A new technique, ‘bleaching-assisted multichannel microscopy’ (BAMM) takes a current long-standing weakness of fluorescence microscopy – photobleaching – and turns it into a strength that improves imaging output by up to three times, with no additional hardware required. Read more about this exciting development from CNBP researchers at the online channel PHYS ORG.
A novel technique developed by researchers at the ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP) will help shine new light on biological questions by improving the quality and quantity of information that can be extracted in fluorescence microscopy.
The technique, ‘bleaching-assisted multichannel microscopy’ (BAMM) takes a current long-standing weakness of fluorescence microscopy – photobleaching – and turns it into a strength that improves imaging output by up to three times, with no additional hardware required.
Reported in the journal ‘Biomedical Optics Express’ (lead author Dr Antony Orth, CNBP Research Fellow at RMIT University), BAMM will help researchers gain biological insights into the intricate processes taking place within living cells. This includes the interplay between proteins and molecules which have the potential to impact a wide range of health areas from fertility, to pain, to heart disease and more.
Publication authors: Antony Orth, Richik N. Ghosh, Emma R. Wilson, Timothy Doughney, Hannah Brown, Philipp Reineck, Jeremy G. Thompson, and Brant C. Gibson.
Below – This figure shows the information-rich cellular images made possible by using the newly reported BAMM technique. The ‘Original’ image shows cells containing multiple fluorescent targets, all having similar colours. This results in a monochrome image. With BAMM, photobleaching rates are colour coded red, green and blue for visualisation, so that each fluorescently labelled structure can be identified even though the fluorophore’s native colour information was never used.
Although many students commence their doctoral studies with the aim of being a university academic, statistics show that the percentage who become professors is only around 0.5%.
The RMIT node of the CNBP hosted an alternative careers workshop with five experts who shared their pathways from doctoral studies into the wider world.
Elliot Taranto completed a PhD in immunology and biology and now works in a technical and sales role at Olympus; Margie Beiharz completed a PhD in zoology and is now a freelance editor; Matthew Lay (pictured top left) undertook his PhD in semiconductor device fabrication and now works as a patent attorney; Shane Huntington’s PhD was in photonics and he is now the Deputy Director of the Melbourne Academic Centre for Health; and Victoria Coleman’s PhD was in semiconductor physics and she now leads the Nanometrology Section at the National Measurement Institute.
The panelists shared their pathways and the opportunities for research, interaction and fulfillment that their careers provided. Often stressed was how the ‘soft skills’ of writing, speaking, and collaboration played key roles in their success.
The session was chaired by CNBP Chief Investigator Andy Greentree.
A fully computational method for extending the depth of field of multicore optical fibers (MOF) imagers has been demonstrated by CNBP researchers in a new paper published in the journal ‘Optics Express’. The work shows that the depth of field can be more than doubled for certain spatial frequencies. Lead author on the publication is CNBP Research Fellow Dr Antony Orth from RMIT University.
Journal: Optics Express.
Publication title: Extended depth of field imaging through multicore optical fibers.
Authors: Antony Orth, Martin Ploschner, Ivan S. Maksymov, and Brant C. Gibson.
Abstract: Compact microendoscopes use multicore optical fibers (MOFs) to visualize hard-to-reach regions of the body. These devices typically have a large numerical aperture (NA) and are fixed-focus, leading to blurry images from a shallow depth of field with little focus control. In this work, we demonstrate a method to digitally adjust the collection aperture and therefore extend the depth of field of lensless MOF imaging probes. We show that the depth of field can be more than doubled for certain spatial frequencies, and observe a resolution enhancement of up to 78% at a distance of 50μm from the MOF facet. Our technique enables imaging of complex 3D objects at a comparable working distance to lensed MOFs, but without the requirement of lenses, scan units or transmission matrix calibration. Our approach is implemented in post processing and may be used to improve contrast in any microendoscopic probe utilizing a MOF and incoherent light.
Australian researchers from the ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP) have developed a 3D printable ‘clip-on’ that can turn any smartphone into a fully functional microscope.
Reported in the research journal ‘Scientific Reports’, the smartphone microscope is powerful enough to visualise specimens as small as 1/200th of a millimetre, including microscopic organisms, animal and plant cells, blood cells, cell nuclei and more.
The clip-on technology is unique in that it requires no external power or light source to work yet offers high-powered microscopic performance in a robust and mobile handheld package.
And the researchers are making the technology freely available, sharing the 3D printing files publicly so anyone – from scientists to the scientifically curious – can turn their own smartphones into microscopes.
Lead developer and CNBP Research Fellow at RMIT University, Dr Antony Orth (pictured), believes the technology has immense potential as a scientific tool, one that is ideal for use in remote areas and for field-work where larger standalone microscopes are unavailable or impractical.
“We’ve designed a simple mobile phone microscope that takes advantage of the integrated illumination available with nearly all smartphone cameras,” says Dr Orth.
The clip-on has been engineered with internal illumination tunnels that guide light from the camera flash to illuminate the sample from behind. This overcomes issues seen with other microscopy-enabled mobile phone devices says Dr Orth.
“Almost all other phone-based microscopes use externally powered light sources while there’s a perfectly good flash on the phone itself,” he explains. “External LEDs and power sources can make these other systems surprisingly complex, bulky and difficult to assemble.”
“The beauty of our design is that the microscope is useable after one simple assembly step and requires no additional illumination optics, reducing significantly the cost and complexity of assembly. The clip-on is also able to be 3D printed making the device accessible to anyone with basic 3D printing capabilities.”
A further advantage noted by Dr Orth is that the clip-on enables both bright-field and dark-field microscopy techniques to be undertaken. Bright-field microscopy is where a specimen is observed on a bright background. Conversely, dark-field shows the specimen illuminated on a dark background.
“The added dark-field functionality lets us observe samples that are nearly invisible under conventional bright-field operation such as cells in media,” he says. “Having both capabilities in such a small device is extremely beneficial and increases the range of activity that the microscope can be successfully used for.”
Dr Orth believes the potential applications for the smartphone microscope are enormous.
“Our mobile microscope can be used as an inexpensive and portable tool for all types of on-site or remote area monitoring.”
“Water quality, blood samples, environmental observation, early disease detection and diagnosis—these are all areas where our technology can be easily used to good effect.”
Dr Orth sees significant benefit in developing countries for the device.
“Powerful microscopes can be few and far between in some regions,” says Dr Orth. “They’re often only found in larger population centres and not in remote or smaller communities. Yet their use in these areas can be essential—for determining water quality for drinking, through to analysing blood samples for parasites, or for disease diagnosis including malaria.”
To ensure that this technology can be utilised the world over, the files for the 3D printing of the microscope clip-on are being made freely available. They are available for download at the CNBP web site – http://cnbp.org.au/online-tools.
“Ideally, a phone microscope should take advantage of the integrated flash found in nearly every modern mobile, avoiding the need for external lighting and power. It should also be as compact and easy to assemble as possible. It is this design philosophy that inspired us in the development of this add-on clip,” says Dr Orth.
The new phone microscope has already been tested by Dr Orth and his CNBP colleagues in a number of areas, successfully visualizing samples ranging from cell culture, to zooplankton to live cattle semen in support of livestock fertility testing.
Below: Cells being viewed by an add-on clip that turns a smartphone into a fully operational microscope.
Tiny 5 nm detonation nanodiamonds glow in different colors and their fluorescence is pH dependent, reports a new paper by CNBP scientists published today in the Nature journal Scientific Reports.
Lead author of the paper Dr Philipp Reineck from RMIT University (Former CNBP Research Fellow and current CNBP Associate Investigator) notes that the research is particulalry exciting as the fluorescence lifetime of the detonation nanodiamonds makes fluorescence lifetime imaging (FLIM) for bioimaging applications feasible.
Journal: Scientific Reports.
Authors: Philipp Reineck, Desmond W. M. Lau, Emma R. Wilson, Nicholas Nunn, Olga A. Shenderova & Brant C. Gibson.
Abstract: Detonation nanodiamonds are of vital significance to many areas of science and technology. However, their fluorescence properties have rarely been explored for applications and remain poorly understood. We demonstrate significant fluorescence from the visible to near-infrared spectral regions from deaggregated, single-digit detonation nanodiamonds dispersed in water produced via post-synthesis oxidation. The excitation wavelength dependence of this fluorescence is analyzed in the spectral region from 400 nm to 700 nm as well as the particles’ absorption characteristics. We report a strong pH dependence of the fluorescence and compare our results to the pH dependent fluorescence of aromatic hydrocarbons. Our results significantly contribute to the current understanding of the fluorescence of carbon-based nanomaterials in general and detonation nanodiamonds in particular.
New CNBP research determines that copper oxide nanocubes are suitable for long-term bioimaging experiments. Lead author on the paper – CNBP PhD student Zafisa Zohora (RMIT University).
Journal: Scientific Reports.
Authors: Nafisa Zohora, Ahmad Esmaielzadeh Kandjani, Antony Orth, Hannah M. Brown, Mark R. Hutchinson & Brant C. Gibson.
Conventional organic fluorophores lose their ability to fluoresce after repeated exposure to excitation light due to photobleaching. Therefore, research into emerging bright and photostable nanomaterials has become of great interest for a range of applications such as bio-imaging and tracking. Among these emerging fluorophores, metal oxide-based nanomaterials have attracted significant attention as a potential multifunctional material with photocatalytic and angeogenisis abilities in addition to fluorescnce applications. However, most of these applications are highly dependent on size, morphology, and chemo-physical properties of individual particles. In this manuscript, we present a method to study the intrinsic optical characteristics of individual copper (I) oxide (Cu2O) nanocubes. When excited at 520 nm using only 11 µW excitation power (1.7 W/cm2), individual nanocubes were observed to emit light with peak wavelengths ~760 nm which is conveniently within the near-infrared 1 (NIR1) biological window where tissue autofluorescence is minimal. Bright and photostable fluorescence was observed with intensities up to 487 K counts/s under constant illumination for at least 2 minutes with a brightness approximately four times higher than the autofluorescence from a fixed cumulus-oocyte complex. With near-IR emission, high fluorescence brightness, and outstanding photostability, Cu2O nanocubes are attractive candidates for long-term fluorescent bioimaging applications.
Fantastic TV talents (and CNBP researchers) Prof Brant Gibson and Dr Philipp Reineck from RMIT University featured on SCOPE TV for kids, 11th November 2017.
Check them out as they discuss the use of diamond nanoparticles in biophotonics to help shed light on cells and the living body!
CNBP researchers Dr Daniel Drumm (lead author pictured) and Prof Andrew Greentree, both at RMIT University, have analysed microscopy in the contexts of Rényi-Ulam games and half-lies, developing a new family of heuristics. Their research is reported in the journal ‘Scientific Reports.’
Journal: Scientific Reports.
Authors: Daniel W. Drumm & Andrew D. Greentree.
Abstract: Finding a fluorescent target in a biological environment is a common and pressing microscopy problem. This task is formally analogous to the canonical search problem. In ideal (noise-free, truthful) search problems, the well-known binary search is optimal. The case of half-lies, where one of two responses to a search query may be deceptive, introduces a richer, Rényi-Ulam problem and is particularly relevant to practical microscopy. We analyse microscopy in the contexts of Rényi-Ulam games and half-lies, developing a new family of heuristics. We show the cost of insisting on verification by positive result in search algorithms; for the zero-half-lie case bisectioning with verification incurs a 50% penalty in the average number of queries required. The optimal partitioning of search spaces directly following verification in the presence of random half-lies is determined. Trisectioning with verification is shown to be the most efficient heuristic of the family in a majority of cases.
Surface chemistry is vital for nanodiamond fluorescence, reports a new paper published by CNBP researchers (lead author Dr Philipp Reineck pictured). The paper was published in the journal ‘ACS Nano’ and is available online.
Journal: ACS Nano.
Publication title: Effect of Surface Chemistry on the Fluorescence of Detonation Nanodiamonds.
Authors: Philipp Reineck, Desmond W. M. Lau, Emma R. Wilson, Kate Fox, Matthew R. Field, Cholaphan Deeleepojananan, Vadym N. Mochalin, and Brant C. Gibson.
Abstract: Detonation nanodiamonds (DNDs) have unique physical and chemical properties that make them invaluable in many applications. However, DNDs are generally assumed to show weak fluorescence, if any, unless chemically modified with organic molecules. We demonstrate that detonation nanodiamonds exhibit significant and excitation-wavelength-dependent fluorescence from the visible to the near-infrared spectral region above 800 nm, even without the engraftment of organic molecules to their surfaces. We show that this fluorescence depends on the surface functionality of the DND particles. The investigated functionalized DNDs, produced from the same purified DND as well as the as-received polyfunctional starting material, are hydrogen, hydroxyl, carboxyl, ethylenediamine, and octadecylamine-terminated. All DNDs are investigated in solution and on a silicon wafer substrate and compared to fluorescent high-pressure high-temperature nanodiamonds. The brightest fluorescence is observed from octadecylamine-functionalized particles and is more than 100 times brighter than the least fluorescent particles, carboxylated DNDs. The majority of photons emitted by all particle types likely originates from non-diamond carbon. However, we locally find bright and photostable fluorescence from nitrogen-vacancy centers in diamond in hydrogenated, hydroxylated, and carboxylated detonation nanodiamonds. Our results contribute to understanding the effects of surface chemistry on the fluorescence of DNDs and enable the exploration of the fluorescent properties of DNDs for applications in theranostics as nontoxic fluorescent labels, sensors, nanoscale tracers, and many others where chemically stable and brightly fluorescent nanoparticles with tailorable surface chemistry are needed.