MQ Photonics Research Centre
Biological Applications and Instrumentation Research Laboratory
Team Members
Dr. Russell Connally (team leader) has a background in biology, chemistry and physics (computers, electronics design and software); his undergraduate degree was in Biotechnology and his thesis "Enhanced Detection of Microorganisms in Autofluorescent Environments through the Application of Time-Resolved Fluorescence Microscopy" was accepted by Academic Senate in November 2004. Russell was awarded a prestigious Vice Chancellors Innovation Fellowship in 2009; his patented GALD technology was recently showcased on ABC’s New Inventors program in 2010.
Dr. Nima Sayadi was awarded an MSc in Organic Chemistry from the University of Shiraz (Iran) in 1997 and gained his PhD from the University of Sydney in 2009 following submission of his thesis titled "Pseudo-Prolines: A Versatile New Tool for the Synthesis of Cyclic Peptides". Nima has extensive experience in the synthesis of peptides using solution and solid phase methods; 2D NMR for the conformational study of peptides; LCMS, GC and GCMS and UV VIS, FTIR, AA.
Dr. Shingo Miyauchi was awarded his PhD in 2010, the theme of his dissertation was the development of a fungal protein expression system targeted for biopharmaceutical and industrial applications. Shingo is currently performing the practical lab work on nucleotide extraction, hybridisation and amplification with Qβ replicase in order to develop a simple and fast detection system for bacteria in horticultural environments. He was appointed under a Physics based fellowship to research a project for Horticulture Australia.
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Mr Tom Lawson is supervised by Dr Russell Connally and is in the final year of his PhD candidature, the tentative title of his thesis is “Detection of Staphylococcus aureus using fluorescence in situ hybridization (FISH)”. Tom has investigated a variety of schemes for the molecular detection of this pathogen and the antibiotic resistant form known as Methicillin-resistant Staphylococcus aureus (MRSA). A principle aim of Tom’s work is to use lanthanide labelled nucleic acid probes to permit time-delayed luminescence imaging specific for S. aureus bacteria.
Background - detection of delayed luminescence
Luminescence is the emission of light arising from an electronically excited state; it is formally categorized as either fluorescence or phosphorescence depending on the nature of the excited state. When the electron in the excited state is paired by a ground state electron of opposite spin, photon emission is characterized by a short lifetime (~10 ns) and is termed fluorescence. Conversely, when photon emission arises from relaxation of an electron from the triplet excited state, where both ground state and excited state electrons share the same spin, transitions are forbidden and lifetimes are typically milliseconds to seconds1. Interestingly, lanthanides show delayed fluorescence in which the triplet state is temporarily involved but transition is from the first excited state. Luminescence lifetimes of hundreds of microseconds to milliseconds are observed for europium and terbium trivalent ions. Lanthanide ions have a low absorbance cross-section and luminescence output is enhanced by chelation of the ions with a sensitizer molecule. Excitation of the attached ion occurs through a chain of events; the sensitizer molecule is excited to the singlet state from which it decays via intersystem crossing and other processes to the triplet state whereon energy transfer occurs to the chelated ion via metal ligand bonds. Crucially, triplet state energy levels of the sensitizer must sit sufficiently above the receiving state of the lanthanide ion to ensure that back transfer via phonon interactions cannot easily occur.
Fluorescence microscopy is a valuable technique that can deliver a huge increase in the ability to detect signals of interest compared with bright-field illumination. Conventionally, the object of interest is labelled with a fluorescent dye and observed through the fluorescence microscope using spectral filters to isolate the desired fluorescence signal. In some cases, the target is embedded in a matrix of intrinsically fluorescent components (autofluorophores) that acts to obscure detection due to the broad spread of the autofluorescence. Spectral selection with optical filters is not useful in these circumstances and as a consequence, target detection becomes arduous. Examples of this include the detection of waterborne pathogens Giardia /Cryptosporidium in the particulate mass collected from large volumes of water; analysis of activated sludge and tissue samples that have been treated with formaldehyde2-4.
For detection of rare targets in autofluorescent environments however, time-gated luminescence (TGL) detection greatly surmounts the capability of conventional fluorescence microscopy. More specifically, the detection of Mycobacterium tuberculosis in respiratory samples is a particularly challenging task; the organism is very small and the samples strongly autofluorescent5,6. This application is one that would benefit greatly from a technique that reduces background autofluorescence. Time-gated luminescence (TGL) techniques rely upon the use of luminophores with lifetimes thousands of times longer (~0.1 to 2 ms) than prompt fluorescent dyes (~20 ns). Regimes to exploit TGL for the suppression of background autofluorescence rely upon a brief, intense pulse of light to excite both prompt and delayed luminescence from the sample. Capture of the radiant emission is delayed for a short period (1-100 µs) to permit prompt fluorescence to decay below the detection threshold. Persistent emission from long-lived luminophores is then captured in the absence of autofluorescence by gating the detector after the resolving period; the principle is shown schematically in Fig. 1. Time gated luminescence (TGL) and time-resolved fluorescence (TRF) techniques are similar in many respects, both exploit differences in fluorescence lifetime to detect the object of interest, but they operate in different time regimes.
Whilst no commercial instruments are available, the mechanisms employed to construct TGL microscopes continue to evolve. The conventional means of implementing TGL microscopy in the lab rely upon the conversion of an epifluorescence microscope through the addition of a sensitive gated camera and a pulsed excitation source 7-15. Chopper wheels are often employed to interrupt the beam from a mercury arc lamp to provide pulsed excitation 16. Other instruments have relied upon the use of high intensity ultra-violet light emitting diodes (UV-LEDs) since they offer very rapid transitions between the on and off state17. Lab-built TGL microscopes have been demonstrated to provide excellent suppression of autofluorescence16,18.
Gated Auto-synchronous Luminescence Detection (GALD) A novel opto-mechanical means of detecting delayed luminescence was first conceived by Dr. Connally in mid 2006, a prototype was demonstrated shortly after but it took another 3 years before the first device could be installed into a fluorescence microscope. The GALD design has since won awards on ABC’s New Inventors and was highly commended in the Engineering Excellence awards of 2010, both in Workplace Health and Safety and Innovations and Inventions categories.
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Applications of the GALD
