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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.


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



Finding a way to shutdown rogue cell replication

24 July 2019:

Almost all cells replace themselves by replicating, but when there are errors in DNA-replication, it can lead to diseases including many cancers.

DNA-replication is complex and involves a host of protein machinery. One of the most important is the protein PCNA, which helps orchestrate the process.

Adelaide University postgraduate student Aimee Horsfall, a member of the ARC Centre of Excellence for Nanoscale Biophotonics (CNBP), was part of the team which analysed the structures of a number of proteins interacting with PCNA.

The work suggests that the 3D shape of these proteins defines how strongly this interaction occurs.

The research is important because, if we can understand what makes the interaction with PCNA stronger, and determine the optimal shape, we can develop a drug that mimics it.

This drug could bind PCNA and stop replication in diseased cells, offering a potential treatment for diseases implicated in erroneous DNA-replication, or as a broad spectrum cancer therapeutic.

Journal: ChemBioChem

Publication Title: Targeting PCNA with peptide mimetics for therapeutic purposes.

Authors: Horsfall AJ, Abell AD, Bruning J.

Abstract: PCNA is an excellent inhibition target to shut down highly proliferative cells and thereby develop a broad spectrum cancer therapeutic. It interacts with a wide variety of proteins through a conserved motif referred to as the PCNA-Interacting Protein (PIP) box. There is large sequence diversity between high affinity PCNA binding partners, with conservation of the binding structure – a well-defined 310-helix. Here, all current PIP-box peptides crystallised with human PCNA are collated to reveal common trends between binding structure and affinity. Key intra- and inter-molecular hydrogen bonding networks which stabilise the 310-helix of PIP-box partners are highlighted, and related back to the canonical PIP-box motif. High correlation with the canonical PIP-box sequence does not directly afford high affinity. Instead, we summarise key interactions which stabilise the binding structure that lead to enhanced PCNA binding affinity. These interactions also implicate the ‘non-conserved’ residues within the PIP-box that have previously been overlooked. Such insights will allow a more directed approach to develop therapeutic PCNA inhibitors.

Keywords: PCNA, peptide mimetics, PIP-box, sliding clamp, DNA replication


Diamonds improve orthopaedic implants

17 July 2019:

3D printing of titanium has made patient-specific orthopaedic implants possible, promising to dramatically improve many people’s quality of life.

But, despite the huge potential, there are still significant problems to overcome, particularly in how the implants integrate with human tissue and bone.

Associate Professor Kate Fox from RMIT University in Melbourne, an Associate Investigator with the CNBP, led the team which, in a previous study, showed that a thin film coating of diamond could provide a better surface for cells to interact.

A new paper, Engineering the Interface: Nanodiamond Coating on 3D-Printed Titanium Promotes Mammalian Cell Growth and Inhibits Staphylococcus aureus Colonization expands on that work.

It describes how applying a nanodiamond (ND) coating on to the 3D printed titanium increased the cell density of both skin bone cells after three days of growth compared to the uncoated 3D printed titanium.

The study also showed an 88% reduction of Staphylococcus aureus – or Golden Staph – adherence to ND-coated substrates compared to those without.

This study, whose lead author is Aaquil Rifai, from RMIT, paves a way to create antifouling structures for biomedical implants.

You can read the paper here.

Journal: ACS Applied Materials & Interfaces

Publication Title:  Engineering the Interface: Nanodiamond Coating on 3D-Printed Titanium Promotes Mammalian Cell Growth and Inhibits Staphylococcus aureus Colonization

Authors: Aaqil Rifai*, Nhiem Tran, Philipp Reineck, Aaron Elbourne, Edwin Mayes, Avik Sarker, Chaitali Dekiwadia, Elena P. Ivanova, Russell J. Crawford, Takeshi Ohshima, Brant C. Gibsonm, Andrew D. Greentree, Elena Pirogova, and Kate Fox*

Abstract:  Additively manufactured selective laser melted titanium (SLM-Ti) opens the possibility of tailored medical implants for patients. Despite orthopedic implant advancements, significant problems remain with regard to suboptimal osseointegration at the interface between the implant and the surrounding tissue. Here, we show that applying a nanodiamond (ND) coating onto SLM-Ti scaffolds provides an improved surface for mammalian cell growth while inhibiting colonization of Staphylococcus aureus bacteria. Owing to the simplicity of our methodology, the approach is suitable for coating SLM-Ti geometries. The ND coating achieved 32 and 29% increases in cell density of human dermal fibroblasts and osteoblasts, respectively, after 3 days of incubation compared with the uncoated SLM-Ti substratum. This increase in cell density complements an 88% reduction in S. aureus detected on the ND-coated SLM-Ti substrata. This study paves a way to create facile antifouling SLM-Ti structures for biomedical implants.

Key Words: nanodiamond, antifouling, 3D printing, biomaterial, implants

Through the looking glass

Mark Hutchinson8 July 2019:

The paper, Stereochemistry and innate immune recognition, opens the door to potential future treatments for sepsis, chronic pain and other conditions that cause inflammation.

The paper’s origins can be traced back nearly 15 years to when CNBP Director Mark Hutchinson began work on a project as a post-doc in the US with Prof Linda Watkins’ team. The goal was to identify the molecular drivers and detection systems involved in causing chronic pain. It began a long journey, in the course of which Mark helped identify one of the detection systems – the Toll Like Receptor 4, or TLR4.

This discovery in turn uncovered a range of other detection and drug action properties of the TLR4 system, including the novel activity of the mirror image structures of a range of chemicals which had previously been thought to lack biological activity.

One of these new discoveries is highlighted in this paper.

For the first time, the mirror image of a well-used receptor blocker, norbinaltorphimine, has been found to be able to block the interaction of TLR4 with MD2, a protein that plays an important part in the body’s immune response.

You can read the paper here.

Journal: FASEB – the Federation of American Societies for Experimental Biology

Publication Title:  Stereochemistry and innate immune recognition: (+)-norbinaltorphimine targets myeloid differentiation protein 2 and inhibits toll-like receptor 4 signaling

Authors:  Xiaozheng Zhang, Yinghua Peng, Peter M. Grace, Matthew D. Metcalf, Andrew J. Kwilasz, Yibo Wang, Tianshu Zhang, Siru Wu, Brandon R. Selfridge, Philip S. Portoghese, Kenner C. Rice, Linda R. Watkins, Mark R. Hutchinson, and Xiaohui Wang

Abstract: Deregulation of innate immune TLR4 signaling contributes to various diseases including neuropathic pain and drug addiction. Naltrexone is one of the rare TLR4 antagonists with good blood-brain barrier permeability and showing no stereoselectivity for TLR4. By linking 2 naltrexone units through a rigid pyrrole spacer, the bivalent ligand norbinaltorphimine was formed. Interestingly, (+)-norbinaltorphimine ((+)-1) showed ∼25 times better TLR4 antagonist activity than naltrexone in microglia BV-2 cell line, whereas (−)-norbinaltorphimine ((−)-1) lost TLR4 activity. The enantioselectivity of norbinaltorphimine was further confirmed in primary microglia, astrocytes, and macrophages. The activities of meso isomer of norbinaltorphimine and the molecular dynamic simulation results demonstrate that the stereochemistry of (+)-1 is derived from the (+)-naltrexone pharmacophore. Moreover, (+)-1 significantly increased and prolonged morphine analgesia in vivo. The efficacy of (+)-1 is long lasting. This is the first report showing enantioselective modulation of the innate immune TLR signaling.

Key Words: norbinaltorphimine; enantioselective modulation; TLR4; MD-2; morphine analgesia

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

What is the potential for CRISPT/Cas Multiplex Biosensing?

29 May 2019:

Recent publication by CNBP PhD student Mr Yi Li and team at the University of New South Wales explores the challenges and opportunities of working with CRISPR /Cas for multiplex detection

Journal: Trends in Biotechnology

Publication TitleCRISPR/Cas Multiplexed Biosensing: A Challenge or an Insurmountable Obstacle?

Authors: Yi Li, Linyang Liu, Guozhen Liu

Abstract:  Performing multiplex detection is still an elusive goal for molecular diagnostics. CRISPR/Cas-based biosensing has demonstrated potential for multiplex detection. Instead of being an insurmountable obstacle, CRISPR/Cas multiplexed biosensing is a realistic challenge with some recent successful applications. Strategic considerations are required to fully explore its potential in multiplex diagnostics.

Key Words:

CRISPR/Cas; multiplex; biosensing; diagnostics; nucleic acid detection


Hemoglobin and its role in the oocyte and early embryo

6 May 2019:

Hemoglobin expression in reproductive cells and the role of hemoglobin on oocyte and early embryo development is the focus of this latest CNBP review paper published in the journal ‘Biology of Reproduction’ (lead author Megan Lim based at the University of Adelaide).

Journal: Biology of Reproduction.

Publication title: Hemoglobin: potential roles in the oocyte and early embryo.

Authors: Megan Lim, Hannah M Brown, Karen L Kind, Jeremy G Thompson, Kylie R Dunning.

Abstract: Hemoglobin (Hb) is commonly known for its capacity to bind and transport oxygen and carbon dioxide in erythroid cells. However, it plays additional roles in cellular function and health due to its capacity to bind other gases including nitric oxide. Further, Hb acts as a potent antioxidant, quenching reactive oxygen species. Despite its potential roles in cellular function, the preponderance of Hb research remains focused on its role in oxygen regulation. There is increasing evidence that Hb expression is more ubiquitous than previously thought, with Hb and its variants found in a myriad of cell types ranging from macrophages to spermatozoa. The majority of non-erythroid cell types that express Hb are situated within hypoxic environments, suggesting Hb may play a role in hypoxia-inducible factor (HIF)-regulated gene expression by controlling the level of oxygen available or as an adaptation to low oxygen providing a mechanism to store oxygen. Oocyte maturation and preimplantation embryo development occur within the low oxygen environments of the antral follicle and oviduct/uterus, respectively. Interestingly, Hb was recently found in human cumulus and granulosa cells and murine cumulus-oocyte complexes (COCs) and preimplantation embryos. Here, we consolidate and analyze the research generated to-date on Hb expression in non-erythroid cells with a particular focus on reproductive cell types. We outline future directions of this research to elucidate the role of Hb during oocyte maturation and preimplantation embryo development and finally, we explore the potential clinical applications and benefits of Hb supplementation during the in vitro culture of gametes and embryos.

New cytokine sensing device developed

1 May 2019:

A molecular imprinted polymer biosensing device (developed on stainless steel) that can successfully detect cytokines has been reported by CNBP researchers. Cytokines are proteins secreted by cells that stimulate surrounding cells into specific action and are important to an organism’s immune responses. The finding was reported in the journal ‘Sensors and Actuators B: Chemical’ with the lead author of the publication being CNBP’s Fei Deng based at UNSW Sydney.

Journal: Sensors and Actuators B: Chemical.

Publication title: Molecularly imprinted polymer-based reusable biosensing device on stainless steel for spatially localized detection of cytokine IL-1β.

Authors: Fei Deng, Ewa M. Goldys, Guozhen Liu.

Abstract: A molecularly imprinted polymer (MIP) based biosensing device on stainless steel (SS) for detection of locally variable concentration of cytokine interleukin-1β (IL-1β) was successfully developed using a sandwich assay scheme. The SS surface was firstly modified with a layer of polydopamine (PDA) followed by the attachment of a layer of poly(ethyleneimine) (PEI) by electrostatic adsorption. Subsequently, the template protein IL-1β was adsorbed on the PEI terminated SS surface due to electrostatic adsorption. A PDA imprinting film was then in-situ synthesized on the surface of the modified SS substrate with incorporated template cytokine. Finally, the template was washed off the SS substrate leaving behind cavities with specific shape and capable of capturing cytokines thus forming a MIP biosensing interface. After exposure to the analyte IL-1β, the MIP biosensing device was incubated with IL-1β detection antibody-modified fluorescent polystyrene beads allowing to determine the amount of captured IL-1β based on fluorescence intensity. The device has been demonstrated to detect IL-1β with low detection limit of 10.2 pg mL−1, and a linear detection range of 25–400 pg mL−1. This MIP biosensing device can be regenerated more than three times with coefficient of variation 2.08%. The device was applied for the detection of IL-1β secreted by rat macrophages, where the good specificity and selectivity were achieved. MIP serves in this device as a superior substitute of antibody with exceptional stability and reusability. The MIP based biosensing technology presented in our work paves a new way for developing a universal and robust sensing platform for the detection of spatially localised small proteins with low physical concentration.