluorescence emission is dependent on the number of amino substituents with the di-substituted compound emitting at longer wavelengths than the mono-substituted compound.

Co-Authors:
Kenneth P Ghiggino, Daniel E Gomez
University of Melbourne,
Parkville, Vic. 3010.

 

 

 

3. Ales Benda

J. Heyrovsky Institute of Physical Chemistry,
Academy of Sciences of the Czech Republic,
Prague, Czech Republic
E-mail: ales.benda@jh-inst.cas.cz

 

Optical Saturation as a Versatile Tool to Enhance Resolution in Confocal Microscopy

 

One of the most actively developing areas in fluorescence microscopy is the achievement of spatial resolution below Abbe’s diffraction limit, which restricts the resolution to several hundreds of nanometers. Most of the approaches in use at this time require a complex optical setup, a difficult mathematical treatment, or usage of dyes with special photophysical properties. In this work, we present a new, to our knowledge, approach in confocal microscopy that enhances the resolution moderately but is both technically and computationally simple. As it is based on the saturation of the transition from the ground state to the first excited state, it is universally applicable with respect to the dye used. The idea of the method presented is based on a principle similar to that underlying saturation excitation microscopy, but instead of applying harmonically modulated excitation light, the fluorophores are excited by picosecond laser pulses at different intensities, resulting in different levels of saturation. We show that the method can be easily combined with the concept of triplet relaxation, which by tuning the dark periods between pulses helps to suppress the formation of a photolabile triplet state and effectively reduces photobleaching. We demonstrate our approach imaging GFP-labeled protein patches within the plasma membrane of yeast cells.

Co-Authors:
Jana Humpolickova,and(1),Joerg Enderlein(2)

1.J. Heyrovsky Institute of Physical Chemistry,
Academy of Sciences of the Czech Republic,
Prague, Czech Republic

2.Third Institute of Physics,
Georg-August-Universitat Goettingen,
Goettingen, Germany

 

 

 

4. Maria dP Crespo-Ortiz

Department of Biochemistry
La Trobe University,
Victoria, 3086

Use of fluorescent sensors to monitor oxidative stress in malaria parasite-infected erythrocytes

The malaria parasite, Plasmodium falciparum, develops within human red blood cells (RBCs), consuming host hemoglobin in order to support its own growth. Hemoglobin from the RBC is transferred to an acidic vacuole (digestive vacuole, DV) for degradation by proteases. The parasites DV, a lysosome-like organelle, is the site of hemoglobin digestion and heme detoxification and, as a consequence, the site of action of chloroquine and other antimalarials. Reactive oxygen species (superoxide and hydrogen peroxide) are by-products of hemoglobin digestion and are believed to exert significant oxidative stress on the parasite.

We used a phosphatidylcholine 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 also examined the ability of artemisinin, a drug currently used in malaria treatment, to induce oxidative stress in the parasite. BODIPY-PC can be readily incorporated into the RBC and parasite membranes. Upon oxidation, a shift in the BODIPY-PC fluorescence from red (fully conjugated) to green (oxidised state) is observed. We detected an increase in the oxidation of the parasite-associated BODIPY 581/591-PC as the intraerythrocytic parasite matures. By contrast BODIPY 581/591-PC associated with the RBC membrane experiences a low level of oxidation even in the later stages of parasite development. Treatment with a pro-oxidant compound caused increased oxidation of the probe in the parasite compartment, but less so in the host cell membrane. Chromatographic analysis of the lipids in infected RBCs shows no evidence for loss of ï¡-tocopherol or the accumulation of lipid hydroperoxides indicating that, despite the increased oxidative stress, the parasite membranes remain protected from substantial lipid oxidation. Treatment of infected RBCs with physiologically relevant levels of artemisinin caused no significant increase in the level of oxidation of BODIPY 581/591 PC.

We used a cell permeant, far red fluorescent nucleic acid-binding dye, SYTO 61, to distinguish between uninfected and infected RBCs in a flow cytometric format, and combined SYTO 61 with 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. Low levels of the oxidised fluorescent form of the reporter (2',7'-dichlorofluorescein, DCF) were detected in ring stage parasites; the DCF signal increased as the intraerythrocytic parasite matured into the trophozoite stage and active hemoglobin digestion occurred. Treatment of infected RBCs with the cysteine protease inhibitor, E-64, which inhibits hemoglobin digestion, decreased the DCF signal. Culturing of parasites in RBCs with 98% catalase inactivation did not affect their growth and resulted in only a modest increase in DCF oxidation suggesting redundancy in the systems for protecting the parasite against oxidative stress

Co-Author:
Leann Tilley(1),(2), Ying Fu(1), Eric Hanssen(1),(2), Tasha Abu-Bakar(1), Roland Stocker(3) and Nick Klonis(1),(2).

1.Department of Biochemistry
La Trobe University,
Victoria, 3086

2.Centre of Excellence for Coherent X-ray Science,
La Trobe University,
Victoria, 3086,

3.Centre for Vascular Research,
School of Medical Sciences,
University of Sydney,
NSW 2006, Australia.

 

 

 

5. Haley Carlson

Department of Chemistry
University of Alberta
Edmonton, 11227 Saskatchewan Drive, Canada
E-mail: hcarlson@ualberta.ca

 

Engineering Circularly Permuted Variants of mCherry for use as Single RFP Biosensors

 

There is significant interest in developing single fluorescent protein (FP)-based constructs where the fluorescence intensity is dependent on external parameters. However, the inherent seclusion of the chromophore from its surrounding environment poses a problem. The creation of circularly permuted variants of avGFP (aquorea Victoria green FP), where the new N- and C- termini are located in close proximity to the chromophore is a potential solution to the problem of creating a single FP-based biosensor that will respond to environmental variables. Our starting template is the engineered variant of Discosoma red FP known as mCherry. We have previously reported the discovery of three circularly permuted variants of mCherry that retained some ability to fold and form the red fluorescent chromophore (1). The three circularly permuted variants that retained a measureable amount of red fluorescene were cp22-, cp184-, and cp193-mCherry. We have since undertaken theirected evolution of the most compliant of these circularly permuted variants (cp193) for improved folding and chromophore maturation efficiency. Somewhat unexpectedly, the substitutions that conferred these properties were found to cluster in the region of the protein close to the new 6-residue loop that ties together the original C- to N- termini. Accordingly, we hypothesized that our evolved circularly permuted variant, in which the C- to N- linker region had undergone evolutionary optimization, would be generally more tolerant of permutation.
Starting from our evolved circularly permuted variant (cp193) we have methodically screened variants with new termini at every position along the β-strand adjacent to the original site of permutation. This effort has led to the discovery of a number of new permutation sites, including some that are near the middle of the β-strand and in close proximity to the chromophore, (193, 195, 196, and 201-207). These sites represent promising locations for the insertion of binding domains with the aim of constructing of red fluorescent single FP-type biosensors. The positions of interest, notably 201 and 202 were among the dimmest of the series so several rounds of error-prone PCR were used to restore the fluorescence. Several mutants with improved brightness contained either the Q213L or L65F mutations, while there were numerous others with various non-repeated mutations. More work will be done to investigate which mutations are truly advantageous to the protein. After a suitably bright FP is selected it will initially be used as the scaffold for the single-FP Ca2+ biosensor, whereby the Ca2+ binding domains M13 and calmodulin will be genetically attached to the FP gene.

 

References:
1.Li, Y. et al., Identification of sites within a monomeric red fluorescent protein that tolerate peptide insertion and testing of corresponding circular permutations , Photochemistry and Photobiology, 2008, 84: 111- 119.

Co-Authors:
Darrel Cotton (1), Sean Liew (2)
1. Department of Medicine and Dentistry, University of Alberta, Canada
2. Department of Chemistry, University of Alberta, Canada

 

 

 

6. Dr. Candida da Fonseca Pereira

1.Centre for Virology, Burnet Institute,
Melbourne, VIC, Australia.

2. Monash Micro Imaging,
Monash University, Clayton, VIC, Australia
E-mail: cfpereira@burnet.edu.au

 

Continuous association of Matrix and Vpr during HIV-1 reverse transcription

 

HIV-1 uncoating is believed to involve the peeling-off the virion matrix shell upon entry to facilitate virus replication.

We have generated fully-infectious virus containing the native HIV-1 glycoproteins, tetracysteine-FlAsH-labeled matrix and mCherry-Vpr, which is known to be associated with the reverse transcription complex.

Contrary to the current dogma, we now show that after receptor-mediated entry the vast majority of matrix proteins remain associated with the reverse transcription complex throughout nucleocytoplasmic transport and reverse transcription.

Our novel dual-labeling fluorescent system will be invaluable to further define the dynamics of the virus-host protein interplay during HIV-1 uncoating.

This work was supported by the Australian National Health and Medical Research Counsel, Portuguese Foundation for Science and Technology and Pfizer Foundation.

Co-Author:
Kate L. Jones (1,5), Paula C. Ellenberg (1), Iain Johnson (6) and Johnson Mak (1,3,4). 1.Centre for Virology, Burnet Institute,
Melbourne, VIC, Australia.
2. Monash Micro Imaging,
Monash University, Clayton,
VIC, Australia
3. Department of Biochemistry and Molecular Biology and
Monash University, Clayton,
VIC, Australia
4. Department of Microbiology,
Monash University, Clayton,
VIC, Australia.
5. School of Medical Science,
Griffith University, (Gold Coast Campus),
Southport, QLD, Australia.
6. Molecular Probes Detection Technologies,
Invitrogen Corporation, Eugene, OR, USA.

 

 

7. Helen Dacres

CSIRO Food Futures Flagship & Division of Entomology,
GPO Box 1700, Canberra, ACT2601, Australia
Helen.dacres@csiro.au

 

Determination of Forster Distances for BRET

 

Forster Resonance Energy Transfer (RET) is the non-radiative transfer of energy from a donor to an acceptor molecule. A critical parameter is the Forster distance (R0) of the RET pair. This is the distance at which 50% of the maximum possible RET efficiency (ERET) is achieved. Sensitive RET-based monitoring of molecular rearrangements requires that the separation of the donor and acceptor RET pair is matched to their Forster distance. Recently, we have shown that bioluminescence resonance energy transfer (BRET), where the donor is a luciferase rather than a fluorophore, provides lower limits of detection and higher sensitivity than conventional fluorescence resonance energy transfer (FRET) [1, 2]. R0 for standard FRET was recently determined experimentally by inserting peptide linker sequences of varying lengths between cyan (CFP) and yellow (YFP) fluorescent proteins [3]. In contrast, R0 has never been determined for BRET. To remedy this knowledge gap, we took a similar experimental approach to that used by Evers et al [3] and, for the first time, the Forster distances for genetically encoded BRET pairs are determined. This information enables rational choices to be made between BRET and FRET in a range of research and diagnostic applications.

 

References:
1. Dacres, H., Dumancic, M.M., Horne, I., Trowell, S.C., 2009. Biosensors and Bioelectronics, 24, 1063 “ 1070.
2. Dacres, H., Dumancic, M.M., Horne, I., Trowell, S.C., 2009. Analytical Biochemistry, 385, 194 “ 202.
3. Evers, T.H., Van Dongen, E.M.E.M., Faesen, A.C., Meijer, E.W., Merckx, M., 2006. Biochemistry, 45, 13183 “ 13192.

Co-Author:
Wang, J., Dumancic, M.M., Trowell, S.C.
CSIRO Food Futures Flagship & Division of Entomology,
GPO Box 1700, Canberra, ACT2601, Australia

 

 

 

8. Michael Strauss

Institute for the Biotechnology of Infectious Diseases,
University of Technology, Sydney (UTS), NSW
E-mail: michael.strauss@student.uts.edu.au

 

Super resolution microscopy (3D-SIM) takes a closer look at the essential bacterial cell division protein, FtsZ

 

FtsZ is an essential bacterial cell division protein which is fundamental to dictating where and when the cell divides. It orchestrates division by self assembling into a cytokinetic structure known as the Z ring at the future division site. This phenomenon is a virtually universal aspect of cell division in bacteria and accordingly it is a potential target for the development of novel antibiotics. However, how the Z ring assembles and how its assembly is regulated in time and space remains unknown, even in well-studied rod-shaped bacteria such as Escherichia coli and Bacillus subtilis. In Staphylococcus aureus, which is a leading cause of hospital- and community-acquired infections around the world, this process remains elusive. The small size of these spherical pathogens (~1 µm in diameter) makes conventional fluorescence microscopy very challenging, if not impossible and FtsZ localization has never been reported in live cells of this organism.

Using immuno-fluorescence microscopy and time lapse microscopy with fluorescent fusion proteins we previously showed that in addition to the Z ring, FtsZ assembles into dynamic helical structures throughout the entire length of B. subtilis cells. This extended helix appears to be a permanent cytoskeleton feature of the cell which is subsequently remodelled in a cell-cycle mediated process to yield a Z ring in B. subtilis. However, the image resolution afforded by conventional fluorescence microscopy cannot adequately visualise the arrangement of FtsZ within these structures. We have therefore visualised them using 3D-SIMTM Super-Resolution Imaging, with the DeltaVision OMX system housed at the UTS Microbial Imaging Facility. The localisation of FtsZ in B. subtilis will be presented for both fixed (immuno-fluorescence) and live (FtsZ-GFP) cells allowing a closer look at the molecular architecture of these FtsZ structures in vivo. In S. aureus, the localisation of FtsZ in live cells will be presented for the first time. Intriguingly, this hints at a similar assembly pathway for Z ring assembly in this important pathogen.

Co- Author:
Lynne Turnbull, Andrew Liew, Torsten Theis, Leigh Monahan, Cynthia Whitchurch and Liz Harry.
Institute for the Biotechnology of Infectious Diseases,
University of Technology, Sydney (UTS), NSW

 

 

 

9. Craig Don Paul

Biochemistry and Molecular Biology;
The Protein Crystallography Unit,
Monasy University, Clayton
E-mail: craig.donpaul@med.monash.edu.au

 

Turning Midori into Orange

 

CGP123 is a bright green fluorescent protein (FP) (λmaxem 505nm, ΦF = 0.699, λmaxabs 495nm, ε ~ 59,000 M-1.cm-1) engineered to withstand prolonged exposure to elevated temperatures. We have used mutagenesis to convert CGP123 into an orange, non-fluorescent, photochromic protein (λmaxem 515nm, ΦF = 0.008, λmaxabs 505nm, ε ~ 97,000 M-1.cm-1). Based on X-ray structural studies a model will be presented to interpret the optical behaviour of these proteins. The orange photochromic protein is suitable for use as photo-switchable dark acceptor with commonly used green fluorescent proteins in fluorescence resonance energy transfer applications.

Co-Author:
Emma, Rodney Devenish, Jamie Rossjohn, Matthew Wilce, Mark Prescott
Biochemistry and Molecular Biology;
The Protein Crystallography Unit,
Monasy University, Clayton

 

 

10. Dr. Michal M. Godlewski

Department of Chemistry and Biomolecular Sciences,
Faculty of Science, Macquarie University,
Sydney, 2109 NSW, Australia;
E-mail: mickgodl@hotmail.com

 

The elusive pulse in protein production in Trichoderma reesei

 

The filamentous fungus Trichoderma reesei has been exploited as a producer of industrial biomass degrading enzymes for decades. Improvement of the production yields and modifications to the secretory profile have been carried out by random mutagenesis and genetic engineering. Apart from the increase in enzyme yields, the introduced changes have altered fungal physiology, and secretory response to facilitate cellular adaptation to overproduction. Attempts to produce heterologus proteins in the fungal cell factory have been only partially successful and the yields have remained below expectations. While most of the efforts have been directed into improving the production yields, less is known of the fungal secretory pathway, its evolution and the progress of proteins through the hyphae. In this work, we have studied the early pattern of expression of the native cellobiohydrolase I (CBHI) and a heterologus xylanase B (XYNB) protein in different strains of the Trichoderma reesei. We found that CBHI production follows a previously unknown pulsing pattern during the first 36 h of growth. This observation was consistent across the wild type to a high-secreting industrial strain. When a misfolded CBHI was expressed, the protein was mostly accumulated within ER, however, some leaked to the suppernatant. In a strain producing a heterologus XYNB the enzyme mainly accumulated in the hyphae. In conclusion, we have demonstrated that expression of the native CBHI protein in T. reesei follows a distinct pulsing pattern, while misfolding of the CBHI prevented the enzyme from exiting ER. Finally, it seems that a foreign protein XYNB produced in T. reesei is mostly sequestered in the hyphae after translocation from the ER. This suggests, that the reported issues with low heterologus protein yield derivate from the bottlenecks in the late secretory pathway, rather than protein misfolding or problems in the ER to Golgi apparatus trafficking.

Co-Author:
Liisa Kautto, Helena Nevalainen,
Department of Chemistry and Biomolecular Sciences,
Faculty of Science, Macquarie University,
Sydney, 2109 NSW, Australia;
E-mail: mickgodl@hotmail.com

 

 

 

11. Dr. Michal M. Godlewski

1.Department of Chemistry and Biomolecular Sciences,
Faculty of Science, Macquarie University,
Sydney, 2109 NSW, Australia;

2.Department of Physiological Sciences,
Faculty of Veterinary Medicine,
Warsaw University of Life Sciences - SGGW,
Warsaw, Poland
E-mail: mickgodl@hotmail.com

 

Correlative microscopy – application of electron microscopy finder‐grids in quantitative scanning cytometry

 

The term correlative microscopy is used to describe a connection between high-throughput, often quantitative screening with high-resolution imaging. The method presented in this poster utilizes high-throughput quantitative population analysis by scanning cytometry to pinpoint the regions of interest (ROI) for further evaluation under high-resolution imaging systems. In the study of TGF‐β1 expression, a major cytokine responsible for sensitization of enterocytes to programmed cell death, cross-sections of piglet small intestine were labelled against TGF‐β1 and overlayed with EM finder‐grids. The samples were scanned by SCAN^R scanning cytometer to determine the index of TGF‐ β1‐responsive enterocytes. From the tissue map generated by SCAN^R, ROI with TGF‐β1‐positive cells were selected and their coordinates stored from the sample overview. ROI were then located under the confocal microscope for visualization of the pattern of the cytokine expression. Results confirmed the paracrine, packet pattern of TGF‐β1‐mediated cell death in the intestine mucosa. The finder‐grids were also utilized for the study of the expression and localization of the fusion protein CBHI‐Venus in the filamentous fungus Trichoderma reesei. CBHI is the main secreted cellulase enzyme of the fungus. T. reesei was cultured for 18 to 28 h after which the hyphae were fixed in 1:1 vol mixture of 70% ethanol with acetone, rinsed in PBS and placed on microscopy slides, and overlayed with finder‐grids. For scanning cytometry, the sample focal plane was maintained over the finder‐grid‐reflected fluorescence, preventing the sample from drifting out of focus. This is especially crucial in the analysis of fungal hyphae, which are often entangled in complex 3D structures. The Venus‐related fluorescence was simultaneously stored and analyzed on a separate, specific fluorescence channel. Results from this study quantitatively confirmed our earlier observations of the pulse pattern of CBHI production in T. reesei. In conclusion, EM finder‐grids have proven a valuable asset in scanning cytometry studies providing a focal plane for the analysis of complex 3D samples and ROI for further high‐resolution imaging

Co-Author:
Piotr Pietrzak(2), Anna Kotunia(3), Romuald Zabielski(2), Marko Nykanen(4), Helena Nevalainen(1)

1.Department of Chemistry and Biomolecular Sciences,
Faculty of Science, Macquarie University,
Sydney, NSW 2109, Australia.

2.Department of Physiological Sciences,
Faculty of Veterinary Medicine,
Warsaw University of Life Sciences - SGGW,
Warsaw, Poland

3. The Kielanowski Institute of Animal Physiology and Nutrition,
Jablonna, Poland

4. Research & Development Office,
Kids Research Institute, Children’s Hospital at Westmead,
Sydney, NSW 2145, Australia

 

 

12. Mr. Daniel Gruszecki

Honours Student w/ BioEnergy Group & Smart Surfaces Group
School of Chemistry, Physics and Earth Science
Flinders University
GPO Box 2100
Adelaide SA 5001
Australia
E-mail: daniel.gruszecki@flinders.edu.au

 

Towards a bioreactor for the mass cultivation of algal species

 

Algae is seen as a possible source for the mass production of commercial biofuel. The key hurdle in realising this technology, will require a substantial improvement in the efficiency of the algae to biofuel production process. A well designed bioreactor will allow control over variables in a controlled environment, allowing an optimisation of the growth process. Initial investigations indicate the need for a multi-disciplinary approach to the problem combining photonics, biology, and engineering. Successful development of an algal bioreactor design involves maximising cell growth rate and cell density, which increases the commercial feasibility of the technology. However, studies of the role of light in algae growth, and how to more effectively deliver radiation, and achieve maximum cell growth in the bioreactor is an essential problem to be resolved. The photosynthetic growth of algae occurs via absorption of photons by light harvesting pigments within the cell walls. This begins an electron transfer process resulting in the growth and reproduction of the culture where a series of chemical compounds. These compounds, known as fatty acid lipids, can be extracted from the algae cell walls and used for the production of biofuel Through investigating algae absorption in the visible spectrum and the growth response of algae to various wavelengths of light, the development of a more effective bioreactor for the mass cultivation of algae is possible. A key parameter in this investigation is the penetration depth of light through culture medium which affects radiation intensity through the medium as culture density increases. This work has focussed on investigating the critical wavelengths for algal growth, with the use of Light Emitting Diodes (LEDs) as a source of illumination. LEDs have been identified as a possible solution for maximising commercial algal growth, providing wavelength specific radiation, a low temperature outpu!t, and a high energy to photon conversion rate in a cost effective manner. Herein, research focuses on the chlorella genus of algae, which is a fresh water green algae identified as a high lipid producing algal strain. Characterisation of the chlorella absorption spectra in primary light harvesting chromophores, indicates that Chlorophyll a and b absorb strongly at 450nm, 490nm, 640nm and 670nm. The wavelengths 450nm, 490nm, 640nm and 670nm appear essential to the growth of chlorella algae. Through analysis of intensity radiation and the penetration of photons via a controlled environment photodiode experiment, optimisations

 

 

13. Christopher Artlett

MQ Photonics, Dept of Physics,
Macquarie University
E-mail: judith@science.mq.edu.au

 

Ultrafast laser ablation of dental enamel

 

Ultrafast pulsed lasers are widely used for precise ablation or laser machining and offer potentially painless treatment for patients with dental caries. While demonstrations of such lasers for dentistry have been reported, the optimal laser parameters for this treatment still need to be determined. We investigated the ablation characteristics, material removal efficiency, and the intra-pulpal temperature rise during ultrafast pulsed laser irradiation for two different laser wavelengths (800 and 400 nm), and analysed the post-treatment quality of the ablated enamel regions.
An ultrafast pulsed Ti sapphire laser (~100 fs pulsewidth, pulse repetition rate 1 kHz, average power up to 200 mW) was used at two different wavelengths to form craters in the enamel of freshly extracted human teeth by irradiating the prepared teeth for periods of up to 6 minutes. During irradiation treatment, the temperature rise inside the dental pulp was monitored by an embedded thermocouple. The depth of the crater and the quality of the prepared crater surface were assessed by optical microscopy and surface profilometry.
The femtosecond ablation characteristics of dental enamel were very similar for both the 400 nm and 800 nm laser wavelengths used. Ablation threshold fluence was significantly lower for the 400 nm wavelength, consistent with theoretical modelling. At higher laser fluences ( > 10 Jcm-2), saturation of the ablated depth was observed, with the 400 nm wavelength experiencing a stronger effect. This was attributed to plasma-shielding effects, either through absorption or reflection. Efficiency of ultra-short pulsed laser ablation was also found to be relatively independent of the laser wavelength for fluences below the saturation threshold. These results are consistent with recent modelling using a multiple rate equation approach.
Thermal measurements by embedded thermocouples within teeth during ablation showed a modest temperature rise (2“ 3ï‚°C) even with high laser fluence over exposure times of 6 minutes with a 1 kHz laser repetition rate. With the laser power reduced to match the fluence range considered to be reasonable for treatments, no discernable temperature rise was evident. No active cooling of any kind was applied. In contrast, the use of longer pulse duration lasers in dental treatment in the past has resulted in reported temperature rises in excess of 20ï‚° C without active cooling, sufficient to permanently damage or kill the nerve.

Post-treatment examination of surfaces and structures produced in dental enamel showed no significant difference due to wavelength. The crater surface was slightly roughened, but not cracked, and was suitable for good adhesion of filling material. After laser ablation there was no evidence of residual thermal damage to surrounding areas of enamel as determined by optical microscopy. An increase in laser power corresponded to an increase in recasting or redepositing of material. Increasing the laser pulse repetition rate had a similar effect, which was much more obvious at higher power levels. It is likely that active cooling would not be required for clinical treatments, and the material removal rates during laser ablation should be comparable with those for conventional mechanical drilling.
Co- Author:
Judith Dawes(1),Graham Marshall(1), Peter Balling(2)
1.MQ Photonics, Dept of Physics,
Macquarie University

2.Dept of Physics,
University of Aarhus,
Denmark

 

 

14. Magnus T. L. Hsu

School of Mathematics and Physics, University of Queensland,
St Lucia QLD 4072, Australia.
E-mail: mhsu@physics.uq.edu.au

 

Quantum limited particle sensing in optical tweezers

 

Particle sensing in optical tweezers systems provides information on the position, velocity and force of the specimen particles. The conventional quadrant detection scheme is applied ubiquitously in optical tweezers experiments to quantify these parameters. We show that quadrant detection is non-optimal for particle sensing in optical tweezers and propose an alternative optimal particle sensing scheme based on spatial homodyne detection. We have developed a formalism for particle sensing in terms of transverse spatial modes and present numerical simulations of the efficacy of both quadrant and spatial homodyne detection. We demonstrate that an order of magnitude improvement in particle sensing sensitivity can be achieved using spatial homodyne over quadrant detection. Experimental results of adaptive spatial homodyne detection for optimal particle sensing are also shown.

Co-Author:
Jian Wei Tay (1) and Warwick P. Bowen (1,2)

1. Jack Dodd Centre for Photonics and Ultracold Atoms,
Department of Physics, University of Otago, Dunedin, New Zealand.

2. School of Mathematics and Physics,
University of Queensland, St Lucia QLD 4072, Australia.

 

 

 

15. David Hvasanov

School of Chemistry
University of New South Wales
Sydney, NSW, 2052, Australia
Email: david.hvasanov@student.unsw.edu.au

 

Membrane-Bound Light Harvesting Bioconjugates as Chloroplast Mimics

 

Cells are highly complex bio-nanoreactors with a complex synthetic medium where multiple multistep reactions occur simultaneously throughout the cell. Cells achieve these reactions with high specificity and efficiency by utilising biological catalysts such as enzymes. In order to prevent interference and degradation of these catalytic pathways, cells undergo compartmentalisation. Compartmentalisation achieves positioning of synthetic pathways at specific sites in the cell (organelles) and regulates the cellular environment by controlling the flux of molecules. Recently, a multistep catalytic cycle was developed which demonstrated enzyme encapsulation with positional control in separate domains using polymersomes [1,2].

In nature, compartmentalisation is demonstrated in plants by the organelle, chloroplast, which is responsible for photosynthesis. The development of enzyme encapsulation to mimic photosynthesis has been previously reported by incorporating adenosine triphosphate (ATP) to convert light energy into ATP chemical energy [3,4].

Previous work in our group have developed bioconjugates, linking iso-1 cytochrome c from Saccharomyces cerevisiaea to a light activated chromophore, ruthenium (II) bisterpyridine complexes which forms a light-harvesting bioconjugate [5]. We were able to demonstrate photoinduced reduction of cytochrome c by the Ru(II) chromophore in the presence of light.

In this work, we present the development of light-harvesting bioconjugates towards synthetic chloroplast mimics through encapsulation, through the use of liposomes and polymersomes. Ruthenium bisterpyridine complexes have been synthesised and covalently attached to the Cys102 residue of iso-1 cytochrome c.

The target is the development of chloroplast mimics via non-covalent and covalent encapsulation of these bioconjugates in vesicles. The location and incorporation of bioconjugate encapsulation in vesicles will be confirmed by fluorescence microscopy using fluorescent dyes conjugated to bioconjugates and model proteins.

 

References:
1. D. M. Vriezema, P. M. L. Garcia, N. S. Oltra, N. S. Hatzakis, S. M. Kuiper, R. J. M. Nolte, A. E. Rowan and J. C. M. van Hest, Angew. Chem. Int. Ed., 46, pp. 7378-7382 (2007).
2. D. M. Vriezema, J. Hoogboom, K. Velonia, K. Takazawa, P. C. M. Christianen, J. C. Maan, A. E. Rowan and R. J. M. Nolte, Angew. Chem. Int. Ed., 42, pp. 772-776 (2003).
3. G. Steinberg-Yfrach, J. L. Rigaud, E. N. Durantini, A. L. Moore, D. Gust and T. A. Moore, Nature, 392, pp. 479-482 (1998).
4. G. Steinberg-Yfrach, P. A. Lindell, S. C. Hung, A. L. Moore, D. Gust and T. A. Moore, Nature, 385, pp. 239-241 (1997).
5. J. Peterson, T. A. Smith and P. Thordarson, Chem. Commun., pp. 1899-1901 (2007).

Co-Author:
J. Peterson and P. Thordarson
School of Chemistry
University of New South Wales
Sydney, NSW, 2052, Australia

 

 

16. Aaron Irving

Monash Institute of Medical Research,
Monash University
E-mail:aaron.irving@med.monash.edu.au

 

PKR: innate immunity and cell biology.

 

The protein kinase PKR is a component of the innate immune system and is a primary sensor of virus infection. Upon activation PKR has been demonstrated to phosphorylate the eukaryotic translation factor 2alpha to halt translation. We have observed PKR also affects basic cellular processes that include motility, phagocytosis, and autophagy. The mechanism for these observations is not clear, but suggest regulation of the actin cytoskeleton. Interestingly, we have identified a protein interaction between PKR and a key regulator of the cytoskeleton. Hence, we have identified a putative link between PKR and fundamental cellular processes that mediate entry and degradation of pathogenic organisms. This scenario suggests a novel mechanism by which innate immune sensors may mediate a response to invasive pathogens.
Co- Author:
Anthony Sadler
Monash Institute of Medical Research,
Monash University

 

 

17. Prof. Hye-Ock Jang

Departments of Dental Pharmacology and Biophysics,
Pusan National University, Beomeo-ri, Mulgeum-eup,
Yangsan-si, Gyeongsangnam-do 626-870,
E-mail: jho9612@pusan.ac.kr

 

Effects of 1-Butanol on Rotational Mobility of n-AS in Neuronal and Model Membranes

 

To provide a basis for studying the molecular mechanism of pharmacological action of 1-butanol, we carried out a study of the membrane action of the general anesthetics. The set of n-(9-anthroyloxy)stearic or palmitic acd (n-AS) probes (n = 2, 6, 9, 12 and 16) have been used to examine gradients in fluorescence polarization. In a does-dependent manner, 1-butanol decreased the anisotropies of 6-AS, 9-AS, 12-AS and 16-AP in the hydrocarbon interior of the synaptosomal plasma membrane vesicles isolated from bovine cerebral cortex (SPMV), and liposomes of total lipids (SPMVTL) and phospholipids (SPMVPL) extracted from the SPMV, but the 1-butanol increased the anisotropy of 2-AS in the membrane interface. The magnitude of rotational mobility in accordance with the carbon atom of phospholipids comprising SPMV, SPMVTL and SPMVPL was in the order at the 16, 12, 9, 6 and 2 position of aliphatic chain present in phospholipids. The sensitivity of increasing or decreasing effect of rotational mobility of the hydrocarbon interior or surface region by 1-butanol differed depending on the carbon atom numbers in the descending order of 16-AP, 12-AS, 9-AS, 6-AS and 2-AS. Furthermore, the sensitivity of increasing or decreasing effect of rotational mobility of the hydrocarbon interior or surface region by the 1-butanol differed depending on the neuronal and model membranes in the descending order the SPMV, SPMVPL and SPMVTL.

Key word: 1-Butanol; Fluorescent probe technique, Hydrocarbon interior, Membrane interface, Rotational mobility
Co-Author:
Dong-Hun Lee(1), Cheol-Ho Lee1, Soo-Cheo(1), Jeong(1), Ju-Seok Park(1), Gye-Beom Shin(1), Won-Il Kim(1), Dong-Soo Jeong(1), Young-Chan Jeon(2), In-Kyo Chung(3), Moon-Kyung Bae(4), Il Yun(1).

1. Departments of Dental Pharmacology and Biophysics,
Pusan National University, Beomeo-ri, Mulgeum-eup,
Yangsan-si, Gyeongsangnam-do 626-870,

2. Departments of Prosthodontics,
Pusan National University, Beomeo-ri, Mulgeum-eup,
Yangsan-si, Gyeongsangnam-do 626-870,

3. Departments of Oral and Maxillofacial Surgery and Clinical Pharmacology
Pusan National University, Beomeo-ri, Mulgeum-eup,
Yangsan-si, Gyeongsangnam-do 626-870,

4. Departments of Oral Physiology and Molecular Biology, School of Dentistry and Research Institute for Oral Biotechnology,
Pusan National University, Beomeo-ri, Mulgeum-eup,
Yangsan-si, Gyeongsangnam-do 626-870,

 

18. Joana Santos

Institute for the Biotechnology of Infectious Diseases,
University of Technology, Sydney, Australia
E-mail: jopatalogica@hotmail.co

 

Live cell imaging: a fluorescent look into the function of a bacterial protein, in Bacillus subtilis

 

Bacterial cell division involves the formation of a septum at midcell, between two replicated chromosomes. It begins with the polymerization of the tubulin-like protein, FtsZ, into a ring, called the Z ring. This ring then constricts as the septum forms. It has been proposed that another cytosolic division protein, FtsA, promotes Z ring formation by directly interacting with FtsZ. However its exact role remains elusive.

A deletion of the ftsA gene in the model Gram positive bacterium, Bacillus subtilis, causes severe cell filamentation and a significant reduction in Z ring formation. However, using immunofluorescence and a synchronous cell cycle we have now shown that this reduction is due to the inability of the Z ring to constrict and thus preventing it from forming at new division sites. Constriction could be delayed either due to a delay in the recruitment of downstream division proteins to the division site, or due to a decrease in the rate of FtsZ turnover in the Z ring, delaying Z ring constriction. To test the first hypothesis we used fluorescent protein fusions to follow the timing of the recruitment of downstream proteins. Our results indicate that the timing of this recruitment is indeed delayed. This suggests that the recruitment of these divisomal proteins to the division site is not strictly dependent on FtsA but it still requires its presence for normal efficiency. To test the second hypothesis, we are currently measuring the in vivo rate of FtsZ turnover in the Z ring, in the presence and absence of FtsA using Fluorescence Recovery After Photobleaching (FRAP). It could be that FtsA plays both roles, and these ideas will be presented.
Co-Author Details:
Elizabeth Harry
Institute for the Biotechnology of Infectious Diseases,
University of Technology, Sydney, Australia

 

 

 

 

19. Betty Kouskousis

1.Centre for Micro-Photonics,
Swinburne University of Technology, Australia

2. Immune Signalling Laboratory,
Peter MacCallum Cancer Centre, Australia
E-mail: bkouskousis@swin.edu.au

 

Nanometre Localisation of Quantum Dots Based on their Stochastic Blinking Properties and (F)PALM Rendering Algorithms.

 

Fluorescence imaging is routinely used to obtain information on the spatial organisation of proteins in cells and other biological tissues. However due to the limit of diffraction imposed by conventional optical techniques, images can only be resolved within 250-300 nm. In response to this, several methods capable of breaking the diffraction barrier have been developed, in particular (fluorescence) photoactivatable localisation microscopy (F)PALM [1, 2]. (F)PALM is based on the serial photoactivation and subsequent bleaching of numerous sparse subsets of photoactivatable fluorescent protein (PA-FP) probes within a specimen. Due to the small subset of PA-FP probes activated, individual molecules can thereby be precisely localised to obtain a highly resolved map (image) with an effective resolution of tens of nanometres. These exciting new technologies are likely to revolutionise cell biology, but are yet unavailable to most biologists.
We describe here experiments towards developing a high-resolution system for biological imaging. As a first step towards developing the algorithms needed for such a system, we have utilised semiconductor nanocrystals or quantum dots (QDs). The attractive optical properties of QDs include narrow emission bands, emission wavelength tuneability with size, photostability and enhanced brightness. The stochastic blinking behaviour of QDs provide opportunities for superresolution without the need for serial activation and bleaching of FA-FPs [3]. In addition QDs provide a simple model for developing the algorithms needed for (F)PALM, as an alternative to PA-FPs, has been explored in this work.
A final high resolution spatial map (image) of randomly dispersed QDs is obtained using independently written localisation and rendering algorithms based on the techniques described by [1, 2]. Furthermore, the results indicate that localisation and photon summing precision rely heavily on the signal to noise (SNR) ratio of the detection system; where the localisation precision was found to rely heavily on the particle density of the sample. We will describe progress in applying (F)PALM and (QD)PALM to biological imaging.

 

Reference:
1.Betzig, E., et al., Imaging Intracellular Fluorescent Proteins at Nanometer Resolution. Science, 2006. 313(5793): p. 1642-1645.
2.Hess, S.T., T.P.K. Girirajan, and M.D. Mason, Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy. 2006. 91(11): p. 4258-4272.
3.Lidke, K.A., et al., Superresolution by localization of quantum dots using blinking statistics. Optics Express, 2005. 13(18): p. 7052-7062.

 

Co-Author Details:
Joel van Embden (1), Dru Morrish (1), Kerrie-Ann McMahon(1,2), Sarah Russell(1,2), Min Gu(1)
1. Centre for Micro-Photonics,
Swinburne University of Technology, Australia

2.Immune Signalling Laboratory,
Peter MacCallum Cancer Centre, Australia

 

 

 

20. Noga Kozer

Ludwig Institute for Cancer Research, Post Office Box 2008,
Royal Melbourne Hospital, Victoria 3050, Australia.
Department of Biochemistry and 1Molecular Biology BIO21 Institute, The University of Melbourne, Melbourne, Victoria 3050
E-mail: noga.kozer@ludwig.edu.au

 

Probing solution conformations of EGFR ectodomain Is it really an open-and-shut case?

X-ray structural studies revealed two conformations of the EGFR ectodomain (ECD) - a compact, tethered conformation in the absence of EGF and an untethered, or extended conformation, in the presence of EGF. To investigate the conformational transitions in solution an EGFR-ECD derivative with a monomeric Red Fluorescent Protein (mRFP) at the N-terminus and an enhanced Green Fluorescent Protein (eGFP) at the C-terminus was created and characterized by fluorescence-detected analytical ultracentrifugation, time-resolved FRET and fluorescence polarization anisotropy. Surprisingly, we found no evidence of a tethered unliganded conformation nor do we observe a large shape change upon ligand binding as predicted by the crystal models. Instead a change in protein dynamics was observed.

Co-Author:
Christine Henderson, Michael F. Bailey1, Antony W. Burgess, Edouard C. Nice, and Andrew H.A. Clayton*
Ludwig Institute for Cancer Research, Post Office Box 2008,
Royal Melbourne Hospital, Victoria 3050, Australia.
Department of Biochemistry and 1Molecular Biology BIO21 Institute, The University of Melbourne, Melbourne, Victoria 3050
E-mail: Andrew.Clayton@ludwig.edu.au*

 

 

21.Dayong Jin

MQ Photonics Research Centre
Department of Physics and Engineering
Faculty of Science
Macquarie University,Sydney
NSW 2109,Australia
E-mail: jin@ics.mq.edu.au

 

Luminescence Silica Nanoparticles for Time-gated Immunofluorescence Cell Imaging

Time-gated luminescence bioimaging based on microsecond-lifetime luminescent biolabels can provide a complete background-free condition for detecting target cells in an autofluorescence biosample matrix. We report our recent success in synthesizing ultra-bright silica nanoparticles with microsecond luminescence lifetimes. These luminescent europium nanoparticles absorb 365nm excitation and emit 617 nm sharp (full width at half maximum <10 nm) luminescence with ~340 μs decay time and a large quantum yield of > 60%. We applied covalent-binding copolymerisation technique using a BHHCT-Eu3+ complex conjugated with 3-aminopropyl(triethoxy)silane, free 3-aminopropyl(triethoxy)silane, and tetraethyl orthosilicate in a water-in-oil reverse microemulsion. By the addition of 3-(trihydroxysilyl)propylmethylphosphonate (THPMP), these nanoparticles (~40 nm in diameter) observed to be well dispersed in aqueous medium. The new nanoparticles were successfully applied to distinguish an environmental pathogen, Giardia lamblia, within a concentrate of environmental water sample using a time-gated luminescence microscope with pulsed UV LED excitation. SEM image was obtained to study the labeled Giardia cells. These results suggest a broad range of potential bioimaging applications where high signal-to-background ratio is required for samples containing high concentration of autofluorescence background.

 

 

22. James R. Mansfield

CRi, Woburn, MA
USA

Taking a closer look: Validating macroscopic optical imaging results with microscopy

 

In vivo optical imaging is a valuable technique for monitoring the distribution of proteins and other biomarkers in living animals quickly and effectively.1,2 However, a critical and sometimes overlooked component of the method development of these experiments is the validation of the results obtained in vivo from intact animals through some other, complementary, technique. Of the two major small animal optical imaging modalities, bioluminescence and fluorescence, only the latter offers the capability to easily validate in vivo results through ex vivo or in situ microscopic imaging. Near-IR-labeled antibodies, particularly for markers that may be overexpressed in cancer, are widely imaged in vivo to determine the biodistribution of a protein or as a means of quantitating tumor size non-invasively. However, as these probes can sometimes bind to necrotic regions in tumors or get trapped in interstitial spaces, it is important to ascertain their b!

iodistribution with appropriately high resolution in order to qualify the in vivo results.
Methods: In vivo fluorescence data from whole animals and from whole organs, both in situ and ex vivo, were compared to the corresponding microscopic images from the same animals. In one model, AF647-labeled anti-EGFR antibodies were injected into mice with subcutaneous tumors and imaged. In another model, fluorescent images of H&E tissue sections from excised tumors from mice that had been injected with Cy5-labeled anti-Her2 were acquired using with multispectral microscopy to generate simulated brightfield images. Such images resemble standard DAB-stained immunohistochemistry samples that can be readily interpreted by pathologists. Finally, an example showing how imaging of whole, excised tumors can be used to validate measured in vivo fluorescence intensity when tissue autofluorescence is correctly unmixed from a multispectral dataset will be shown.

Results: Microscopic imaging indicated that the EGFR labeling pattern was often consistent with specific binding to the EGFR antigen. However, in some instances, binding of the label to necrotic regions was demonstrated, affecting the validity of whole-tumor intensity estimations: strong labeling in some tumors could be shown to be due to prominent binding to necrotic regions rather than to viable tumor. In vivo tumor intensity signals, however, agreed closely with ex vivo whole-organ results.

Conclusions: In vivo imaging, while useful and practical, requires careful validation of the model system being imaged to ensure correct interpretation of results. Two good methods of validation are the imaging of tissues, either in situ or as whole, excised organs, and the high-resolution imaging of tissue sections on a microscope. Of the popular optical imaging modalities, only fluorescence imaging is readily amenable to such validation.

References:
1. Graves EE, et al., Curr Mol Med. 2004 Jun;4(4):419-30. 2. Choy G et al., Mol Imaging. 2003 Oct 2(4):303-12.
Co-Author:
Dawn L. Nida (2),Rebecca Richards-Kortum (2),Richard M. Levenson (1)
1. CRi, Woburn, MA;
2. Rice University, Houston, TX

 

 

 

 

Poster Session 2 - Thursday 26th November 2009

3.30 pm-5.00 pm

 

 

23. Massimiliano Massi

Department of Chemistry
Curtin University of Technology
Kent Street, Bentley 6845 WA, Australia
E-mail: m.massi@curtin.edu.au

 

Design of Phosphorescent Probes Based on Organometallic Re(I) Tetrazolate Complexes

 

Compared to purely organic compounds, luminescent heavy metal complexes (e.g. Re and Ir) possesses advantageous photoluminescence properties. These include larger Stokes shifts (larger than 150 nm) and longer excited state lifetimes (from tens of nanoseconds up to millisecons range), both of which can be finely tuned by means of chemical design. These advantages allow easy differentiation between the luminescence of the probe and the interfering autofluorescence, thus reducing typical issues associated with the use of fluorescent organic labels. The downside of bioprobes based on heavy metals is their potential toxicity. Recent studies have shown however that often toxicity is not associated with a particular metal centre per se, but with the choice of certain ligands. We have recently started investigating whether organometallic complexes bearing tetrazolate ligands could be promising candidates for optical imaging, owing to the phosphorescent properties of the metal as well as the high biocompatibility of the tetrazole functional group. We have prepared and investigated the photophysical properties of a series of (biim)(CO)3ReL complexes (biim = bipyridine, phenanthroline; L = aryltetrazolate). Preliminary applications of these compounds in optical imaging ar

Co-Author:
Massimiliano Massi, Morry Silberstein
Department of Chemistry
Curtin University of Technology
Kent Street, Bentley 6845 WA, Australia

 

 

 

24. Guillaume Maucort

PhD Student
Centre for Biophotonics and Laser Science
School of Mathematics and Physics
The University of Queensland
St Lucia, 4072, QLD

The Queensland Brain Institute
St Lucia, QLD 4072
Australia
E-mail : maucort@physics.uq.edu.au

 

Biophysical measurements of neurosecretory vesicle microenvironment during stimulation of exocytosis

 

Neurosecretory vesicles are important carriers loaded with neurotransmitters in neurons and neurosecretory cells that underpin neuronal and hormonal communication. In response to stimulation, neurosecretory vesicles undergo a journey in the cytosol of the neurosecretory cells to the plasma membrane where they fuse in a process called exocytosis. Changes in the dynamics of vesicles due to local viscosity variation have been hypothesized to tightly control exocytosis by regulating the number of vesicles diffusing to the plasma membrane. Although much work has been done in understanding the dynamics of the cortical actin cytoskeleton in this process, no direct measurement of the viscoelasticity of the microenvironment surrounding the secretory vesicles has been attempted before.

Here we propose two complementary experimental strategies to study general and particular dynamics of neurosecretory vesicles in cultured bovine chromaffin cells.

The first experiment uses 3D confocal image stacks of a cell of interest to extract the 3D trajectory. Chromaffin cells are transfected by electroporation with hGH-GFP which is stored within secretory vesicle and imaged by confocal microscopy. Using image analysis we track these multiple vesicles in three dimensions before and after stimulation of the cells by nicotine. Such an experiment allows us to track multiple vesicles at the same time and then to obtain the major trends in positions (relative to the membrane), trajectories and speed of the vesicles.

The second experimental strategy relies on real-time physical tracking of a single vesicle. The fluorescent sample is imaged by a fast camera and the signal is analysed in real time. By selecting a region of interest including a vesicle, specially developed software including a feedback loop allows us to follow the vesicle in three dimensions using a piezo-driven stage. This real time tracking added to the fast camera allows us to measure the power spectrum of Brownian motion of the vesicle during its journey and thus to detect any changes in the mechanical properties of its surroundings.

The three main paradigms are caged diffusion, directed diffusion and free diffusion. In previous work, changes in behaviour have been observed after stimulation, depending on the location of the vesicle within the cell. Actin filaments of the cytoskeleton may be the vectors of such changes in behaviour and greatly influence the dynamics of vesicles moving through the cell.

The effect on the dynamics of exocytosis of different secretagogues (as KCl and nicotine), the actin filament disruption, and the effect of relevant drugs can be monitored with these non-invasive techniques and will be used to quantify the change in the microenvironment surrounding neurosecretory vesicles.
Co-Authors :
P.Wen(1), T.Nieminen(2), N.Heckenberg(2), F.Meunier(1) and H.Rubinsztein-Dunlop(2)

1) The Queensland Brain Institute
St Lucia, QLD 4072
Australia

2) Centre for Biophotonics and Laser Science
School of Mathematics and Physics
The University of Queensland
St Lucia, 4072, QLD

 

 

25. Dr. Dalibor Mijaljica

Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Faculty of Medicine, Nursing and Health Sciences
Monash University,Building 13D,Wellington Road,
Clayton Victoria 3800,Australia
E-mail: Dalibor.Mijaljica@med.monash.edu.au

 

Monitoring autophagy of organelles in yeast using fluorescent protein tags

 

Autophagy ('self-eating') is an evolutionary conserved process for the degradation of intracellular components including entire organelles by delivery to the vacuole. The mitochondrion, peroxisome, endoplasmic reticulum and even the nucleus are subject to a form of selective autophagy called organellophagy. Despite considerable intensive research efforts by various groups our mechanistic knowledge of organellophagy remains limited.
We have developed a qualitative and quantitative method for monitoring autophagy of the nucleus (nucleophagy) and mitochondrion (mitophagy) using Rosella, a biosensor comprised of a fast maturing pH-stable red fluorescent protein (DsRed.T3) fused to a pH-sensitive green fluorescent protein variant (pHluorin). Its mode of action relies upon differences in pH between the nucleus or mitochondrion and the vacuole. Rosella targeted to organelles fluoresces both red and green. Under nitrogen starvation, alterations in fluorescence emission allow unambiguous distinction between material delivered to the vacuole (loss of green fluorescence at acidic pH) and that localised to the organelle (retention of both red and green fluorescence). Additionally, during conditions of starvation, Rosella allows morphological changes in the nucleus to be followed in various autophagy-defective yeast mutants that appear to represent trapped intermediates in autophagic process.

 

Co-Author:
Carlos J Rosado, Mark Prescott, Rodney J Devenish

Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Faculty of Medicine, Nursing and Health Sciences
Monash University,Building 13D,
Wellington Road,Clayton Victoria 3800,Australia
E-mail:Carlos.Rosado@med.monash.edu.au
Mark.Prescott@med.monash.edu.au
Rod.Devenish@med.monash.edu.au

 

 

26. Mr. Nitin Mohan

ESS Dept. National Tsing-Hua university Taiwan ,
TIGP-NSTP, Institute of Atomic and Molecular sciences,
Academia Sinica,
email : nitinramo@gmail.com

 

Single Particle Optical Characterization of Nanodiamonds and In vivo Bio-Imaging in C.elegans

 

A current trend in fluorescent probe technology is to expand the role of fluorophores that emit light in the far red and near infrared region. The negatively charged nitrogen-vacancy defect center, (N-V)–, in type Ib diamond is one of such fluorophores and has drawn much attention in recent years. The center exhibits some distinct fluorescence features such as extended red emission at ~700 nm and is highly photostable, i.e. no photoblinking and photobleaching. These properties, along with the diverse surface functionalizability and non-toxic nature of the nanomaterial, have made fluorescent nanodiamond (FND) a promising candidate among other conventional markers, i.e. organic dyes, fluorescent proteins, and quantum dots, for biological applications. Despite these excellent photophysical and biochemical features, the size of FND is also an important parameter to characterize. Decreasing its size, clearly, will increase the mobility of the fluorescent probe inside a cell! , minimize the alteration of the properties of targeted molecules, and enhance its translocation into cell nuclei. However, the size-controlled production of FNDs is not so well developed as that of other probes. Here we report procedures of production and isolation of 10-nm FND particles utilizing ion irradiation and differential centrifugation methods.

Particle size analysis of the isolated 10-nm FNDs was performed by Dynamic Light Scattering. Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM) measurements were done to further confirm the size distributions at single particle level. Optical characterization of single 10-nm FNDs by confocal microscopy revealed the photostability of (N-V)- within the diamond lattice. The defect centers were quantified to be 3 per crystal on an average by photon correlation technique. Dynamics of the single 10-nm FNDs in solution was studied by Fluorescence correlation spectroscopy (FCS), where the particle size and fluorescence intensity could be measured simultaneously. Also we compared the normalized autocorrelation functions of the intensity fluctuations of DsRed-Monomer (a 4-nm monomeric red fluorescent protein) and 10-nm FND particles excited under the same experimental conditions (λex = 532 nm and Iex = 80 kW/cm2), and the average fluorescence intensity of single FND is about ~8-fold higher than that of a single DsRed-Monomer. And finally we demonstrate that brighter and smaller FNDs could be used as FRET donors with suitable acceptors.

Moving on, we explore the possibilities of using FNDs for long term bio-imaging in vivo. The extended far red fluorescence along with long term photo stability and non cytotoxic in nature makes it the ideal candidate for imaging the long term biochemical processes in vivo. We choose Caenorhabditis-elegans as our in vivo system because of its size amenable to optical microscopy, short life cycle, rapid growth, apparent simplicity and well defined behaviors. Fluorescent nanodiamonds are incorporated into wild type C elegans by feeding them directly with FND solution. In our primary observations with bright field and epifluorescence microscopy, the FND particles remain with in the lumen of the C- elegans and could image the whole digestive system of the organism for several days. With proper surface fictionalization with BSA protein and dextran we find FNDs absorbed into the intestinal cells from lumen by endocytosis. The second choice of incorporating FND particles into the organism is by microinjection into the syncytial gonads of gravid hermaphrodites according to the standard protocol. Later on due to the cytoplasmic streaming in C elegan's gonad FND would get embedded in the embryos. And eventually FND is dispersed within the newly hatched worms. We also study the toxicity and oxidative stress induced by FND and figure out the stress response pathway in C elegans.

Co-Author:
Mr. Y.-K. Tzeng, Mr. Chao-Sheng Chen Ms. L. Yang, Mr. Y.-Y. Chen, Dr. Y. Y. Hui, Mr. C.-Y. Fang and Prof. H.-C. Chang,
Institute of Atomic and Molecular Sciences,
Academia Sinica ,Taipei 106 (Taiwan)
Email : hcchang@po.iams.sinica.edu.tw)

 

 

 

27. DylanM Owen

Centre for Vasular Research,
University of New South Wales,
Sydney, Australia
E-mail:dylan.owen@unsw.edu.au

 

PALM Microscopy and Cluster Analysis of Raft and Non-Raft Proteins at the Cell Surface

 

Photo-activatable localization microscopy (PALM) is a single-molecule imaging technique to generate super-resolution images, typically of thin optical-sections acquired by total-internal reflection (TIRF) (evanescent wave) laser excitation. Proteins of interest tagged with a photo-switchable or photo-activatable fluorescent protein were expressed in human cell lines (HeLa). By activating less than one molecule per diffraction-limited volume and imaging until it is bleached, the point-spread-function (PSF) centroid can be calculated with great precision (<10 nm). By repeating this step many times, a super-resolution image is reconstructed. These images are then subjected to cluster analysis using Ripley’s K-function and Getis and Franklin’s L-function techniques. These determine statistically whether a given molecular distribution is clustered, relative to the completely-spatially-random (CSR) case. From this, parameters such as the cluster size, distribution, density and shape can be determined. The method is also extendable to 2-channel PALM data and co-clustering analysis.
Membrane lipid microdomains (lipid rafts) present in the plasma membrane of mammalian cells are cholesterol and sphingolipid-enriched areas of high lipid order. They are thought to play an important role in the clustering and segregation of various signaling proteins based on the molecules’ affinity for ordered or disordered membranes, determined at least in part, by the membrane proteins’ lipid-anchor type. Two candidates of raft-mediated segregation are the T-cell –receptor early signaling pathway kinase Lck (raft-associated) and G-protein coupled receptor-associated kinase Src (non-raft-associated).
Here, we perform PALM microscopy and statistical cluster analysis of photo-activatable fluorescent fusion constructs of the acylated tails (membrane-targeting sequence) of Lck and Src in HeLa cells, including 2-channel co-clustering analysis. Cells are also treated with cyclodextrin (which destroys rafts by sequestering cholesterol from the plasma membrane) and Latrunculin B (which disrupts the actin cytoskeleton). Finally the cells are treated with Cholera toxin subunit B which cross-links lipid microdomains by binding to the putative raft-constituent – the ganglioside GM1. We will present these data and discuss the suitability of PALM imaging for the identification of membrane domains.
Co-Author:
J. Rossy, D. Williamson, C. Rentero, K. Gaus.
Centre for Vasular Research,
University of New South Wales,
Sydney, Australia

 

 

 

28. Sandeep Menon Perinchery

Department of Physics and Engineering;
Macquarie University, Sydney 2109 NSW, Australia
E-mail: sandeep@science.mq.edu.au

 

Insights into autofluorescence of human urine

 

Human urine is a complex medium, containing a number of natural fluorophores, most of which are tryptophan metabolites. Due to these fluorophores, normal urine has strong fluorescence. However, despite of the biological variability the spectral characteristics of undiluted human urine show relatively low autofluorescence at short UV (250-300 nm) excitation. We have established key factors contributing to this effect, by studying key fluorophores and other major components in the urine. We found that the leading effects reducing the UV fluorescence include quenching of fluorophores by ammonium, high concentration of indoxyl sulfate producing the inner filter effect and concentration quenching.

In our study, we have also investigated the difference in the autofluorescence spectrum of healthy human urine samples from individuals with bacteriuria. The findings indicate the potential of autofluorescence spectrum of human urine to be developed as a simple and rapid diagnostic tool.
Co-Author:
Unnikrishnan Kuzhiumparambil(2), Subramanyam Vemulpad(3) and Ewa M Goldys (1,4)

(1)Department of Physics and Engineering;
Macquarie University, Sydney 2109 NSW, Australia

(2)Depatment of Chemistry and Biomolecular Sciences, Macquarie University, Sydney 2109 NSW, Australia

(3)Faulty of Science, Macquarie University, Sydney 2109 NSW, Australia

(4)Coordinator, ARC/NHMRC Network "Fluorescence Applications in Biotechnology and Life Sciences", Macquarie University, Sydney 2109 NSW, Australia

 

 

 

29. Varun K A S

Physics Department,
Macquarie University, Sydney,
NSW 2109, Australia
E-mail: varun@physics.mq.edu.au

 

An investigation into the nonspecific binding of commercially available quantum dots

Quantum dots are fluorescent nanoparticles that have wide spread applications in biological imaging and tracking[1]. Several chemistries for conjugating biologically active molecules, for example peptides or antibodies, to these quantum dots are currently known, and have certain advantages and disadvantages. A major drawback with such conjugated nanoparticles is the non specific binding of their unconjugated counterparts, giving rise to reduced statistical discrimination between internalisation pathways.
We find that quantum dots with carboxyl functional groups on the surface adhere to the cell membranes, and are eventually taken up by the cells, whereas amino quantum dots with a PEG spacer provide us with a good control with almost zero nonspecific binding. Studies have shown that PEG has a major role in reducing non specific binding of such nanoparticles[2]. One of the widely used quantum dot bio-functionalisation strategy is the combination of streptavidin coated quantum dots and biotinylated peptides/antibodies, which relies on the high affinity of streptavidin for biotin [3-5]. Interestingly we find that streptavidin coated quantum dots are taken up by the cells non-specifically in a time scale of 30 min when introduced in nanomolar extracellular concentrations, reducing the ability to distinguish specific from nonspecific intake. We demonstrate correlations between surface chemistry and the uptake of such particles by the cells in vitro to provide improved biological imaging and understanding of pathway mediated trafficking.

 

References
1.Zhou M, Ghosh I. Quantum dots and peptides: A bright future together. Biopolymers. 2007;88(3).
2.Chang E, Yu WW, Colvin VL, Drezek R. Quantifying the Influence of Surface Coatings on Quantum Dot Uptake in Cells. Journal of Biomedical Nanotechnology. 2005;1:397-401.
3.Chen B, Liu Q, Zhang Y, Xu L, Fang X. Transmembrane Delivery of the Cell-Penetrating Peptide Conjugated Semiconductor Quantum Dots. Langmuir. 2008;24(20):11866-71.
4.Biju V, Itoh T, Anas A, Sujith A, Ishikawa M. Semiconductor quantum dots and metal nanoparticles: syntheses, optical properties, and biological applications. Anal Bioanal Chem. 2008;391(7):2469-95.
5.Wu XY, Liu HJ, Liu JQ, Haley KN, Treadway JA, Larson JP, et al. Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots. Nat Biotechnol. 2003 Jan;21(1):41-6.

Co-Author Details:
Jinjun Sun(1), Eun Ju Kim(3), Tim Kelf(1), Vikram Jagannath Tallapragada(2), David Inglis(1), Krystyna Drozdowicz-Tomsia(1), Ewa Goldys(1), Ann Goodchild(2), Andrei Zvyagin(1)

1) Physics Department,
Macquarie University, Sydney,
NSW 2109, Australia

2) The Australian School of Advanced Medicine,
Level 1, Dow Corning Building, 3 Innovation Road, Macquarie University,
NSW 2109, Australia

3)Daegu University, College of Education,
Department of Science Education-Chemical Education Major,
Jillyang, Gyeongsan, Gyeonbuk 712-714, S. Korea
E-mail: azvyagin@ics.mq.edu.au

 

 

30. Dr. Sivananthan Sarasanandarajah

Sivananthan Sarasanandarajah
Medical Scientist, Department of Medical Physics,
Royal Adelaide Hospital, SA 5000, Australia
Formerly PhD student, Department of Physics & Astronomy,
University of Canterbury, Christchurch, New Zealand.
E-mail: siva.sarasanandarajah@health.sa.gov.au

 

Identifying Components (fluorophores) of the Ultra violet-Visible Fluorescence of Bacillus globigii

 

Fluorescence is being considered as a technique for the rapid detection and identification of pathogenic spores used in bioterrorism. The potential to extract large amounts of resonance information from multiwavelength measurements makes UV-Vis fluorescence spectroscopy a powerful and sensitive characterization tool. Dipicolinic acid (DPA), calcium dipicolinate (CaDPA) and tryptophan are major components of bacterial spores. Bacillus Globigii (BG) spores were suspended in deionized water in a quartz cuvet. The absorbance and the fluorescence from 200 to 600 nm. DPA and CaDPA were prepared separately with deionized water. Tryptophan is added to the mixture of these compounds and the absorbance and the fluorescence spectra were measured with these compounds aqueous and dried before and after exposure to UV light. Finally, this simulant mixture and the BG suspension were exposed to UV light and the absorbance and fluorescence were re-measured. All these measurements were done at room temperature. We identify DPA, CaDPA as probable major components of BG spore fluorescence along with tryptophan. We show that these components in a nearly dry state, with CaDPA as the major component, are consistent with fluorescence measurements of BG. Changes in the fluorescence of BG spores after exposure to UV light is consistent with changes in the DPA, CaDPA and Tryptophan fluorescence upon UV exposure.

Acknowledgements: Lou Reinisch,PhD ; Easaw Chacko,PhD; Joseph Kunnil,PhD; Burt Bronk,PhD

 

 

31. Galiya Sharafutdinova

School of Mathematical and Physical Sciences, University of Newcastle, University Drive, Callaghan, NSW, 2308,
E-mail:galiya.sharafutdinova@studentmail.newcastle.edu.au

 

A New Scanner Design for Two-Photon Fluorescence Fast Imaging Microscopy

 

Signal quality in scanned laser induced two-photon fluorescence microscopy depends of the spatial and temporal characteristics of the excitation femtosecond pulsed laser beam at the microscope objective focus. It is well known that group-velocity dispersion negatively impacts the excitation pulse temporal characteristics, however, the scan engine contribution has been overlooked, though it contributes both spatial and temporal distortion to the excitation laser pulses and the sample fluorescence signal is reduced non-uniformly across the scan field as a result. A new scanner design with two flat scanning mirrors and two off-set parabolic reflectors between them as an afocal relay provides ideal scan engine performance for a fast imaging two-photon system. Using OSLO optical modelling software, the advantages of the new system in comparison with three known scanners at the intermediate image plane are shown in terms of minimised spot area, wavefront error, pulse time broadening and a fluorescence intensity reduction factor is introduced. These results suggest that the application of the novel scanner in two-photon imaging microscope will increase the two-photon excited signal and improve image contrast and signal-to-noise ratio.

Co-Author Details:
John Holdsworth(1)*, and Dirk van Helden(2)
1. School of Mathematical and Physical Sciences, University of Newcastle, University Drive, Callaghan, NSW, 2308,
2. School of Biomedical Sciences, University of Newcastle, University Drive, Callaghan, NSW, 2308, Australia
E-mail: john.holdsworth@newcastle.edu.au*

 

 

32. Raz Shimoni

(a) Centre of Micro-Photonics
Swinburne University of Technology
Melbourne, Australia

(b) Peter MacCallum Cancer Centre
St Andrews Place, East Melbourne, Australia
E-mail: raz.shimoni@petermac.org

 

Identification of an optimal RICS setup by a novel automated RICS software

 

Time-lapse fluorescence imaging has revolutionized studies of biology in the last 15 years. In addition to the now routine tracking of bulk fluorescence, for instance of a protein moving into the nucleus in response to an extracellular signal, technologies are now emerging that enable much more sophisticated analysis of the motion and interactions of proteins within living cells.

Introduced by E. Gratton and M. Digman, Raster Image Correlation Spectroscopy (RICS) is a novel approach to assess diffusion coefficient of fluorescent macromolecules by using confocal laser scanning microscopy [1]. Since protein-protein interactions and interactions of proteins with various cellular components can alter the diffusion coefficient of proteins in living cells, measuring the rates of diffusion in living cells can provide indications of biological activity and might be an important key to elucidate biological processes.

The principle of RICS is that the autocorrelation function of a specific region of interest (ROI) statistically characterizes the frequency of the fluctuations in the emission intensity that is collected from the confocal volume while the laser beam raster scan the sample. Since the frequency of the fluctuations is related to the diffusion coefficient, fitting the corresponding experimental autocorrelation into a physical model can give the average diffusion coefficient for the ROI.

In order to utilize RICS, specific parameters during the image acquisition and post-acquisition analysis are required [2]. Hence, our aim was to identify the optimal RICS setup and to establish a RICS routine to measure diffusion coefficients of proteins in living cells. In order to achieve this goal we developed a novel automated software based on RICS theory with which to analyze confocal images and to measure diffusion coefficients of the fluorophores.

In this poster, we show how the experimental setup was characterized under a range of settings. In addition, we show the identification of new effects in RICS that have not been reported in any published RICS literature. The optimal framework for accurate measurements was successfully achieved by using free diffusing fluorescence proteins as control. A precise comparison between the diffusion coefficients of the control to the protein of interest was used for calibration, and smoothed diffusion maps were generated, demonstrating the potential of RICS.

References:
1. M. A. Digman, P. Sengupta, P. W. Wiseman, C. M. Brown, A. R. Horwitz, E. Gratton, "Fluctuation Correlation Spectroscopy With A Laser-Scanning Microscope: Exploiting The Hidden Time Structure" Biophysical Journal, v. 88, p. 33-36, 2005.
2. C. M. Brown, R. B. Dalal, B. Hebert, M. A. Digman, A. R. Horwitz, E. Gratton, "Raster Image Correlation Spectroscopy (RICS) For Measuring Fast Protein Dynamics And Concentrations With A Commercial Laser Scanning Confocal Microscope" Biophysical Journal, v. 229, p. 78-91, 2008.

Co-Author:
Z. Bomzon (c), M. Gu (a), M. Ludford-Menting (b), S. Ellis (b), S. Russell (a,b)

(a) Centre of Micro-Photonics
Swinburne University of Technology
Melbourne, Australia

(b) Peter MacCallum Cancer Centre
St Andrews Place, East Melbourne, Australia

(c) Technion, Israel Institute of Technology
Haifa, Israel
E-mail: sarah.russell@petermac.org

 

 

33. Mushtaq A. Sobhan

Department of Physics and Engineering,
Macquarie University, North Ryde 2109 NSW,
Australia
E-mail : msobhan@science.mq.edu.au

 

Visible fluorescence from gold nanoparticles

 

In this work we investigate the fluorescence of commercially available gold nanoparticles of various sizes ranging from 5 to 50 nm. The nanoparticles show visible fluorescence (400-445 nm) with excitation in the UV wavelength range (295-340 nm). We observe a blue shift in the both emission and excitation spectrum with varying nanoparticle size. Nanoparticle in the size range from 5 to 15 nm showed two peaks in the emission spectrum, while size>15nm had single peak emission. However the emission peak widths stayed constant with varying sizes. The effect of stabiliser citrate on the character of the spectrum was examined. We further investigated the effect of surface charge on these nanoparticles by measuring the zeta potential. To compare these results, we also measured the fluorescence of femtosecond laser ablated gold nanoparticles produced in pure deionised water. Our results in pure water show that the emission peaks of the nanoparticles are observed at ~410 nm when excited with 300 nm wavelength.

Co-Author
Ewa M. Goldys
Department of Physics and Engineering,
Macquarie University, North Ryde 2109 NSW,
Australia
E-mail : goldys@science.mq.edu.au

 

 

 

34. Zhen Song

Department of Engineering and Physics,
Macquarie University, North Ryde 2109 NSW,
Australia
Email: song@ics.mq.edu.au

 

Evaluation of Skin Permeability of ZnO Nanoparticles by Two-Photon Excitation Microscopy

Cosmetic formulations include nano-scale ingredients, such as TiO₂ or ZnO nanoparticles (NPs) as efficient UV light absorber or filter. Concerns have been raised that there might be potential hazard of topically applied NPs penetrate through stratum corneum into deeper layers of human skin and diffuse further into the circulatory system. Once this occur, it would be impossible to be clear out during the routine metabolic process of human being. ZnO sized 30nm or larger have been claimed to stay mainly in the superfacial layer of human skin or hair follicle. In our investigation, ZnO NPs of a broader size range, of 3nm – 100nm and surface coatings of various hydrophobic and surface charge properties, were applied to human and porcine skins, and their permeability was studied by means of fluorescence scanning confocal and nonlinear optical imaging. In order to quantify ZnO signal on the autofluorescence background of skin and second harmonic signal of collagen, the linear and nonlinear optical properties of ZnO NP were characterized. A blue shift of absorption band due to the quantum confinement effect of the ZnO NP was observed. The linear and nonlinear absorption cross-sections and quantum yield of ZnO NPs were determined by its quantitative comparison with the tabulated parameters of the organic laser dyes.

Co-Author
Timothy A Kelf(a), Alexey Popov(b), Adrei Zvyagin(a)
a) Department of Engineering and Physics,
Macquarie University, North Ryde 2109 NSW,
Australia

b)Optoelectronics and Measurement Techniques Laboratory,
University of Oulu, P.O. Box 4500, 90014 Oulu,
Finland

 

 

35. Lilian L Soon

Australian Key Centre for Microscopy and Microanalysis,
Australian Microscopy and Microanalysis Research Facility (AMMRF),
University of Sydney, Sydney, NSW, 2006, Australia.
E-mail: l.soon@usyd.edu.au

 

Photoconversion and imaging the dynamics of the cellular translocation of ROM

 

Reduced On-random Motile (ROM) is a scaffold protein, containing two adjacent protein-protein interacting domains: a PDZ (postsynaptic density protein-95/discs-large /ZO-1) domain and a LEU (leucine-rich) domain and a PDZ domain binding region at the C-terminus. In this investigation, we report GFP-ROM occurring in vesicles and at the plasma membrane (PM) of H1299 lung cancer cells. Transfection of truncation mutants consisting either the PDZ or LEU domain showed that the LEU domain of ROM was localised to both PM and a fraction of ROM-labelled vesicles, whereas, the PDZ domain localised to the PM and the nucleus but not to vesicles. The data suggested that PM and vesicular localisation of ROM were differentially regulated by the LEU and PDZ domains. There was therefore, spatial and molecular segregation of ROM function in the two compartments. ROM was subcloned into a plasmid carrying a photoconvertible protein (Phamret). The photoconversion experiments verified the diffusion of ROM from the cytoplasm to the PM at membrane ruffling sites and conversely from the PM to the cytoplasm. The transport of ROM to the PM occurred at a speed of 338.2±25.7 μm2 /sec and its localization at the PM was stable for 0.11±0.02 sec.

Co- Author:
Lingyan Yang (1), Ching Yu Wooi (1), Tomoki Matsuda (2), Takeharu Nagai (2) and Lilian L Soon (1)

1.Australian Key Centre for Microscopy and Microanalysis,
Australian Microscopy and Microanalysis Research Facility (AMMRF),
University of Sydney, Sydney, NSW, 2006, Australia.

2. Laboratory for Nanosystems Physiology,
Research Institute for Electronic Science,
Hokkaido University Kita 20, Nishi 10 Kita-ku,
Sapporo 001-0020, Japan

 

 

 

36. Wei Deng

Department of Engineering and Physics,
Macquarie University, North Ryde 2109 NSW,
Australia
E-mail : deng@ics.mq.edu.au

 

Enhanced Flow Cytometry Based Bead Immunoassays by Using Metal Nanostructures

 

While the principle of fluorescence enhancement of metal nanostructures is well known, the utility of this effect in practical methodologies used in analytical laboratories remains to be established. In this work we explore the advantage of fluorescence enhancement for flow cytometry. We report the observation of metal-enhanced fluorescence emission of fluorophores located on the surface of silica beads coated with nanostructured silver, suitable for flow cytometry detection. The fluorescence enhancement was investigated using a model AlexaFluor 430 IgG immunoassay and AlexaFluor 430 labeling. Approximately 8.5-fold and 10.1-fold higher fluorescence intensities at 430 nm excitation were, respectively, observed from silvered ~ 400 nm and 5 µm silica beads deposited on glass as compared to the control sample. The 400 nm and 5 µm beads were compatible with flow cytometry readout, although lower enhancement factors of 3.0 and 3.7 were obtained. We show that such values are consistent with less favourable overlap of the plasmon resonance in silver nanostructures with 488 nm excitation wavelength used in the flow cytometry experiment. We thus demonstrated that the silvered silica beads are able to provide intensified fluorescence signals in flow cytometry which can improve the sensitivity of flow cytometry based bio-assay systems.

Co-Author:
Krystyna Drozdowicz-Tomsia, Dayong Jin and Ewa M Goldys
Department of Engineering and Physics,
Macquarie University, North Ryde 2109 NSW,
Australia
E-mail : goldys@ics.mq.edu.au

 

37. Yingying Su

The Electron Microscope Unit, Australian Key Centre for Microscopy & Microanalysis (AKCMM), The University of Sydney, NSW 2006, Australia
Email: y.y.su@usyd.edu.au

 

Introducing Live Cell Imaging into Events of Colorectal Cancer Cells with Actin-Binding Reagents

 

Previous research proves that actin cytoskeleton plays a crucial role in maintaining cell shapes and mediating cell motility. Also, malignant cells migrate into surrounding connective tissue require actin dynamics-polymerization and depolymerization of actin filament [1, 2]. Interestingly, alterations in membrane composition and actin organization have been linked to programmed cell death pathways. We found in a preliminary study, by using actin-binding reagents, a relationship between actin-mediated fine structural changes and an increase in the number of apoptotic colorectal cancer (CRC) cells [3]. Our recent study on detergent resistant domains (DRMs) shows that DRMs play an important role in transmembrane signaling and interacting with actin-binding drugs. We hypothesize that this specialized membrane domains connected to the actin cytoskeleton are playing an important role in anti-cancer drug uptake and / or anti-cancer drug resistance [4].
Our aim is via treatment of cancer cells with different anti-actin drugs, to study the connection between actin cytoskeleton and DRMs by combining live cell imaging and electron microscopy. By investigating the actin cytoskeleton roles in cell response to anti-actin drug, we could discover possible low-toxicity-high-efficiency anti-cancer drugs in the future.
In this report we will show about our preliminary results we obtained on CRC cells treated with actin-binding drugs by using the combined live cell and electron microscopy imaging approach. Briefly, we found that actin-binding drugs induces significant changes in membrane domain distribution and actin organization at different concentrations over time, leading to further programmed cell death.

References:
1.J. Rao, and N. Li, Curr. Cancer Drug Target, 4, (2004), pp.345-54.
2.J.S. Condeelis, et al., Semin Cancer Biol, 11(2), (2001), pp. 119-28.
3.F. Braet, et al., Springer New York, (2008), pp.37-49.
4.K. Jahn and F. Braet, Micron, 39, (2008), pp.1393-1397.

Co-Author Detail:
Kristina Jahn, Ya-Na Wu, Renee Whan, Lilian Soon, Filip Braet
The Electron Microscope Unit, Australian Key Centre for Microscopy & Microanalysis (AKCMM), The University of Sydney, NSW 2006, Australia

 

 

38. Jinjun Sun

Physics Department,
Macquarie University, Sydney,
NSW 2109, Australia
E-mail: jsun@science.mq.edu.au

 

Quantum Dot assisted somatostatin tracking in rat pancreases and pituitary tumor cells

 

Somatostatin (SST), as a peptide also an agonist produced by neuroendocrine neurons of the periventricular nucleus of the hypothalamus, gets involved in the endocrine system and affects neurotransmission as well as cell proliferation by interacting with G-protein-coupled somatostatin receptors and inhibiting the release of numerous secondary hormones. Single molecule tracking of SST is important for the studies of diagnostic and treatment methodology, such as drug delivery, which can revolutionise the clinical administration of patients with neuroendocrine tumors.
In order to realize SST tracking, quantum dot (QD), a fluorescent anti-photobleaching semiconductor nanoparticle, is linked to SST to be internalized via agonist-receptor complex into cells which express SST receptors. With this SST-QD complex, we can track the SST endocytosis in the rat tumor cells, AR42J, GH4C1 and AtT20, under laser scanning confocal microscope. The combination of nanoparticles bioconjugation and confocal imaging, provide us the promising methodology to examine the details of some neuropeptide, similar to SST, trafficking into the cells, which is good tool to study drug delivery in cells and even small animals. Intriguing information in the rational design of biocompatible drug carriers will be induced through the research on such fluorescent peptide-QD complex which is a superior alternative to magnetic and radioactive imaging contrast agents in preclinical drug screening, validation and delivery research (Smith, Duan et al. 2008).

Reference:
Smith, A. M., H. Duan, et al. (2008). "Bioconjugated quantum dots for in vivo molecular and cellular imaging." Advanced Drug Delivery Reviews 60(11): 1226-1240.

Co- Author:
Varun K A S(1), Eun Ju Kim(3), Tim Kelf(1), Vikram Jagannath Tallapragada(2), Ann Goodchild(2), David Inglis(1), Krystyna Drozdowicz-Tomsia(1), Ewa Goldys(1), Andrei Zvyagin(1)
1) Physics Department,
Macquarie University, Sydney,
NSW 2109, Australia

2) The Australian School of Advanced Medicine,
Level 1, Dow Corning Building, 3 Innovation Road, Macquarie University,
NSW 2109, Australia

3)Daegu University, College of Education,
Department of Science Education-Chemical Education Major,
Jillyang, Gyeongsan, Gyeonbuk 712-714, S. Korea
E-mail: azvyagin@ics.mq.edu.au

 

 

 

39. A-Young Sung

Department of Optometry and vision Sciences,
Daebul University,
526-702, Jeonnam
South Korea
E- mail:angel123sg@yahoo.com

 

Preparation and photo-optical/imaging analysis of UV-blocking contact lens material containing TiO2 nanoparticles

TiO2 nanoparticles are commonly used as a UV-absorbing or UV-blocking agent. Light is essential for the human brain to identify objects, parts of the eye such as the cornea, retina and lens can be damaged when exposed directly to an excessive amount of UV rays which may also cause various eye diseases such as cataract and macular degeneration [1, 2].
The mixed reagents such as MMA(methylmethaceylate), NVP(N-vinylpyrrolidone), HEMA(2-hydroxyethylmethacrylate), TiO2 nanoparticles, and 0.1wt% AIBN (2,2'-azobisisobutyronitrile; a radical initiator) were placed in a cast mould and heated for 40 min at the 70~100 ℃ temperature range for preparing the contact lens shape of polymeric thin material. The prepared polymeric lens material was then subject to the photo-optical/imaging analysis using various hightech instruments including FE-SEM, HR-TEM, and UV-vis spectrophotometer. Refractive index, optical transmittance, water content, and advanced electron microscopic images were obtained from the analysis.
The polymeric lens material with dispersed TiO2 nanoparticles has a smooth surface, confirmed by FE-SEM and HR-TEM. The polymeric material showed the transmittance of 6% and 51%, respectively, for UV-A and UV-B, leading to a UV-blocking effect. The visible light transmittance of the material was in the 77~89% range, satisfying the requirement for medical lens materials. The refractive indices, ndry and nwet, were found to be 1.507 and 1.432, respectively. The water content was calculated to be 43.09%.

Reference:
1. Gies, P. H., C. R. Roy, S. Toomey, and A. McLennan, MutationResearch 1998, 442(1), 15.
2. Takai, D., S. H. Park, Y. Takada, S. Ichinose, M. Kitagawa, and M. Akashi, Free Radic. Res. 2006, 40(11), 1138.

Co-Authors:
Tae-Hun Kim(1), Ki-Hun Ye(1), Hyun-Soo Choi(1), Hee-Gweon Woo(2)

1. Department of Optometry and vision Sciences,
Daebul University, Jeonnam, 526-702, South Korea
2. Deptartment of Chemistry, Chonnam National University, Gwangju,
500-757, South Korea

 

 

 

 

40. A-Young Sung

Department of Optometry and vision Sciences,
Daebul University,
526-702, Jeonnam
South Korea
E- mail:angel123sg@yahoo.com

Preparation and photo-optical/imaging analysis of antibacterial contact lens material containing silver nanoparticles

Silver nanoparticles show antimicrobacterial activity [1-3]. Recently, silver nanoparticles have been applied to various fields due to antibiosis and high conductivity.
The mixture of chemical reagents such as NVP(N-vinylpyrrolidone), MMA(methylmethacrylate), BMA(buthylmethacrylate), MA(methacrylic acid), AA(acryl amide), Silicone to make conventional contact lens polymeric materials were mixed with AgNO3. The final mixture was then copolymerized at 70 oC for 40 min using a temperature-controlled water bath. The analysis for obtaining antimicrobial activity, optical properties, and electron microscopic images were performed.
Both solid culture and liquid culture medium were used for the antimicrobial test. The contact lens polymeric materials containing silver exhibit good antibiosis. The size of silver nanoparticles found to be 10~20 nm by FE-SEM and HR-TEM. The water content and visible light transmittance were measured to be 32.35% and 88.34%, respectively. The refractive indices, ndry and nwet, were found to be 1.517 and 1.453, respectively. It is found that the copolymer with antimicrobial and photo-optical properties is suitable for high functional ophthalmological medical lens.

References:
1. Lansdown, A. B. Curr Probl Dermatol. 2006, 33, 17.
2. Kim, T. N.; Feng, Q. L.; Kim, J. O.; Wu, J.; Wang, H.; Chen, G. C.; Cui, F. Z.; J. Mater. sci. Mater. Med. 1998, 9, 129.
3. Gang, H. Y.; Jung, M. J.; Jung, Y. K. The Korean Society for Biotechnology and Bioengineering 2000, 15(5), 521.

Co-Authors:
Ki-Hun Ye(1), Tae-Hun Kim(1), Hee-Gweon Woo(2)
1. Department of Optometry and vision Sciences,
Daebul University, Jeonnam, 526-702, South Korea
2. Department of Chemistry, Chonnam National University, Gwangju,
500-757, South Korea

 

 

 

41. Dr. Pall Thordarson

School of Chemistry, The University of New South Wales,
Sydney, NSW 2052, Australia.
E-mail: p.thordarson@unsw.edu.au

 

Long-lived red emission from NIR multiphoton excited lanthanide-doped self-assembled gels

 

The unique luminescent properties of trivalent lanthanide coordination complexes make them attractive candidates as new molecular probes for biological imaging. Europium(III) complexes emit in the visible region and display long luminescent decay times (> tens of microseconds ) and have been applied in cellular imaging, including selective imaging of cancerous tissues. Unfortunately, most lanthanide complexes need excitation with ultraviolet (UV) light (< 300 nm) which is not desirable for biological materials. Multi-photon excitation provides an interesting alternative as two or three photons, with half or a third of the energy of the corresponding one-photon, are used to excite the chromophore. The lower energy of near infrared (NIR) multi-photon excitation greatly reduces cell damage and also penetrates much further into a biological tissue than would be the case if visible or UV excitation was used. Lanthanide complexes, and other chromophores that respond well to NIR-multi-photon excitation, are an interesting target for these applications. The long emission lifetimes of lanthanides make them also a good target material within the emerging area of time-gated spectroscopy and microscopy.] The ability of these time-gated techniques to reduce auto-fluorescence, in combination with NIR-multi-photon excitation of lanthanides, could result in significant advances in biological and medicinal imaging and spectroscopy.
Self-assembled gels are a new class of “smart” materials that are showing a great promise for applications in drug delivery and tissue engineering, in combination with their ability to respond to external stimuli such as pH changes or addition of anions. We show here for the first time that it is possible to “dope” self-assembled gels with lanthanide ions such as Europium(III). Furthermore, these Eu(III) doped show all the classical spectroscopic properties expected for organic lanthanide complexes, indicating that efficient energy transfer is taking place from the gel-matrix to the Eu(III). Finally we show that these materials can be excited using NIR (780 nm) multiphoton laser sources, resulting in the classical long-lived red emission (612 nm) that usually arises from organic Eu(III) complexes.

Co-Author:

Dr. Sabrina Dehn,(1), Katie W. K. Tong(1), Dr. Raphael G. C. R. Clady(1), Dr. Dylan M. Owen(2), A/Prof. Katharina Gaus(2), A/Prof. Filip Braet(3)

1.School of Chemistry, The University of New South Wales,
Sydney, NSW 2052, Australia.
2.Centre for Vascular Research, The University of New South Wales,
Sydney, NSW 2052, Australia.
3.Australian Key Centre for Microscopy and Microanalysis,
The University of Sydney, NSW 2006, Australia.

 

42. Sam Wallace

School of Chemistry and Physics
University of Adelaide
sam.wallace@adelaide.edu.au

Quantification of myosin in cardiomyocytes using multiphoton microscopy

 

The ability to quantify the myosin content of cardiomyocytes will lead to a better understanding of the link between low fetal birth weight and increased risk of heart disease in adult life(1). The use of nonlinear optical processes such as second harmonic generation (SHG) and two photon excitation fluorescence (TPEF) provide high contrast and molecular specificity which are crucial for quantitative optical microscopy. The SHG signal can provide images of the myosin filaments in the cardiomyocyte(2), whilst the two photon excitation fluorescence signal is used to determine the total cell area(3, 4). We determine the dependence of measured myosin area on the signal to noise ratio (SNR) of the image to find the real myosin content. We find the average myosin content does not change between mononucleated and binucleated cardiomyocytes. Cardiomyocytes from the left ventricle have a lower average myosin content of 70.8% compared to the right ventricle 76.4% (p-value < 0.08). We also find that there appears to be no difference in myosin content between cells from healthy fetuses compared with cells from fetuses that have undergone intrauterine insults.

 

References:
1.J. G. Eriksson, International Journal of Obesity 30, S18 (Dec, 2006).
2.S. V. Plotnikov, A. C. Millard, P. J. Campagnola, W. A. Mohler, Biophys. J. 90, 693 (January 15, 2006, 2006).
3.J. H. Burrell et al., Anatomical Record Part a-Discoveries in Molecular Cellular and Evolutionary Biology 274A, 952 (Oct, 2003).
4.S. J. Wallace, J. L. Morrison, K. J. Botting, T. W. Kee, Journal of Biomedical Optics 13, (Nov-Dec, 2008).

Co-Author:
Janna Morrison(2),Kimberely Botting(2),Tak Kee(1)

1. School of Chemistry & Physics,
University of Adelaide, South Australia.

2. Sansom Institute, Early Origins of Adult Health Research Group,
University of South Australia, South Australia

 

43. Kim M. Hajek

Centre for Biophotonics and Laser Science,
School of Mathematics and Physics,
The University of Queensland, QLD 4072, Australia
E-mail: kmhajek@physics.uq.edu.au

 

superCARS: Proposed widefield super-resolved coherent anti-Stokes Raman scattering (CARS) microscope

 

Over the last ten years, coherent anti-Stokes Raman scattering (CARS) microscopy has grown in popularity as an imaging modality for the life sciences. Its principal appeal is that specific biological components can be imaged without the need for fluorescent labelling. Specifically, CARS microscopy generates intrinsic contrast from the target species through a four-wave-mixing interaction. This advantage comes at the price of meeting energy and phase-matching conditions, of which the latter is the more onerous. Looking at the CARS process in more detail, an anti-Stokes signal beam ω_AS, k_AS) results from the nonlinear interaction of two pump beams (ω_P, k_P) and one Stokes beam (ω_S, k_S) with a molecular vibrational transition (Ω), such that, with . . In addition to this energy condition, the beams must satisfy the phase-matching condition,where and l is the interacti , with on length. For the most part, phase matching is ensured by tight focusing of collinear input beams into the sample, yielding a very short interaction length; signal is generated from one point only, which must be scanned in order to build up an image of the sample. An alternative phase-matching geometry has, however, been implemented by Heinrich et al. [1], to generate a CARS signal across a wide field, obviating the need for scanning. Here, is minimised by delivering the Stokes beam to the sample through the (imaging) objective, while the pump beams are incident at high angle (around 76°) from the other side.
We propose adapting this configuration to realise a super-resolved CARS (superCARS) microscope, with a factor of two-to-three in resolution enhancement over standard widefield and collinear CARS configurations. Such a microscope would offer the intrinsic species-specific contrast generation of CARS, at a better resolution than commonly available in biological imaging (i.e. confocal microscopy). While certain fluorescence imaging techniques do offer even higher resolution, the appeal of our system lies in its combination of desirable features: high resolution, intrinsic contrast of target species, and widefield imaging. In this poster, we will set out our proposed scheme for adding super-resolution to widefield CARS microscopy. We will further simulate the implementation of this scheme and thus the expected resolution improvement offered by a superCARS microscope, and finally, discuss a means of realising this scheme experimentally.

 

Reference:
1. Heinrich, C., S. Bernet, and M. Ritsch-Marte, "Wide-field coherent anti-Stokes Raman scattering microscopy." Applied Physics Letters, 2004. 84(5): p. 816-18.

Co-Author:
Brad Littleton, Timothy J. McIntyre, Halina Rubinsztein-Dunlop
Centre for Biophotonics and Laser Science,
School of Mathematics and Physics,
The University of Queensland, QLD 4072, Australia

 

 

 

 

 

44. Cynthia B Whitchurch

Microbial Imaging Facility,
Institute for the Biotechnology of Infectious Disease,
University of Technology, Sydney
E-mail: Cynthia.Whitchurch@uts.edu.au

 

Super-resolution imaging of microorganisms using 3D Structured Illumination Microscopy

 

n conventional microscope systems, image resolution is limited by the angle of light that can successfully traverse the light path and enter the objective lens. While objectives can be built with very high numerical apertures, a limit is reached when light can no longer cross the interfaces between different refractive indices. At this point, Brewster’s angle is achieved and additional light and information (resolution) are not able to be captured by the objective lens. This angle ultimately limits the resolution of conventional microscope systems to at best 250 nm. Because of this limitation, optical microscopy techniques have not proven to be a useful tool for examining the cell biology of microorganisms due to their small size.

“Super-Resolution” is the term used to define an imaging system’s capacity to exceed the resolution limit imposed by physics and the wavelength of light and provide resolutions that are significantly better than the diffraction limit. In recent years, several super-resolution imaging methods have been developed. These methods allow precise visualisation and measurement of features that are not able to be resolved with conventional microscopy techniques.

The Applied Precision DeltaVision OMX imaging system uses 3D-structured illumination microscopy (3D-SIM) technology. The DeltaVision OMX system surpasses the 250nm resolution barrier by a factor of 2-3 in X, Y and Z dimensions. This technology will resolve features previously invisible to conventional microscopy. The DeltaVision OMX works with traditional fluorochromes eliminating the need to genetically engineer novel or difficult photoswitchable probes as is required for other super-resolution technologies and can image up to 30 µm into the sample depending on the wavelength used. The DeltaVision OMX is also engineered to deliver superior live-cell imaging performance through true simultaneous capture of up to 4 channels at high frame rates. The Microbial Imaging Facility in the University of Technology, Sydney has recently installed the second commercially released DeltaVision OMX 3D-SIM instrument in the world, and the only 3D-SIM OMX super-resolution microscope in the Southern Hemisphere. We are particularly interested in applying this exciting technology to examine the cell biology of microorganisms, and to study the interactions of microbial pathogens with host cells and tissues.

Co-Author:
Lynne Turnbull
Microbial Imaging Facility,
Institute for the Biotechnology of Infectious Disease,
University of Technology, Sydney