Tag Archives: Martin Ploschner

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

A kids focus at Sydney Science Festival

10 August 2018:

Over one hundred primary school children saw CNBP and Macquarie University researchers Dr Martin Ploschner (pictured) and Dr Annemarie Nadort present fun-filled light-focused science demonstrations at the Australian Museum as a part of National Science Week and the Sydney Science Festival for 2018.

Dr Martin Ploschner demonstrated how every-day items such as soap, detergent, money, and identity documents will glow or fluoresce when UV light is shone on them. Also demonstrated was the ‘glow affect’ from natural organisms such as scorpions, green leaves and bacteria on pistachios.

Dr Annemarie Nadort showed the children how they could see a network of blood vessels in their own tongue with a special microscope camera, facilitating an understanding of the human body and the tools needed to be able to see within it.

“The kids were amazed by seeing the continuous flow of red blood cells in the vessels. They were described as being like ‘in a rollercoaster’ or ‘like little ants walking on paths’, said Dr Nadort.

“It was great to see the excitement and interest from kids as young as six at our stand. Hopefully we managed to play a small role in promoting an ongoing interest in science in these bright and eager minds,” she said.

Below – Dr Annemarie Nadort and Dr Martin Ploschner demonstrate the wonders of science to children at the Australian Museum.

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!”

Outreach fun linked to science

10 June 2017:

Dr Martin Ploschner, CNBP Researcher, has taken his outreach skills to the Czech and Slovak School, Sydney where he performed several hands-on science shows for approximately 100 students, all aged 10 and under.

The show connected fun light-based activities with CNBP science and included the creation of gigantic fluorescent bubbles as well as the use of fluorescent screens that were able to be used as canvas that could be ‘painted on’ with light.

“I had a great time at the school and the activities were very well received,” says Martin.

“The younger kids had fun and the older children asked a lot of questions about the science behind the show. As an added bonus, I was invited back for further school open days as well!”

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!

Physicsinthepub_photo

Newest CNBP recruit publishes in Nature Photonics

Martin Ploschner web14 July 2015:

Our newest CNBP recruit, Dr Martin Ploschner, has just been published in the prestigious journal Nature Photonics.

The research, titled “Seeing through chaos in multimode fibres” is a study from his previous role at the University of Dundee and could open up a new era of superfast communications technology and lead to breakthroughs in the treatment of medical conditions.

The paper can be accessed here: http://rdcu.be/dmRm

The University of Dundee media release available here: http://www.dundee.ac.uk/news/2015/breakthrough-in-fibre-optics-opens-up-possibility-of-better-understanding-of-disease-and-communication-revolution.php

CNBP’s newest Research Fellow

Martin Ploschner web1 July 2015:

CNBP welcomes its newest Research Fellow – Dr Martin Ploschner.

Located at Sydney’s Macquarie University, Martin will be working in Prof Ewa Goldys’ team, working on the development of novel imaging tools (using computer holography) for imaging of fragile biological systems (such as early stage mouse embryos). He will also be developing new illumination methods based on compressing sensing for applications in biology.

Looking forward to his time with the CNBP, Martin’s previous role was at the University of Dundee, Scotland, working with Dr Tomas Cizmar. His work  involved the development of new techniques that pave the way for the use of multimode fibre as a miniature substitute to a standard microscope objective.

Welcome to the CNBP team Martin!