Macquarie University
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Development of time-gated luminescence bio-imaging instruments

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posted on 2022-03-28, 12:51 authored by Xianlin Zheng
The general goal for biosensing is to realize ultra-sensitive, high-contrast, rapid and high throughput detection, localization and quantification of trace amounts of biomolecules and diseased cells of rare types within complex samples. Luminescent probes, such as lanthanidecomplexes and photon upconversion nanomaterials, hold the potential to realize this goal due to their unique optical properties, which include long luminescence lifetimes in the microsecond region and as well sharp spectral emission spectra. Application of the time-gated and time-resolved techniques based on these probes will provide background-free detection conditions. But such a potential has been seriously limited by the availability of suitable instruments The focus of my PhD research program is the development and the design of new instruments to realize time-domain detection towards advanced optical characterizations of luminescent materials and their analytical applications in biosensing and bioimaging. Specifically, my research project aims to advance and translate the time-gated luminescence detection technique into three prototype instruments -the multi-colour microscope, the invivo imaging system, and the high-speed scanning cytometry The development of the first instrument, a multi-colour microscope, in this work had the specific aims of (a) increasing the detection efficiency, (b) improving the compatibility and stability, and (c) reducing the cost and complexity associated with time-gated luminescence microscopes. These aims were met by designing and engineering a high-efficiency excitation unit that is based on a high power and high repetition rate Xenon flash lamp, to provide broadband illumination for simultaneously exciting multiple lanthanide luminescent probes. The time-gated detection module of this apparatus is optimised by using a fast-rotating optimal chopper with a pinhole on the edge of the chopper blade to realise a high switching speed. The modular design has been proved to offer high compatibility and stability when installed to a commercial inverted microscope, and high-contrast dual-colour imaging has been demonstrated in this work by imaging two types of micro-organisms stained by a red-emitting europium complex and a green-emitting terbium complex, respectively, using this setup. The second instrument, an in-vivo imaging system, extended the time-gated technique usedi n background-free small animal imaging. In this work, the mechanics and optics of the time-gated detection unit of this instrument was re-designed so that this instrument became more suited to the visualization of upconversion nanoparticles when it was used as the optical contrast agents. Their ability to be excited at 980 nm and emit at 800 nm are advantageous for deep-tissue imaging of animal models because near-infrared light lies in the biological transparent window' where haemoglobin and proteins demonstrate a low absorption. In this work, it was shown that an excitation module housing up to eight 980 nm fibre-coupled diode could be engineered, and a synchronization circuit to generate time-delayed pulses with sufficient driving capacity could be designed. Capable of significantly reducing the optical background and the thermal accumulation, the system integrating near-infrared optical imaging and time-gated technique was demonstrated that could visualise upconversion nanoparticles injected into a Kunming mouse in vivo. The third instrument, a high-speed scanning cytometry, aimed to achieve high-precision pinpointing of individual luminescent targets at high sample throughput for quantitative measurements. Based on the orthogonal scanning automated microscopy (OSAM) method previously developed by members of the Author's research group, the next-generation referenced-OSAM (R-OSAM) was further engineered by integrating two linear encoders and an autofocus unit to provide the positional reference and compensate the sample tilt in real time. It has been evaluated using micrometre-scale luminescent beads incorporating down-converting lanthanide complexes or upconversion nanoparticles, crystalline microplates, colour-barcoded microrods, and quantitative suspension array assays. Through the course of this work, a range of device modules have been demonstrated with high detection efficiency, low cost, improved stability and compatibility to modify commercial systems. These create new opportunities for chemistry and biology laboratories to access advanced time-gated luminescence techniques for material characterisation and biosensing applications This work is structured as a thesis by publication. The three result chapters are presentedin the form of four peer-reviewed journal papers.


Table of Contents

Chapter 1: Introduction -- Chapter 2: Multi-colour time-gated luminescence microscopy -- Chapter 3: Time-gated animal imaging system -- Chapter 4: Encoder-assisted scanning microscopy -- Chapter 5: Conclusions and perspectives


Bibliography: Pages 135-138 Thesis by publication.

Awarding Institution

Macquarie University

Degree Type

Thesis PhD


PhD, Macquarie University, Faculty of Science and Engineering, Department of Physics and Astronomy

Department, Centre or School

Department of Physics and Astronomy

Year of Award


Principal Supervisor

James A. Piper

Additional Supervisor 1

Dayong Jin

Additional Supervisor 2

Yiqing Lu


Copyright Xianlin Zheng 2016. Copyright disclaimer:




1 online resource (xviii, 138 pages)

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