Imaging

Hyperspectral imaging of autofluorescence in cells and tissues

Autofluorescent colours and spatial patterns in cells and tissues allow us to non-invasively probe biological processes in situ. They provide a window into biology, because native autofluorescent molecules, most notably NADH and flavins, play a major role in cellular energy production which is central for life. Using autofluorescence (AF), cells and tissues can be interrogated using only light, without any extraneous chemical interference¹^,². Autofluorescence imaging is carried out by using hyperspectral microscopy systems (hardware and software) developed by CNBP researchers. We also developed hyperspectral ophthalmology imaging systems for rapid research translation.

Potential Uses and Applications

Hyperspectral image of autofluorescence (Courtesy E. Goldys)

Using hyperspectral imaging we have been able to bring deep new insights into key biological processes such as neurodegeneration², chronic pain⁶ and pioneering results in reproduction,^{7, 8} aging⁹, and immunity such as the first-ever non-invasive monocyte analysis, reflecting immune physiology¹⁰. These new capabilities are particularly well suited for the non-invasive AF-based assessment of physiological function, in ways which have never previously been possible.

Contact

Prof Ewa Goldys: e.goldys@unsw.edu.au

Key publications

1. M. E. Gosnell, A. G. Anwer, S. B. Mahbub, S. M. Perinchery, D. W. Inglis, P. P. Adhikary, J. A. Jazayeri, M. A. Cahill, S. Saad, C. A. Pollock, M. L. Sutton-McDowall, J. G. Thompson and E. M. Goldys, ‘Quantitative Non-Invasive Cell Characterisation and Discrimination Based on Multispectral Autofluorescence Features’, Scientific Reports 6 (2016). DOI: 10.1038/srep23453

2. M. E. Gosnell, A. G. Anwer, J. C. Cassano, C. M. Sue and E. M. Goldys, ‘Functional hyperspectral imaging captures subtle details of cell metabolism in olfactory neurosphere cells, disease-specific models of neurodegenerative disorders’, Biochimica Et Biophysica Acta-Molecular Cell Research 1863 (1), 56-63 (2016). DOI: 10.1016/j.bbamcr.2015.09.030

6. V. Staikopoulos, M. E. Gosnell, A. G. Anwer, S. Mustafa, M. R. Hutchinson and E. M. Goldys, in Spie Biophotonics Australasia, edited by M. R. Hutchinson and E. M. Goldys (2016), Vol. 10013.

7. M. L. Sutton-McDowall, M. Gosnell, A. G. Anwer, M. White, M. Purdey, A. D. Abell, E. M. Goldys and J. G. Thompson, ‘Hyperspectral Microscopy Can Detect Metabolic Heterogeneity within Bovine Post-Compaction Embryos Incubated under Two Oxygen Concentrations (7% Versus 20%)’, Human Reproduction 32 (10), 2016-2025 (2017). DOI: 10.1093/humrep/dex261

8. M. L. Sutton-McDowall, L. L. Y. Wu, M. Purdey, A. D. Abell, E. M. Goldys, K. L. MacMillan, J. G. Thompson and R. L. Robker, ‘Non-esterified fatty acid-induced endoplasmic reticulum stress in cattle oocytes alters cell metabolism and developmental competence’ Biology of Reproduction 94 (1) (2016). DOI: 10.1095/biolreprod.115.131862

9. R. L. Michael J. Bertoldo, Wing-Hong Jonathan Ho, Angelique H. Riepsamen, Xing L. Jin, Kaisa Selesniemi, Dale M. Goss, Saabah Mahbub, Jared M. Campbell, Abbas Habibalahi, Wei-Guo Nicholas Loh, Neil A. Youngson, Jayanthi Maniam, Ashley S.A. Wong, Dulama Richani, Catherine Li, Yiqing Zhao, Maria Marinova, Lynn-Jee Kim, Laurin Lau, Rachael M Wu, A. Stefanie Mikolaiczak, Toshiyuki Araki, David G. Le Couteur, Nigel Turner, Margaret J. Morris, Kirsty A. Walters, Ewa Goldys, Christopher O’Neill, Robert B. Gilchrist, David A. Sinclair, Hayden A. Homer, Lindsay E. Wu, ‘NAD(+) Repletion Rescues Female Fertility during Reproductive Aging’, Cell Reports (2019). DOI:10.1016/j.celrep.2020.01.058

Imaging needles

Imaging needles are smart needles that can see where they are going. Each needle contains a tiny imaging probe that scans the tissue as it is inserted. We have developed imaging needles based of two optical technologies. Optical coherence tomography can visualise the microstructure of tissue and also detect blood vessels. Fluorescence can provide insight about the molecular composition of tissue. The miniaturised imaging probe that lies at the heart of the needle can also be deployed in a flexible transparent catheter instead of a rigid needle. This provides us with flexible probes can be inserted inside a blood vessel or an airway, imaging deep within the body.

3D printed miniature endoscope optics inside a blood vessel. (Image: Florian Sterl, Sterltech Optics)

Potential use and application areas:

Smart brain biopsy needle for safer neurosurgery. Our needle can detect when it is in cancer and warn the neurosurgeon of ‘at risk’ blood vessels.
Meat quality in livestock. Our imaging needles can measure the percentage of intramuscular fat in meat, which is a strong indicator of eating quality.
Improved detection of heart disease. When deployed in a flexible catheter instead of a needle, our imaging probes visualise the atherosclerotic plaques that cause heart disease.

Key contacts

Prof. Robert McLaughlin: robert.mclaughlin@adelaide.edu.au
Dr. Jiawen Li: jiawen.li01@adelaide.edu.au

Key publications

Ramakonar, B.C. Quirk, R.W. Kirk, J. Li, A. Jacques, C.R.P. Lind, R.A. McLaughlin, “Intraoperative detection of blood vessels with an imaging needle during neurosurgery in humans,” Science Advances, 4(eaav4992), 2018. DOI: 10.1126/sciadv.aav4992
Li, S. Thiele, B.C. Quirk, R.W. Kirk, J.W. Verjans, E.Akers, C. Bursill, S.J. Nicholls, A.M. Herkommer, H. Giessen, R.A. McLaughlin, “Ultrathin monolithic 3D printed optical coherence tomography endoscope for preclinical and clinical use,” Light: Science & Application, 9(124), 2020. DOI: 10.1038/s41377-020-00365-w

Multimodal imaging

Multimodal imaging, a combination of imaging modalities, can provide richer information and overcome the limitations of the independent imaging techniques. This approach has the potential to provide profound new information from a broad range of samples ranging from biological to solid-state materials and photonic devices. Imaging modalities such as Coherent anti-Stokes Raman spectroscopy (CARS), Stimulated Raman spectroscopy (SRS) and Fluorescence-lifetime imaging microscopy (FLIM) are being combined with two photon, confocal fluorescence, and quantum microscopy. This broad range of multimodal imaging techniques can examine label-free vibrational signatures in materials, characterise individual atoms with optical signatures, and be used to unravel the spectral fingerprint of molecules. This powerful imaging tool kit will unlock new knowledge across the fields of physics, chemistry, biology, and beyond.

Potential uses and applications

Single photon characterisation
Label-free bioimaging
Autofluorescence imaging
In-vivo imaging
Lifetime mapping

Key contacts

Prof Brant Gibson: brant.gibson@rmit.edu.au
Dr Brian Yang: brian.yang@rmit.edu.au

Key publication

Orth, R. N. Ghosh, E. R. Wilson, T. Doughney, H. Brown, P. Reineck, J. G. Thompson and B. C. Gibson, ‘Super-multiplexed fluorescence microscopy via photostability contrast’, Biomedical Optics Express, Vol. 9, Issue 7, pp. 2943-2954 (2018) DOI: 10.1364/BOE.9.002943

Quantum microscopy

Movie showing the localisation of two single photon emitters. The open circles show the location of three, Hanbury Brown and Twiss detectors, the plus signs are the locations of the emitters, the blue and orange dots show the results of 500 reconstructions of the emitter locations. The contours show the effective point spread function for the protocol and all lengths are in units of the standard deviation of the point spread function. As the time progresses, the reconstructions become better as shown, beating the diffraction limit in all cases shown here.

Quantum microscopy is a new approach to microscopy that uses the classical and quantum properties of light to improve resolution. Our method provides new ways to localise single photon active fluorophores. This diffraction unlimited scheme can potentially combined with other super-resolution schemes (for example STED) to improve resolution in living cells.

Potential Uses and Applications

Bioimaging where phototoxicity limits resolution

Contact

Prof Andrew Greentree: andrew.greentree@rmit.edu.au

Key publication

G. Worboys, D. W. Drumm, and A. D. Greentree, Quantum multilateration: Subdiffraction emitter pair localization via three spatially separate Hanbury Brown and Twiss measurements, Physical Review A 101, 013810 (2020). DOI:10.1103/PhysRevA.101.013810

Super-resolution microscopy

CNBP has unique capabilities for imaging single lanthanide upconversion nanoparticles, with optical resolution ~50nm in the case of STimulated Emission Depletion (STED) microscopy or 180nm for Superlinear Excitation Emission (SEE) microscopy. Both techniques are based on confocal microscopy with 980nm laser excitation, in the case of STED requiring also a second annular depletion beam at 810nm aligned with the excitation beam. The CNBP Super-resolution laboratories house 3 confocal microscopy systems, the first a custom-made system which can switch from SEE to STED modes, the second a custom-made confocal system for single-nanoparticle optical characterisation, the third a commercial (Olympus FC3000) confocal microscope incorporating 980nm excitation.

Super res image — Imaging performance of upconversion nanoparticles by means of the super-resolution techniques SEE and STED, in comparison with a diffraction-limited confocal image.

Potential Uses and Applications

High-resolution bioimaging
Developments of hybrid optoelectronic materials-upconversion nanoparticles in thin film polymers

Contact

Dr Simone De Camillis: simone.decamillis@mq.edu.au
Dr Yiqing Lu: yiqing.lu@mq.edu.au
Emeritus Professor Jim Piper: jim.piper@mq.edu.au

Key publication

LIU Y, LU Y, YANG X, ZHENG X,WEN S, WANG F, VIDAL X, ZHAO J, LIU D, ZHOU Z, MA C, ZHOU J, PIPER JA, PENG X and JIN D, Amplified stimulated emission in upconversion nanoparticles for super-resolution nanoscopy, Nature 543 (2017) DOI: 1038/nature21366
DENKOVA D, PLOSCHNER M, DAS M, PARKER LM, ZHENG X, LU Y, ORTH A, PACKER NH and PIPER JA, 3D sub-diffraction imaging in a conventional confocal configuration by exploiting super-linear emitters, Nature Communications 10 (1) (2019) 1-12 DOI: 10.1038/s41467-019-11603-0
PLOSCHNER M, DENKOVA D, DE CAMILLIS S, DAS M, PARKER LM, ZHENG X, LU Y, OJOSNEGROS S and PIPER JA, Simultaneous super-linear excitation-emission and emission depletion allows imaging of upconversion nanoparticles with higher sub-diffraction resolution, Optics Express 28, 24308 (2020) DOI: 10.1364/OE.400651
DE CAMILLIS S, REN P, CAO Y, PLOSCHNER M, DENKOVA D, ZHENG X, LU Y, and PIPER JA, Controlling the non-linear emission of upconversion nanoparticles to enhance super-resolution imaging performance, Nanoscale 12, 20347 (2020) DOI: 10.1039/D0NR04809G

3D Microendoscopy

Microendoscopy colour cores (courtesy A. Orth)

Optical fibre bundle microendoscopes are widely used for visualizing hard-to-reach areas of the human body. These ultrathin devices often forgo tunable focusing optics because of size constraints and are therefore limited to 2D imaging modalities. Ideally, microendoscopes would record 3D information for accurate clinical and biological interpretation, without bulky optomechanical parts.

In our research, we have demonstrated that the optical fibre bundles commonly used in microendoscopy are inherently sensitive to depth information. We use the mode structure within fibre bundle cores to extract the spatio-angular description of captured light rays—the light field—enabling digital refocusing, stereo visualization, and surface and depth mapping of microscopic scenes at the distal fibre tip. Our approach is resilient to fibre bending, making it attractive for a wide range of applications.

Potential use and application areas:

Endoscopy
Surgical procedures
Industrial imaging
Fluorescence imaging

Key contacts

Prof Brant Gibson: brant.gibson@rmit.edu.au
Dr Antony Orth: antony.orth@gmail.com

Key publications

Orth, M. Ploschner, E. R. Wilson, I. S. Maksymov, B. C. Gibson, ‘Optical fiber bundles: ultra-slim light field imaging probes’, Science Advances, 5, no. 4 (2019). DOI:10.1126/sciadv.aav1555
Orth, M. Ploschner, I. Maksymov, and B. C. Gibson, ‘Extended depth of field imaging through multicore optical fibers’, Optics Express, Vol. 26, Issue 5, pp. 6407-6419 (2018). DOI:10.1364/OE.26.006407

Time-gated microscopy/lifetime imaging

Time-gated microscopy of long-lifetime (microsecond to millisecond) luminescent bioprobes enables effective suppression of scattered excitation light and autofluorescence resulting in greatly enhanced signal-to-background for detection of weak luminescence emission from the bioprobes. CNBP has extensive experience in development and use of long-lifetime lanthanide molecular and custom-designed nanoparticle probes as well as development of time-gated luminescence (TGL) instrumentation, including wide-field TGL microscopy and high-speed scanning TGL microscopy (Orthogonal Scanning Automated Microscopy-OSAM). A key further development of this technique is Lifetime Imaging where bioprobes of specific lifetimes in the microsecond to millisecond range can be imaged separately to allow mapping of lifetime-multiplexed probes.

Potential use and application areas:

Biomedical diagnostics-infectious diseases, cancer and vascular malformations
Detection of environmental pathogens
Security labelling

Key contacts

Dr Yiqing Lu: yiqing.lu@mq.edu.au
Dr Xianlin Zheng: xianlin.zheng@mq.edu.au
Dr Nima Sayyadi: nima.sayyadi@mq.edu.au
Emeritus Professor Jim Piper: jim.piper@mq.edu.au

Key publications

LU YQ, LU J, ZHAO JB, CUSIDO J, RAYMO FM, YUAN JL, YANG S, LEIF RC, HUO YJ, PIPER JA, ROBINSON P, GOLDYS E and JIN D, On-the-fly decoding luminescence lifetimes in the microsecond region for lanthanide-encoded suspension arrays, Nature Communications 5 (2014) Art.No. 3741 DOI: 1038/ncomms4741
SAYYADI N, JUSTINIANO I, CONNALLY RE, ZHANG R, SHI B, KAUTTO L, EVEREST-DAS AV, JUAN Y, WALSH BJ, JIN D, WILLOWS RD, PIPER JA and PACKER NH, Sensitive Time-Gated Immunoluminescence Detection of Prostate Cancer Cells Using a TEGylated Europium Ligand, Analytical Chemistry 88 (2016) 9564-9571 DOI: 10.1021/acs.analchem.6b02191
FAN Y, WANG P, Lu Y, WANG R, ZHOU L, Zheng X, Li X, PIPER JA and ZHANG F, Lifetime-engineered NIR-II nanoparticles unlock multiplexed in vivo imaging, Nature Nanotechnology 13 (2018) 941-946 DOI: 10.1038/s41565-018-0221-0

Smartphone microscope

CNBP researchers have developed a 3D printable ‘clip-on’ that can turn any smartphone into a fully functional microscope. Read more from the science paper and download the relevant 3D printable files here. Notes relating to the 3D printing process are also available.

TOOLS & CAPABILITIES