Category Archives: MQ

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.

Link: https://www.nature.com/articles/s41467-019-11603-0

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
Email: libraryseminars@thehills.nsw.gov.au
Phone: 02 9761 4510
https://www.scienceweek.net.au/exploring-medical-brain-research/

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 https://www.scienceweek.net.au/neuroscience-at-night/ 
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.

CNBP research wins Young Scientist Award

18 July 2019:

CNBP research fellow Dr Lindsay Parker, of Macquarie University, has won an award for the best research paper from an investigator under 40, at an international conference in Rome.

Lindsay’s work is aimed at better understanding molecules ex-pressed in the brain during pain, brain diseases and brain cancer. This could lead to improved precision drugs that specifically target only the unhealthy cells in the brain.

She won a “Young Scientist Award” at the 41st PIERS (Photonics & Electromagnetics Research Symposium) held at the University of Rome in June.

Her paper, “Utilising Glycobiology for Fluorescent Nanodiamond Uptake and Imaging in the Central Nervous System” was in the category “Remote Sensing, Inverse Problems, Imaging, Radar & Sensing”.

The paper, in collaboration with RMIT University and the University of Colorado Boulder, investigated the ability of lectin-coated fluorescent nanodiamonds to recognise specific central nervous system cell types.

The prize included cash, and an invitation to the Symposium Banquet held at Palazzo Brancaccio. Lindsay also received travel awards from MQ University Primary Carer Support for Conference Attendance ($2000) and MQ Research Centre for Diamond Science and Technology ($1000) which meant her partner and baby William were also able to be in Rome with her as she worked.

While she was in Europe, Lindsay took the opportunity to give invited talks at the Czech Academy of Sciences in Prague and at the University of Groningen in Netherlands while visiting two other labs working in similar research areas to her synthetic nanochemistry expert Dr Petr Cigler and nanobiotechnology expert A/Prof Romana Schirhagl.

Shedding light on golden staph

3 July 2019:

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.

Dr Nima Sayyadi in the lab

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.

Luminescent In Situ Hybridization (LISH)

New probe to detect hydrogen peroxide

10 June 2019:

A team of CNBP researchers have published a new paper discussing the design and application of a micro fabricated needle-like probe to measure hydrogen peroxide.  This new microfluidic tool has applications for monitoring dynamic chemical reactions in analytical chemistry and biological systems.

Journal: RSC Advances

Publication Title: Microfabricated needle for hydrogen peroxide detection

Authors: Shilun Feng, Sandhya Clement, Yonggang Zhu, Ewa M. Goldys and David W. Inglis

Abstract:  A microfabricated needle-like probe has been designed and applied for hydrogen peroxide (H2O2) sampling and detection using a commercial, single-step fluorescent H2O2 assay. In this work, droplets of the assay reagent are generated and sent to the needle tip using a mineral-oil carrier fluid. At the needle tip, the sample is drawn into the device through 100 mm long hydrophilic capillaries by negative pressure. The sampled fluid is immediately merged with the assay droplet and carried away to mix and react, producing a sequence of droplets representing the H2O2 concentration as a function of time. We have characterized the assay fluorescence for small variations in the sample volume. With the calibration, we can calculate the concentration of H2O2 in the sampled liquid from the size and intensity of each merged droplet. This is a microfluidic data-logger system for on-site continuous sampling, controlled reaction, signal storage and on-line quantitative detection. It is a useful tool for monitoring dynamic chemical reactions in analytical chemistry and biological applications.

Key words: Microfluidics, probe, H2O2, analytics chemistry

Binding mechanisms of solid-binding peptides

5 April 2019:

A new review article by CNBP PhD student Rachit Bansal and CNBP Associate Investigators (Anwar Sunna, Andrew Care and Tiffany Walsh) reports on experimental tools to study the binding mechanism of synthetic peptides to solid materials. The review provides insights into the role of these peptides as molecular building blocks for nanobiotechnology.

Journal: New Biotechnology.

Publication title: Experimental and theoretical tools to elucidate the binding mechanisms of solid-binding peptides.

Authors: Rachit Bansal, Andrew Care, Megan S. Lord, Tiffany R. Walsh, Anwar Sunna.

Abstract: The interactions between biomolecules and solid surfaces play an important role in designing new materials and applications which mimic nature. Recently, solid-binding peptides (SBPs) have emerged as potential molecular building blocks in nanobiotechnology. SBPs exhibit high selectivity and binding affinity towards a wide range of inorganic and organic materials. Although these peptides have been widely used in various applications, there is a need to understand the interaction mechanism between the peptide and its material substrate, which is challenging both experimentally and theoretically. This review describes the main characterisation techniques currently available to study SBP-surface interactions and their contribution to gain a better insight for designing new peptides for tailored binding.

Henan University visit

Prof Jim Piper2 April 2019:

CNBP Chief Investigators at Macquarie University, Prof Jim Piper and Prof Nicole Packer, as well as CNBP Associate Investigator Dr Bingyang Shi have met with delegates from Henan University led by Prof. Yang Zhonghua, Deputy Vice Chancellor and Henan University Vice President.

Henan University, founded in 1912, is located in Kaifeng, China and is known globally for its strength in the Biology discipline. Discussed at the meeting were CNBP research areas and projects, as well as the potential for collaboration. Prof. Piper and Prof. Packer were invited to visit Henan University for further talks later in the year.

L to R – Prof Nicole Packer, Prof. Yang Zhonghua, Prof Jim Piper and Dr Bingyang Shi.

 

 

Diagnosing eye surface cancer

24 Mar 2019:

A new automated non-invasive technique for diagnosing eye surface cancer (ocular surface squamous neoplasia or OSSN) has been developed by CNBP researchers and collaborators. The technique has the potential to reduce the need for biopsies, prevent therapy delays and make treatment far more effective for patients.

Read more in a news item on the Australian Medical Association website.

Visualizing neuroinflammation

21 March 2019:

A new time-gated microscopy approach has been reported by CNBP researchers that will help neurobiologists better visualize neurokine signaling (and other) molecules in cells or tissue samples. Lead author of the publication is CNBP researcher Dr Lindsay Parker, Macquarie University.

Journal: Journal of Neuroinflammation.

Publication title:  Visualizing neuroinflammation with fluorescence and luminescent lanthanide-based in situ hybridization.

Authors: Lindsay M. Parker, Nima Sayyadi, Vasiliki Staikopoulos, Ashish Shrestha, Mark R. Hutchinson and Nicolle H. Packer.

Abstract: 

Background
Neurokine signaling via the release of neurally active cytokines arises from glial reactivity and is mechanistically implicated in central nervous system (CNS) pathologies such as chronic pain, trauma, neurodegenerative diseases, and complex psychiatric illnesses. Despite significant advancements in the methodologies used to conjugate, incorporate, and visualize fluorescent molecules, imaging of rare yet high potency events within the CNS is restricted by the low signal to noise ratio experienced within the CNS. The brain and spinal cord have high cellular autofluorescence, making the imaging of critical neurokine signaling and permissive transcriptional cellular events unreliable and difficult in many cases.

Methods
In this manuscript, we developed a method for background-free imaging of the transcriptional events that precede neurokine signaling using targeted mRNA transcripts labeled with luminescent lanthanide chelates and imaged via time-gated microscopy. To provide examples of the usefulness this method can offer to the field, the mRNA expression of toll-like receptor 4 (TLR4) was visualized with traditional fluorescent in situ hybridization (FISH) or luminescent lanthanide chelate-based in situ hybridization (LISH) in mouse BV2 microglia or J774 macrophage phenotype cells following lipopolysaccharide stimulation. TLR4 mRNA staining using LISH- and FISH-based methods was also visualized in fixed spinal cord tissues from BALB/c mice with a chronic constriction model of neuropathic pain or a surgical sham model in order to demonstrate the application of this new methodology in CNS tissue samples.

Results
Significant increases in TLR4 mRNA expression and autofluorescence were visualized over time in mouse BV2 microglia or mouse J774 macrophage phenotype cells following lipopolysaccharide (LPS) stimulation. When imaged in a background-free environment with LISH-based detection and time-gated microscopy, increased TLR4 mRNA was observed in BV2 microglia cells 4 h following LPS stimulation, which returned to near baseline levels by 24 h. Background-free imaging of mouse spinal cord tissues with LISH-based detection and time-gated microscopy demonstrated a high degree of regional TLR4 mRNA expression in BALB/c mice with a chronic constriction model of neuropathic pain compared to the surgical sham model.

Conclusions
Advantages offered by adopting this novel methodology for visualizing neurokine signaling with time-gated microscopy compared to traditional fluorescent microscopy are provided.

New CNBP researcher at Macquarie University

25 January 2019:

CNBP is happy to announce its newest Research Fellow based at Macquarie University, Dr Simone De Camillis.

Simone will be working with CNBP Chief Investigator Prof. Jim Piper and his team to further explore the UCNP super-resolution technology for advanced biological applications, as well as collaborate across the wider CNBP community.

Simone completed his PhD at Queen’s University Belfast where he investigated ultrafast electrodynamics in nucleosides and aromatic amino acids. Studying the photo-chemistry of the building blocks of life is a fundamental step for understanding processes leading to mutation, damage and the alteration of the biological functions of the relative macro-molecule.

He has also worked as experimental physicist at the French Research Centre CEA in the optical characterisation of HgCdTe Infrared array detectors used for space and astrophysics applications.

His expertise includes femtosecond and attosecond laser technology, pump-probe interferometric systems, ultrafast molecular dynamics and time-of-flight mass spectroscopy.

Welcome to the team Simone!