Monthly Archives: August 2019

Super-resolution method could bring nanoscale microscopy to every lab

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.


Science on show – what’s on in National Science Week

7 August 2019:

The CNBP and its researchers are taking part in a wide range of activities for National Science Week.

This Thursday 8 August researcher Dr Wei Deng from UNSW Sydney will explain how nanotechnogy is changing how we treat cancer, as part of Inspiring Australia’s Talking Science series.

It will be held at the Max Webber Library, in Blacktown, Sydney. More details here.

On Sunday, 11 August, Adelaide University’s Lyndsey Collins-Praino will host Kids Navigate Neuroscience, an event at which children aged 4-10 can explore how the brain works in a fun and hands-on way by participating in a series of interactive neuroscience exhibits.

You can find out more about the event here. Bookings are essential and can be made through Eventbrite.

On Tuesday 13 August explore medical brain research by joining Dr Lindsay Parker, a researcher at Macquarie University, as she discusses how she is trying to create better medicines for Alzheimer’s, chronic pain and brain cancer, by only targeting the unhealthy cells in the brain.

This event is part of Inspiring Australia’s Talking Science series as part of National Science Week. Bookings available now. Contact details:
Castle Hill Library
The Hills Shire Library Service
Phone: 02 9761 4510

There is a fun evening next Friday, 16 August, at the Adelaide Medical School, University of Adelaide, where you can explore the neuroscience of sex, drugs and salsa dancing.

A series of interactive exhibits will address questions such as, what role does the brain play in sexual attraction? Can you salsa dance your way to a healthy brain? How does the brain perceive different flavours when drinking wine, and how can pairing wine with different foods alter this perception?

More details here and bookings are through Eventbrite.

Also next Friday, 16 August, the whole family is invited to see some amazing short videos on a massive screen in a free National Science Week Event hosted by STEMSEL Foundation Braggs Lecture Theatre, University of Adelaide AI Light Science Spectacular.

You will find out how the eye works, how NASA finds planets in other solar systems and how detected the edge of the Universe.

You will also explore light, from nanoscale biophotonics with CNBP research fellow Dr Roman Kostecki to exploring the Universe with Dr Jerry Madakbas, a photonics physicist who builds night vision sensors for NASA.

You can book through Eventbrite.

Also on Friday night:

What role does the brain play in sexual attraction? Can you salsa dance your way to a healthy brain? How does the brain perceive different flavours when drinking wine, and how can pairing wine with different foods alter this perception?

These days, you can’t seem to walk through the aisle of a grocery store without being bombarded by newspaper and magazine headlines touting the latest and greatest breakthrough in neuroscience research. But how can you tell fact from fiction?

Join us for this Big Science Adelaide event, held at the Adelaide Health and Medical Sciences (AHMS) building at the University of Adelaide, where we’ll explore the answers to these questions and many more!

More details at 
Finally, CNBP researchers will be taking part in Science in the Swamp, a fun, free family festival of science displays, shows and activities on Sunday 18 August in Centennial Park, Sydney.

Join scientists as they show what amazing superpowers you find in nature – super sight, super hearing, super strength and camouflage are only some of the capabilities on show.

Be sure to put on your cape and dress up as your favourite superhero for this great event. You can find out more details here.

Nanoparticle discovery another step towards personalised medicine

1 August 2019:

A team led by the CNBP’s Dr Guozhen Liu has developed intelligent biodegradable polymer nanoparticles, which can help monitor a cell-signalling protein, or cytokine, widely expressed in cancer cells. The technique can help with earlier diagnostics and even treatment and represents another step towards personalised nanomedicine.

The research integrates a specific fluorogen – a molecule that generates fluorescence and can be used for protein monitoring – with PLGA nanoparticles for the first time.

The fluorogen in question is a so-called “aggregation-induced emission” fluorogen, known as an AIEgen. Aggregation-induced emission (AIE), has become an important area of research since its discovery around 20 years ago. It describes an abnormal phenomenon, in which some compounds show greater fluorescence as they aggregate than when in solution, as is more common. These AIEgens provide superior advantages for biosensing and bioimaging.

The integration of the nanoparticle and the AIEgen could become an important tool in the relatively new field of medicine known as “theranostics” – a combination of “therapy” and “diagnostics” made possible through the use of nanoparticles and an important transition towards personalised medicine.

Dr Liu’s discovery, for example, detects high levels of the cytokine VEGF-A found in tumor cells, and monitors simultaneous photothermal therapy (PTT), in which heat is used to kill cancer cells, and magnetic resonance imaging (MRI) as part of a whole package of early diagnostics and treatment of cancer cells.

It could be used in the future as a smart drug delivery system, with cancer drugs loaded in the nanoparticles for controlled and sustained release targeted precisely to a tumor.
In the future, Dr Liu believes it will be possible to develop the next generation of intelligent nanoparticles which can continually monitor cytokines and cytokine-triggered drug delivery while also carrying out deep tissue imaging.

Dr Liu is an ARC Future Fellow and Senior Lecturer at Graduate School of Biomedical Engineering at UNSW.

You can read the paper here.

Journal: Nanomedicine

Publication Title: AIEgen based poly(L-lactic-co-glycolic acid) magnetic nanoparticles to localize cytokine VEGF for early cancer diagnosis and photothermal therapy

Authors: Ma, K (Ma, Ke); Liu, GJ (Liu, Guo-Jun); Yan, LL (Yan, Lulin); Wen, SH (Wen, Shihui); Xu, B (Xu, Bin); Tian, WJ (Tian, Wenjing); Goldys, EM (Goldys, Ewa M.); Liu, GZ (Liu, Guozhen)

Abstract: Aim: We demonstrated a novel theranostic system for simultaneous photothermal therapy and magnetic resonance imaging applicable to early diagnostics and treatment of cancer cells. Materials & methods: Oleic acid-Fe3O4 and triphenylamine-divinylanthracene-dicyano were loaded to the poly(L-lactic-co-glycolic acid) nanoparticles (NPs) on which anti-VEGF antibodies were modified to form anti-VEGF/OA-Fe3O4/triphenylamine-divinylanthracene-dicyano@poly(L-lactic-co-glycolic acid) NPs. The 1H nuclear magnetic resonance (NMR), mass spectra, fluorescence, UV absorption, dynamic light scattering, transmission electron microscope and inductively coupled plasma mass spectrometry tests were used to characterize the NPs, and the bioimaging was illustrated by confocal laser scanning microscope (CLSM) and in vivo MRI animal experiment. Results: This system was capable to recognize the overexpressed VEGF-A as low as 68pg/ml in different cell lines with good selectivity and photothermal therapy effect. Conclusion: These ultrasensitive theranostic NPs were able to identify tumor cells by fluorescence imaging and MRI, and destroy tumors under near infrared illumination.

Author Keywords: AIEgen; cytokines; MRI; PDT; PLGA nanoparticle; PTT; theranostics