When the Reserve Bank of Australia wanted to develop new security technologies for bank notes, Prof Jim Piper’s Advanced Imaging research group in the ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP) at Macquarie University had an answer: timecoded nanoparticles. Continue reading
Friday 16 August:
CNBP researchers have unlocked the potential to transform microscopy at the nanoscale from a costly, complex option to an everyday laboratory tool, available in every lab.
The technique, described in a paper by lead authors Dr Denitza Denkova and Dr Martin Ploschner, which has been dubbed upconversion super-linear excitation-emission – or uSEE – microscopy, can be used not only for observation but also for the activation of biological structures with super-resolution.
This opens new avenues in optogenetics for precise activation of neurons in the brain or for targeted delivery of drugs with increased sub-cellular precision.
Standard optical microscopes can image cells and bacteria but not their nanoscale features which are blurred by a physical effect called diffraction.
Optical microscopes have evolved over the last two decades in order to bypass this diffraction limit; however, these so-called super-resolution techniques typically require expensive and elaborated instrumentation or imaging procedures.
“We have identified a particular type of fluorescent markers, upconversion nanoparticles, which can enter into a regime where light emitted from the particles grows abruptly – in a super-linear fashion – when increasing the excitation light intensity,” Martin says. “Our key discovery is that if this effect is exploited under the right imaging conditions, any standard scanning optical microscope can spontaneously image with super-resolution.”
The discovery addresses a key challenge for microscopy – the so-called diffraction limit. This prevents optical microscopes from seeing very small features clearly as, when the size and distance between the features start reaching the nanoscale range, they begin to blur together and appear as one.
And that is a problem for biologists to observe nanoscale samples – which is what researchers tackling some of our toughest health challenges need to do all the time.
Little wonder then that accessing the world that lies beyond this diffraction limit has become a holy grail for optical microscopy researchers over the past two decades.
In 2014, the Nobel Prize in Chemistry was awarded to three scientists, who developed three different techniques, capable of tricking physics to overcome the diffraction limit.
This landmark work set the scene for an explosion of so-called super-resolution techniques, which have led to revolutionary discoveries.
So far, however, all of these methods have had significant drawbacks. They are far from user-friendly and require either complicated and costly equipment or elaborated image processing, which often leads to imaging artefacts.
When it comes to 3D imaging, there are even more complications.
All the methods until now also require increasing the illumination power to increase the resolution – but that presents particular problems in the world of biology, where excessive light can harm a fragile specimen.
Denitza’s and Martin’s team took a novel approach to the problem. They wanted to make super-resolution possible on a confocal microscope, without set-up modifications or image processing, so that it would be available for use in any lab at practically no extra cost.
Their key discovery was that they could use a standard scanning optical microscope as a 3D super-resolution machine by imaging “upconversion” nanoparticles, potentially bound to the biological structure being studied. Unlike other super-resolution methods, uSEE microscopy offers better resolution at lower powers, and so minimises the damage to biological samples.
But it is not just the amount of light. Its colour also influences the photo-damage and the resolution. For example, UV- light is more harmful, but since it yields a better resolution, most of the super-resolution methods work in the UV and visible wavelengths.
However, in recent years biologists have become increasingly interested in using near-infrared light. It is less harmful and also allows imaging deeper in the tissue. But it does require a sacrifice in resolution, and the field of super-resolution has a very limited pool of fluorophores and techniques which work in the near-infrared regime.
Conveniently, the upconversion nanoparticles, on which the fluorescent markers employed in uSEE microscopy are based, are excited in the desired near-infrared colour spectrum. They are becoming increasingly popular as biological markers as they offer numerous other advantages for biology, including stable optical performance and possibility for multi-colour imaging.
Numerous papers have been published in the recent years about imaging of such particles for bio-applications. However, the effect of spontaneous super-resolution remains overlooked, mainly because the composition of the particles has not been fine-tuned for this application or the particles were not imaged under suitable conditions.
The CNBP team identified a particular nanoparticle composition which provides a strong improvement of the resolution. To make it easier for the end-user, the researchers developed a theoretical framework to optimise the particles and the imaging parameters for their own laboratory setting.
The concept of this method has been around for decades, and several groups have tried to put it into practice, but they either couldn’t identify fluorescent labels with adequate photo-physics, or the imaging conditions were not suitable to achieve bio-imaging in a convenient laboratory setting.
The CNBP team has shown for the first time that the technique can be used in a 3D biological environment, with biologically convenient particles which are both easy to work with and do not harm the samples.
This new methodological toolbox has the potential to go beyond the applications for which it has so far been used. It can be extended to a much broader imaging context, opening new avenues in the research of super-linear emitters and combining them with other imaging modalities to improve their performance.
Journal: Nature Communications
Publication Title: 3D sub-diffraction imaging in a conventional confocal configuration by exploiting super-linear emitters
Authors: Denitza Denkova, Martin Ploschner, Minakshi Das, Lindsay M. Parker, Xianlin Zheng, Yiqing Lu, Antony Orth, Nicolle H. Packer & James A. Piper
Abstract: Sub-diffraction microscopy enables bio-imaging with unprecedented clarity. However, most super-resolution methods require complex, costly purpose-built systems, involve image post-processing and struggle with sub-diffraction imaging in 3D. Here, we realize a conceptually different super-resolution approach which circumvents these limitations and enables 3D sub-diffraction imaging on conventional confocal microscopes. We refer to it as super-linear excitation-emission (SEE) microscopy, as it relies on markers with super-linear dependence of the emission on the excitation power. Super-linear markers proposed here are upconversion nanoparticles of NaYF4, doped with 20% Yb and unconventionally high 8% Tm, which are conveniently excited in the near-infrared biological window. We develop a computational framework calculating the 3D resolution for any viable scanning beam shape and excitation-emission probe profile. Imaging of colominic acid-coated upconversion nanoparticles endocytosed by neuronal cells, at resolutions twice better than the diffraction limit both in lateral and axial directions, illustrates the applicability of SEE microscopy for sub-cellular biology.
A groundbreaking new technique will slash the time it takes to detect potentially lethal golden staph infection from two days to just two hours.
Researchers from the ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP) targeted the bacterium with a luminescent DNA probe.
“This allows us to find the “needle in the haystack” because only the “needle” lights up,” says Dr Nima Sayyadi, Research Fellow at the Macquarie University node of the CNBP and lead author on the paper.
Golden staph, or Staphylococcus aureus, lives on the skin or in the nose. It is usually harmless, but if it enters the skin through a cut it can cause a range of infections, which in some cases are fatal.
In the most at-risk patients, such as the elderly, it is vital to identify the infection and begin treatment with appropriate antibiotics as soon as possible. However, current identification techniques require culturing cells for up to two days to provide a positive infection result.
The new approach, known as Time-Gated Luminescent in Situ Hybridization (LISH), takes just two hours and could have a range of other applications. While it cannot yet separately identify drug resistance strains of golden staph, researchers are working on it.
CNBP scientists are also working on a range of transformational research projects based on the luminescence based detection of single cells in human body fluid samples, which will help them label antibodies and molecules as well as DNA.
“We’ve also done work in prostate cancer and bladder cancer where the target cell can be quickly and easily identified in urine samples,” says Project Lead and CNBP node leader at Macquarie University, Professor James Piper AM.
The research was reported in the journal Molecules, which you can read here.
Researchers have developed a new form of nanoparticle and associated imaging technique that can detect multiple disease biomarkers, including those for breast cancer, found in deep-tissue in the body.
Reported in the science journal ‘Nature Nanotechnology’, the research opens up a new avenue in minimally invasive disease diagnosis and will potentially have widespread use both for biomedical research and for clinical applications.
“The use of nanoparticles for bio-imaging of disease is an exciting and fast-moving area of science,” says research author Dr Yiqing Lu (pictured) at the ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University.
“Specially designed nanoparticles can be placed in biological samples or injected into specific sites of the body and then ‘excited’ by introduced light such as that from a laser or an optical fibre,” he says.
“Disease biomarkers targeted by these nanoparticles then reveal themselves, by emitting their own specific wavelength signatures which are able to be identified and imaged.”
A major limitation however is that only a single disease biomarker at a time is able to be distinguished and quantified in the body using this type of detection technique.
“Detection of multiple biomarkers (known as multiplexing) in the body has been a major challenge for researchers,” says Dr Lu.
“The tissue environment is extremely complex—full of light absorbing and scattering elements such as blood, muscle and cartilage. And introducing multiple nanoparticles to a site, operating at multiple wavelengths to detect multiple biomarkers, produces too much interference. It makes it extremely difficult to determine accurately if a range of disease biomarkers are present.”
What Dr Lu and the research team have done to solve this issue has been to engineer innovative nanoparticles that emit light at the same frequency (near infrared light) but that are able to be coded to emit light for set periods of time (in the microsecond-to-millisecond time range).
“It is the duration of the light-emission and the biomarker reaction to this timed amount of light (known as luminescence lifetime) that produces a clearly identifiable molecular signature,” he says.
“Multiple disease biomarkers can be clearly identified and imaged based on this approach as there are no overlapping wavelengths interfering with the reading.”
“This enables high-contrast optical biomedical imaging that can detect multiple disease biomarkers all at the one time.” says Dr Lu.
In an exciting breakthrough in laboratory testing, the innovative nanoparticles have been able to detect multiple forms of breast cancer tumours in mice.
“We’re extremely excited where this work is taking us,” says Professor Fan Zhang at Fudan University (China) and joint-lead author on the research publication.
“We were able to successfully detect and identify key biomarkers for a number of different sub-types of breast cancer.”
“This technique has the potential to provide a low-invasive method of determining if breast cancer is present, as well as the form of breast cancer, without the need to take tissue samples via biopsy.”
“Ultimately our novel nanoparticles will enable quantitative assessment for a wide range of disease and cancer biomarkers, all at one time. The technique will be able to be used for early-stage disease screening and potentially utilised in integrated therapy,” says Professor Fan Zhang.
Professor Jim Piper, CNBP node leader at Macquarie University and also an author on the paper is similarly upbeat with the results that have been obtained.
“This is a major advance in a long-term effort at our Centre at Macquarie University to develop innovative techniques for simultaneous detection of multiple disease markers in humans and animals,” he says.
“Next steps in our research collaboration are to further refine the nanoparticles, to examine issues related to a clinical roll-out of the technology and to explore further applications and disease areas where this technique could be best utilised.”
Reported in the prestigious journal ‘Nature Nanotechnology’, the international team of researchers involved with the study are based at the ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University and Fudan University, China.
Notably, the work is an extension of previous nanoparticle-imaging research undertaken by Dr Lu at Macquarie University which has been awarded a patent in the United States and China, and which has already been licensed with commercial partners.
Journal: Nature Nanotechnology.
Publication title: Lifetime-engineered NIR-II nanoparticles unlock multiplexed in vivo imaging.
Authors: Yong Fan, Peiyuan Wang, Yiqing Lu, Rui Wang, Lei Zhou, Xianlin Zheng, Xiaomin Li, James A. Piper & Fan Zhang.
As silver sponsor at the annual STA ‘Science meets Business’ event held in Sydney, November 9th 2017, CNBP was extremely well represented, supporting a push to improve engagement and collaboration between the research sector and Australian industry.
In addition to having numerous Centre scientists in attendance – those with a strong interest and focus on commercialisation and translation of research, CNBP also had senior personnel speak and present in a variety of capacities.
This included CNBP Director Prof Mark Hutchinson (pictured top left), who together with Andrew Grant (Availer) discussed CNBP’s commercialisation success and the taking of ideas from ‘boom to the showroom.’ Deep dive (idea creation), value-add solutions, solving pain points and interesting new jobs were all touched upon in a quick fire exchange of views.
Additionally, Centre Investigator and Miniprobes founder Prof Robert McLaughlin participated in the ‘soapbox sesssion’ where three competitively-selected ‘soapbox leaders’ made compelling pitches, sparking robust discussion as they quizzed delegates for perspectives on new ideas to create useful collaboration.
“It was great to be at this years ‘Science meets Business’, bringing CNBP science and innovation to industry and learnings back again,” concluded Prof Hutchinson. “I look forward to hearing about other successful collaborations at next year’s STA event.”
Researchers at the ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University, the University of Technology Sydney (UTS), Peking University and Shanghai Jiao-tong University have made a breakthrough in the development of practical super-resolution optical microscopy that will pave the way for the detailed study of live cells and organisms, on a scale 10 times smaller than can currently be achieved with conventional microscopy.
Reported in Nature, the international team of researchers has demonstrated that bright luminescent nanoparticles can be switched on and off using a low-power infrared laser beam, and used to achieve images with a super resolution of 28nm.
Professor Jim Piper (pictured), leader of the research team at Macquarie University and the CNBP sees these nanoparticles as having new unique properties. “These allow researchers to see well beyond normal limits of standard microscopes. It will let you see deeper and more clearly at the cellular and intra- cellular level—where proteins, antibodies and enzymes ultimately run the machinery of life.”
The research featured in BioPhotonics World.
Our researchers and collaborators have made a breakthrough in the development of practical super-resolution optical microscopy that will pave the way for the detailed study of live cells and organisms, on a scale 10 times smaller than can currently be achieved with conventional microscopy.
Reported in Nature, it was demonstrated that bright luminescent nanoparticles can be switched on and off using a low-power infrared laser beam, and used to achieve images with a super resolution of 28nm (about 1/36 the wavelength of light).
Find out more by accessing the paper online.
Title: Amplified stimulated emission in upconversion nanoparticles for super-resolution nanoscopy.
Authors: Yujia Liu, Yiqing Lu, Xusan Yang, Xianlin Zheng, Shihui Wen, Fan Wang, Xavier Vidal, Jiangbo Zhao, Deming Liu, Zhiguang Zhou, Chenshuo Ma, Jiajia Zhou, James A. Piper, Peng Xi & Dayong Jin.
Prof Jim Piper, CNBP Macquarie University Node Leader has been awarded Honorary Life Membership of the Australian Optical Society (AOS) following the organisation’s annual general meeting, held in 2015.
The award recognizes Jim’s contribution to the society over many years. His signature was on the Memorandum of Association that established the AOS as an association in 1983. He took over the AOS presidency in 1984-1985, and was the recipient of the AOS W.H. (Beattie) Steel Medal – in 1997.
Also awarded life membership at the December 2015 meeting were Macquarie University’s Prof. Brian Orr and Prof. Ross McPhedran from the University of Sydney.
All continue to be involved with the AOS on an ongoing basis.
CNBP Director, Macquarie University Node, Prof Jim Piper has attended the ‘Enabling Technologies Technical Exchange Meeting 2015’, recently held in Arlington, Virginia.
The event, held under the auspices of the 2015 United States-Australia Joint Commission Meeting on Science and Technology, looks to enhance strategic scientific dialogue between the two countries as well as promote joint efforts in addressing complex problems facing the world today.
This year’s theme was ‘Enabling Technologies’ with Prof Piper giving two well received talks – ‘Supporting Structures for Australian Discovery Research’ and ‘Time-coded Luminescent Nanoparticles for High Contrast Detection of Specific (sub) Cellular Targets’.
Congratulations to Professor Jim Piper who has been elected president of Science Technology Australia (STA).
Science & Technology Australia represents Australia’s scientists and technologists – promoting their views on a wide range of policy issues to government, industry and the community.