Tag Archives: PeerRev

New light source developed for fluorescent microscopy imaging

Aziz Rehman16 October 2017:

A tuneable and programmable integrating sphere light source for wide-field fluorescent microscopy imaging, employing nine light-emitting diodes (LEDs), has been successfully demonstrated and reported by CNBP researchers in a new paper (lead author Aziz ul Rehman pictured).

The paper, published in ‘Photodiagnosis and Photodynamic Therapy’ is accessible online.

Journal: Photodiagnosis and Photodynamic Therapy.

Publication title: Programmable LED-Based Integrating Sphere Light Source for Wide-Field Fluorescence Microscopy.

Authors: Aziz ul Rehman, Ayad G.Anwer, Ewa M.Goldys.

Abstract: Wide-field fluorescence microscopy commonly uses a mercury lamp, which has limited spectral capabilities. We designed and built a programmable integrating sphere light (PISL) source which consists of nine LEDs, light-collecting optics, a commercially available integrating sphere and a baffle. The PISL source is tuneable in the range 365–490 nm with a uniform spatial profile and a sufficient power at the objective to carry out spectral imaging. We retrofitted a standard fluorescence inverted microscope DM IRB (Leica) with a PISL source by mounting it together with a highly sensitive low- noise CMOS camera. The capabilities of the setup have been demonstrated by carrying out multispectral autofluorescence imaging of live BV2 cells.

Nanorubies for targeted bio-imaging

12 October 2017:

Researchers from the CNBP have released a new paper that examines the use of nanorubies for targeted bio-imaging activity. The work (lead author Varun Sreenivasan pictured) is trans-disciplinary in nature, drawing on the Centre’s collective knowledge in physics, pharmacology, chemistry, material science and embryology. The paper, published in ACS Applied Materials and Interfaces is accessible online.

Journal: ACS Applied Materials and Interfaces.

Publication title: Development of Bright and Biocompatible Nanoruby and its Application to Background-free Time-gated Imaging of G-protein Coupled Receptors.

Authors:  Varun K. A. Sreenivasan, Wan Aizuddin W Razali, Kai Zhang, Rashmi R Pillai, Avishkar Saini, Denitza Denkova, Marina Santiago, Hannah Brown, Jeremy Thompson, Mark Connor, Ewa M. Goldys, and Andrei V Zvyagin.

Abstract: At the forefront of development of fluorescent probes for biological imaging applications are enhancements aimed at increasing their brightness, contrast, and photostability, especially towards demanding applications of single molecule detection. In comparison with existing probes, nanorubies exhibit unlimited photostability and a long emission lifetime (3.7 ms), which enable continuous imaging at single-particle sensitivity in highly scattering and fluorescent biological specimens. However, their wide application as fluorescence probes has so far been hindered by the absence of facile methods for scaled-up high volume production and molecularly-specific targeting. The present work encompasses large scale production of colloidally stable nanoruby particles, demonstration of their biofunctionality and negligible cytotoxicity, as well as validation of its use for targeted biomolecular imaging. In addition, optical characteristics of nanorubies are found to be comparable or superior to state-of-the-art quantum dots. Protocols of reproducible and robust coupling of functional proteins to the nanoruby surface are also presented. As an example, NeutrAvidin-coupled nanoruby show excellent affinity and specificity to µ-opioid receptors in fixed and live cells, allowing wide-field imaging of G-protein coupled receptors with single particle sensitivity.

New med-tech zinc sensor developed

27 September 2017:

A new zinc sensor has been developed and reported by CNBP researchers, which will allow for a deeper understanding of the dynamic roles that metal ions play in regulating health and disease in the living body.

The research, published in the journal ‘ACS Omega’ reports that the newly designed chemical sensor can detect and measure zinc levels in cells. It also has the functionality and portability to take continuous or repeated measurements within a single biological sample.

“This makes the sensor potentially suitable for use in future diagnostic tools that could open up entirely new windows into the body,” says lead author of the research Dr Sabrina Heng (pictured), Research Fellow at the ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), at the University of Adelaide.

Read the full CNBP media release here and the publication here.

Journal: ACS Omega.

Publication title: A Rationally Designed Probe for Reversible Sensing of Zinc and Application in Endothelial Cells.

Authors: Sabrina Heng, Philipp Reineck, Achini K. Vidanapathirana, Benjamin J. Pullen, Daniel W. Drumm, Lesley J. Ritter, Nisha Schwarz, Claudine S. Bonder, Peter J. Psaltis, Jeremy G. Thompson, Brant C. Gibson , Stephen J. Nicholls, and Andrew D. Abell.

Abstract: Biologically compatible fluorescent ion sensors, particularly those that are reversible, represent a key tool for answering a range of fundamental biological questions. We report a rationally designed probe with a 6′-fluoro spiropyran scaffold (5) for the reversible sensing of zinc (Zn2+) in cells. The 6′-fluoro substituent overcomes several limitations normally associated with spiropyran-based sensors to provide an improved signal-to-background ratio and faster photoswitching times in aqueous solution. In vitro studies were performed with 5 and the 6′-nitro analogues (6) in HEK 293 and endothelial cells. The new spiropyran (5) can detect exogenous Zn2+ inside both cell types and without affecting the proliferation of endothelial cells. Studies were also performed on dying HEK 293 cells, with results demonstrating the ability of the key compound to detect endogenous Zn2+ efflux from cells undergoing apoptosis. Biocompatibility and photoswitching of 5 were demonstrated within endothelial cells but not with 6, suggesting the future applicability of sensor 5 to study intracellular Zn2+ efflux in these systems.

New technique to aid bladder cancer diagnosis

25 September 2017:

A new and innovative automated computer technique has been developed by CNBP researchers that is able to significantly aid in the diagnosis of bladder cancer.

The technique—which allows suspect lesion images to be quickly and effectively analysed and then classified for cancer risk, has been reported in the medical journal ‘Urologic Oncology’.

“What we’ve done is develop a computer program to carry out an automated analysis of cystoscopy images,” says lead author of the research, Dr Martin Gosnell, Researcher at the ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP) at Macquarie University and Director at Quantitative Pty Ltd.

Cystoscopy is one of the most reliable methods for diagnosing bladder cancer explains Dr Gosnell.

“Images are taken of the bladder and its insides for suspicious lesions during a routine clinical patient evaluation. Dependent on the findings, this initial scan can then be followed up by a referral to a more experienced urologist, and a biopsy of the suspicious tissue can be undertaken.”

The issue says Dr Gosnell is that the clinician examining the initial images makes a visual judgement based on their professional expertise as to the next steps of action that should be undertaken—such as the need to take a biopsy for subsequent pathological analysis.

“Potential errors and unnecessary further interventions may result from the subjective character of this initial visual assessment.”

“What we’ve done,” says Dr Gosnell, “is to create an automated image analysis technique which can identify tissue and lesions as either high-risk or minimal-risk.”

Read the full CNBP media release and the science paper here.

Journal: Urologic Oncology.

Publication title: Computer-assisted cystoscopy diagnosis of bladder cancer.

Authors: Martin E. Gosnell (pictured top), Dmitry M. Polikarpov, Ewa M. Goldys, Andrei V. Zvyagin and David A. Gillatt.

Abstract:

Objectives

One of the most reliable methods for diagnosing bladder cancer is cystoscopy. Depending on the findings, this may be followed by a referral to a more experienced urologist or a biopsy and histological analysis of suspicious lesion. In this work, we explore whether computer-assisted triage of cystoscopy findings can identify low-risk lesions and reduce the number of referrals or biopsies, associated complications, and costs, although reducing subjectivity of the procedure and indicating when the risk of a lesion being malignant is minimal.

Materials and methods

Cystoscopy images taken during routine clinical patient evaluation and supported by biopsy were interpreted by an expert clinician. They were further subjected to an automated image analysis developed to best capture cancer characteristics. The images were transformed and divided into segments, using a specialised color segmentation system. After the selection of a set of highly informative features, the segments were separated into 4 classes: healthy, veins, inflammation, and cancerous. The images were then classified as healthy and diseased, using a linear discriminant, the naïve Bayes, and the quadratic linear classifiers. Performance of the classifiers was measured by using receiver operation characteristic curves.

Results

The classification system developed here, with the quadratic classifier, yielded 50% false-positive rate and zero false-negative rate, which means, that no malignant lesions would be missed by this classifier.

Conclusions

Based on criteria used for assessment of cystoscopy images by medical specialists and features that human visual system is less sensitive to, we developed a computer program that carries out automated analysis of cystoscopy images. Our program could be used as a triage to identify patients who do not require referral or further testing.

Below: Dr Martin Gosnell and Prof Ewa Goldys.

First reversible ‘turn-off’ sensor for Glutathione

6 September 2017:

The first reversible ‘turn-off’ sensor for Glutathione has been reported by CNBP researchers in a paper published in the science journal Biosensors.

The paper is accessible online (open access).

Dr Sabrina Heng notes:

γ-Glutamyl-cysteinyl-glycine (GSH) plays a critical role in maintaining redox homeostasis in biological systems and a decrease in its cellular levels is associated with disease. Many diseases including Parkinson’s, cancer, heart diseases and Alzheimer’s are indicated by a decrease in GSH levels. In this case, a ‘turn on’ sensor would result in reduced fluorescence relative to healthy cells. An important advance would come from the development of a sensor that is measurably turned off by GSH and back on by a lower level of GSH. This would then provide an opportunity to sense reduced GSH levels during the onset of important diseases.

With that in mind we have rationally designed, to the best of our knowledge, the first reversible, reaction-based ‘turn-off’ probe that is suitable for sensing decreasing levels of GSH, a situation known to occur at the onset of various diseases.  We have demonstrated that the sensor can be used to detect changes of intracellular GSH in live HEK 293 cells to provide a potentially regenerable sensor for monitoring lower levels of intracellular GSH as associated with the onset of important diseases.

Journal: Biosensors.

Publication title: A Rationally Designed Reversible ‘Turn-Off’ Sensor for Glutathione.

Authors: Sabrina Heng (pictured), Xiaozhou Zhang, Jinxin Pei and Andrew D. Abell.

Abstract: γ-Glutamyl-cysteinyl-glycine (GSH) plays a critical role in maintaining redox homeostasis in biological systems and a decrease in its cellular levels is associated with diseases. Existing fluorescence-based chemosensors for GSH acts as irreversible reaction-based probes that exhibit a maximum fluorescence (‘turn-on’) once the reaction is complete, regardless of the actual concentration of GSH. A reversible, reaction-based ‘turn-off’ probe (1) is reported here to sense the decreasing levels of GSH, a situation known to occur at the onset of various diseases. The more fluorescent merocyanine (MC) isomer of 1 exists in aqueous solution and this reacts with GSH to induce formation of the ring-closed spiropyran (SP) isomer, with a measurable decrease in absorbance and fluorescence (‘turn-off’). Sensor 1 has good aqueous solubility and shows an excellent selectivity for GSH over other biologically relevant metal ions and aminothiol analytes. The sensor permeates HEK 293 cells and an increase in fluorescence is observed on adding buthionine sulfoximine, an inhibitor of GSH synthesis.

Exploring small-sized nanoflakes

29 August 2017:

Size-dependent structural and electronic properties of MoSmonolayer nanoflakes, of sizes up to 2nm, have been investigated by CNBP researchers using density-functional theory (DFT). The paper, published in Scientific Reports is accessible online.

Journal: Scientific Reports.

Publication title: A study of size-dependent properties of MoSmonolayer nanoflakes using density-functional theory.

Authors: M. Javaid (pictured), Daniel W. Drumm, Salvy P. Russo & Andrew D. Greentree.

Abstract: Novel physical phenomena emerge in ultra-small sized nanomaterials. We study the limiting small-size-dependent properties of MoS2 monolayer rhombic nanoflakes using density-functional theory on structures of size up to Mo35S70 (1.74 nm). We investigate the structural and electronic properties as functions of the lateral size of the nanoflakes, finding zigzag is the most stable edge configuration, and that increasing size is accompanied by greater stability. We also investigate passivation of the structures to explore realistic settings, finding increased HOMO-LUMO gaps and energetic stability. Understanding the size-dependent properties will inform efforts to engineer electronic structures at the nano-scale.

New technique to aid IVF embryo selection

28 August 2017:

Researchers at the ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP) have successfully developed an advanced new imaging technique, which can help assess the quality of early-stage embryos.

The research, reported in the journal ‘Human Reproduction’ has the potential to significantly benefit the IVF industry of the future, improving assisted reproduction outcomes for women.

“We use a special type of imaging to show differences in the metabolism and chemical make-up of embryos before they’ve been implanted,” says lead author Dr Mel Sutton-McDowall (pictured).

“This technique can give us an objective measure of which embryo to choose as part of the IVF process.”

This ‘hyperspectral imaging’ measures light that cells naturally produce during their normal activities. The light or ‘autoflorescence’ produced changes according to the chemical reactions or metabolism going on in the cell.

Being able to measure embryo metabolism is viewed by many researchers as one of the most important factors as to whether a particular IVF program will be successful.

However, says Dr Sutton-McDowall, fertility specialists take a largely subjective approach in deciding which embryos should be used.

“Pre-implantation screening of embryos generally takes place under a normal optical microscope. Although it’s quite easy to discern poor embryos (due to differences in uniformity), it is far harder for the clinician to determine objectively, the viability of the other embryos,” she says.

“The challenge is how to choose the single healthiest embryo out of this group to maximise the chances of pregnancy.”

Dr Sutton-McDowall sees the use of hyperspectral imaging as a new tool that can be combined with other diagnostic methods to provide a more accurate and objective embryo viability assessment.

“The benefit of hyperspectral imaging is that it can capture information-rich content of inspected objects. It analyses every pixel in an image for its light intensity at differing wavelengths,” she says

“This lets us drill down and analyse the hyperspectral signature of each individual embryo, looking for known or anomalous characteristics. It lets us discriminate between embryos, but also measuring metabolic differences within individual embryos. We predict that embryos that have cells with homogeneous (uniform) metabolic profiles are the healthier ones.”

To date, this imaging technology has only been tested on cattle embryos but Dr Sutton-McDowall notes that the technique is extremely promising.

“It offers benefits of being a non-invasive imaging approach that provides real-time information to the clinician,” she says.

The paper is accessible online.

Journal: Human Reproduction.

Publication title: Hyperspectral microscopy can detect metabolic heterogeneity within bovine post-compaction embryos incubated under two oxygen concentrations (7% versus 20%).

Authors: Melanie L. Sutton-McDowall, Martin Gosnell, Ayad G. Anwer, Melissa White, Malcolm Purdey, Andrew D. Abell, Ewa M. Goldys, Jeremy G. Thompson.

Abstract: 

STUDY QUESTION
Can we separate embryos cultured under either 7% or 20% oxygen atmospheres by measuring their metabolic heterogeneity?

SUMMARY ANSWER
Metabolic heterogeneity and changes in metabolic profiles in morula exposed to two different oxygen concentrations were not detectable using traditional fluorophore and two-channel autofluorescence but were detectable using hyperspectral microscopy.

WHAT IS KNOWN ALREADY
Increased genetic and morphological blastomere heterogeneity is associated with compromised developmental competence of embryos and currently forms the basis for embryo scoring within the clinic. However, there remains uncertainty over the accuracy of current techniques, such as PGS and time-lapse microscopy, to predict subsequent pregnancy establishment.

STUDY DESIGN, SIZE, DURATION
The impact of two oxygen concentrations (7% = optimal and 20% = stressed) during post-fertilisation embryo culture was assessed. Cattle embryos were exposed to the different oxygen concentrations for 8 days (D8; embryo developmental competence) or 5 days (D5; metabolism measurements). Between 3 and 4 experimental replicates were performed, with 40–50 embryos per replicate used for the developmental competency experiment, 10–20 embryos per replicate for the fluorophore and two-channel autofluorescence experiments and a total of 21–22 embryos used for the hyperspectral microscopy study.

PARTICIPANTS/MATERIALS, SETTING, METHODS
In-vitro produced (IVP) cattle embryos were utilised for this study. Post-fertilisation, embryos were exposed to 7% or 20% oxygen. To determine impact of oxygen concentrations on embryo viability, blastocyst development was assessed on D8. On D5, metabolic heterogeneity was assessed in morula (on-time) embryos using fluorophores probes (active mitochondria, hydrogen peroxide and reduced glutathione), two-channel autofluorescence (FAD and NAD(P)H) and 18-channel hyperspectral microscopy.

MAIN RESULTS AND THE ROLE OF CHANCE
Exposure to 20% oxygen following fertilisation significantly reduced total blastocyst, expanded and hatched blastocyst rates by 1.4-, 1.9- and 2.8-fold, respectively, compared to 7% oxygen (P < 0.05), demonstrating that atmospheric oxygen was a viable model for studying mild metabolic stress. The metabolic profiles of D5 embryos was determined and although metabolic heterogeneity was evident within the cleavage stage (i.e. arrested) embryos exposed to fluorophores, there were no detectable difference in fluorescence intensity and pattern localisation in morula exposed to the two different oxygen concentrations (P > 0.05). While there were no significant differences in two-channel autofluorescent profiles of morula exposed to 7% and 20% oxygen (main effect, P > 0.05), morula that subsequently progressed to the blastocyst stage had significantly higher levels of FAD and NAD(P)H fluorescence compared to arrested morula (P < 0.05), with no change in the redox ratio. Hyperspectral autofluorescence imaging (in 18-spectral channels) of the D5 morula revealed highly significant differences in four features of the metabolic profiles of morula exposed to the two different oxygen concentrations (P < 0.001). These four features were weighted and their linear combination revealed clear discrimination between the two treatment groups.

LIMITATIONS, REASONS FOR CAUTION
Metabolic profiles were assessed at a single time point (morula), and as such further investigation is required to determine if differences in hyperspectral signatures can be detected in pre-compaction embryos and oocytes, using both cattle and subsequently human models. Furthermore, embryo transfers should be performed to determine the relationship between metabolic profiles and pregnancy success.

WIDER IMPLICATIONS OF THE FINDINGS
Advanced autofluorescence imaging techniques, such as hyperspectral microscopy, may provide clinics with additional tools to improve the assessment of embryos prior to transfer.

Nano-diamond arrays on glass

23 August 2017:

Researchers from CNBP’s RMIT University node (lead author Ashleigh Heffernan), have published a paper demonstrating a directed self-assembly method to position nanodiamonds on glass. The method, allowing for the statistical quantification of fluorescent nanoparticles provides a step towards fabrication of hybrid photonic devices for applications from quantum cryptography to sensing.

The paper is accessible online.

Journal: Scientific Reports.

Publication title: Nanodiamond arrays on glass for quantification and fluorescence characterisation.

Authors: Ashleigh H. Heffernan, Andrew D. Greentree & Brant C. Gibson.

Abstract: Quantifying the variation in emission properties of fluorescent nanodiamonds is important for developing their wide-ranging applicability. Directed self-assembly techniques show promise for positioning nanodiamonds precisely enabling such quantification. Here we show an approach for depositing nanodiamonds in pre-determined arrays which are used to gather statistical information about fluorescent lifetimes. The arrays were created via a layer of photoresist patterned with grids of apertures using electron beam lithography and then drop-cast with nanodiamonds. Electron microscopy revealed a 90% average deposition yield across 3,376 populated array sites, with an average of 20 nanodiamonds per site. Confocal microscopy, optimised for nitrogen vacancy fluorescence collection, revealed a broad distribution of fluorescent lifetimes in agreement with literature. This method for statistically quantifying fluorescent nanoparticles provides a step towards fabrication of hybrid photonic devices for applications from quantum cryptography to sensing.

Want a better camera? Just copy bees!

4 July 2017:

Check out the latest buzz about bees and their extra light-sensing eyes! CNBP CI Prof Andy Greentree is coauthor on a new paper in PNAS, which identifies how the eyes and brains of honeybees work together, to process colour information.

“If we can design technology to mimic the way bees do this, we’ll be able to create better cameras and image-processing systems for drones and robots,” say the researchers in an article on the science news channel ‘The Conversation‘.

 

Enhancement of the NV quantum yield

3 July 2017:

Researchers from CNBP’s RMIT University node (lead author CNBP PhD student Marco Capelli pictured), have had a paper published in the journal ‘Nanoscale’.

The researchers report an enhancement of the nitrogen-vacancy (NV) quantum yield by up to 7% in bulk diamond caused by an external magnetic field.

The paper is accessible online.

Journal: Nanoscale.

Publication title: Magnetic field-induced enhancement of the nitrogen-vacancy fluorescence quantum yield .

Authors: M. Capelli, P. Reineck, D. W. M. Lau, A. Orth, J. Jeske, M. W. Doherty, T. Ohshima, A. D. Greentree and B. C. Gibson.

Abstract: The nitrogen-vacancy (NV) centre in diamond is a unique optical defect that is used in many applications today and methods to enhance its fluorescence brightness are highly sought after. We observed experimentally an enhancement of the NV quantum yield by up to 7% in bulk diamond caused by an external magnetic field relative to the field-free case. This observation is rationalised phenomenologically in terms of a magnetic field dependence of the NV excited state triplet-to-singlet transition rate. The theoretical model is in good qualitative agreement with the experimental results at low excitation intensities. Our results significantly contribute to our fundamental understanding of the photophysical properties of the NV defect in diamond.