Tag Archives: Denitza Denkova

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

CNBP science at the Calyx

12 September 2017:

CNBP researchers Dr Denitza Denkova and Dr Martin Ploschner took their luminescence and fluorescence science expertise to the general public, at a special after-hours event known as ‘Science at the Calyx’ at the Royal Botanic Garden in Sydney.

Presenting to an audience of approximately sixty people, the CNBP scientists focused on giving members of the public information about the origins of luminescence and examples of it being used – from everyday life to medical applications, and the amazing natural phenomenon of bioluminescence which can be found in plants, animals and fungi.

Following the hour long talk, there were demonstrations including the use of fluorescent bubbles, a ‘magical’ fluorescent screen, the showing of several fluorescent specimens and an examination of fluorescence in money and documents for security purposes. There was also as ample time for attendees to talk to the researchers about their work with fluorescent molecules and nanoparticles.

According to Dr Denkova, the event was highly rewarding.

“There was plenty of opportunity for personal interaction which was embraced by attendees. Many had an interest in the medical applications related to fluorescence, but there were also great questions on practical everyday activities – such as how to paint bikes with fluorescent paint to help improve road safety. Following the talk, people had the chance to walk around the beautiful garden with a UV torch in their hand to discover for themselves which plants are fluorescent. Both Martin and myself enjoyed communicating our science to a wider public!”

Physics in the pub!

cnbplogosquare120 June 2016:

Attendees at Sydney’s ‘Physics in the pub’ event were treated to a magical light-inspired show by CNBP researchers Martin Ploschner, Denitza Denkova and Varun Sreenivasan. Together they wowed the audience at the Three Wise Monkeys Hotel, using little more than UV light, fluorescent paint and other handy fluorescing materials.

Their act, one of a number on the night, aimed to take science out of the laboratory,  to take it to the public, and to make it educational, entertaining and fun in equal measure!

All three researchers enjoyed the experience of showcasing their science in a relaxed and informal environment, and quickly overcame any potential stage nerves to flaunt their fluro-physics to a full-house of engaged and interested members of the public.

Well done to all three – a short video of the fun-filled show can be viewed online!


MQ Uni hosts Coolangatta school students

192_denitza_denkova_WP12 February 2016:

CNBP Research Fellow Denitza Denkova has helped host a school visit to Macquarie University’s Physics and Astronomy Department.

The Year 12 science students, from Kingscliff High School, South Coolangatta, were shown a number of experiments and videos relevant to their Higher School Certificate study.

Explained were principles encompassing particle and wavelength duality, as well as the significance of the recent announcement related to the discovery of gravitational waves – a major event in the world of physics.

Supporting Science Experience 2016

Varun Sreenivasan_web15 January 2016:

CNBP researchers  Varun Sreenivasan and Denitza Denkova extended their outreach skills in support of the ConocoPhillips Science Experience program that took place at Macquarie University 13-15th January 2016.

The national program, focused on  Year 9 and 10 students who have an interest in science, is rolled-out across universities and tertiary institutions across Australia. Students who attend are provided with the opportunity to engage in a wide range of fascinating science activities under the guidance of scientists who love their work.

As a part of this program, Varun and Denitza contributed to the ‘Physics and Chemistry Magic Show’ which ran for an hour at Macquarie University’s Science Faculty. Demonstrated were concepts such as the ‘Stroboscopic effect’, the physics behind a ‘singing wineglass’ and the fun that can be had with Sulfur Hexafluoride.

“Through this event, we were able to share our excitement of science with youngsters, hopefully motivating them to do science as they grow up,”said Varun.

“We also improved our presentations and quick problem solving skills too!”


CNBP visits Belmont High School

Outreach-1ab13 November 2015:

CNBP researchers Andy Greentree, Denitza Denkova and Lindsay Parker took their science to the students on Friday Nov 13th, visiting Belmont High School for a fun filled day of outreach, incorporating science demonstrations, talks, informal discussion and chat.

First up was a 75 minute stage show in front of approximately 80 students from Years 8 and Year 9. Light as a concept was first explained, its wave and particle nature providing the basis for the session. A leaf-blower, table tennis balls, lasers, strobe lights and running water were also used, demonstrating the nature of light and its reflective, refractive and diffractive properties.

Nanodiamonds, iPhones, invisibility cloaks and glow sticks also made an appearance with the challenges and opportunities of looking deeply into the body and CNBP’s research mission clearly outlined.

This was followed by morning tea with the teaching staff and then a further talk and Q&A session with the school’s Year 12 chemistry class.

Thank you to all the students and teachers involved on the day. Great fun and extremely rewarding!

Nonlinear Physics Centre visit

192_denitza_denkova_WP4 May 2015:

CNBP Research Fellow Denitza Denkova, has given an invited seminar talk at the Nonlinear Physics Centre, part of the Australian National University (ANU), located in Canberra. The seminar focused on her PhD research, ‘Optical characterization of plasmonic nanostructures – imaging of the magnetic field of light’. Also discussed were her current research activities on super resolution, as well as potential collaboration possibilities. Denkova was also taken on a tour of the Centre’s lab facilities as part of a well received visit.

New Arrivals at the CNBP Macquarie Node

IMG_20150304_153824033_256 March 2015:

The power of three.

The CNBP Macquarie Node is growing rapidly with the arrival of three new Research Fellows last week.

Guozhen Liu has arrived from the Central China Normal University. She has over 10 year’s research experience at various universities, primarily in the areas of nanobiofabrication, biosensing and electrochemistry. She also has over 6 years’ institute experience, working on diabetes products. She will be working on cytokine assays, important in various parts of the CNBP. She will join Ewa’s team.

Kai Zhang, is a visiting scholar from Jilin University, China. Her background is in polymer chemistry and physics and her research field is the synthesis and application of composite nanoparticles, especially in drug delivery and gene delivery. She is supported by the China Scholarship Council and will be working with Ewa Goldys on molecular assays.

Denitza Denkova has recently finished her PhD at KU Leuven, Belgium, in the area of near-field microscopy and plasmonic nanoparticles. Here, she will be working with Prof. Jim Piper and Prof. Dayong Jin on the Olympus project, and will be responsible for the development and implementation of a low-power, low-cost STED microscopy system.

Welcome to all three of you – It’s great to have you all on board.