Tag Archives: microscopy

Label-free real-time tracking of individual bacterium

Images from the optical microscope showing the tracks of bacteria interacting with a surfaceAntimicrobial resistant (AMR) infections are already the third leading cause of death in the USA and are predicted to kill 50 million people per year by 2050.  It is the next pandemic starting already.  We have been using our capability to track nanoparticles in an optical microscope [see ‘Slow moving nanoparticles‘ on December 13th, 2017 and ‘Nano biomechanical engineering of agent delivery to cells‘ on December 15th, 2021] to track individual bacterium as they interact with surfaces to form biofilms.  Bacterial biofilms are complex colonies of bacteria that are highly resistant to antimicrobial agents and can cause life-threatening infections.  We have used our label-free, real-time tracking capabilities to explore the dynamics and adhesion of bacteria to surfaces and found that viable bacteria adhered to the surface but continue to move with rotary or sliding motions depending on the mechanics of their attachment to the surface.  Bacteria that were killed by contact with the surface did not move once they were attached to the surface.  The image shows examples of these motions from our paper published last month.  Our ability to detect these differences in the dynamics of bacteria will allow us to detect the onset of the formation of biofilms and to quantify the efficacy of antimicrobial surfaces and coatings.

Image: Figure 4 – Tracks (yellow lines) of the sections (purple circles) of four E. coli bacteria experiencing: (a) random diffusion above the surface; (b) rotary attachment; (c) lateral attachment; (d) static attachment. The dynamics of the four bacteria was monitored for approximately 20 s. The length of the scale bars is 5 μm. From Scientific Reports, 12:18146, 2022.

Source:

Giorgi F, Curran JM & Patterson EA, Real-time monitoring of the dynamics and interactions of bacteria and the early-stage formation of biofilms, Scientific Reports, 12:18146, 2022.

Thinking out of the box leads to digital image correlation through space

This is the third in a short series of posts on recent engineering research published by my research group.  Actually, two have already been published: ‘Salt increases nanoparticle diffusion‘ on April 22nd, 2020; and ‘Spatio-temporal damage maps for composite materials‘ on May 6th, 2020 and then I got distracted.  This third one arose from the same project as the time-damage maps which was sponsored by the United States Air Force.  The time-damage maps allow us to explore the evolution of failure in complex materials; however, we already know that damage tends to initiate from imperfections or flaws in the microstructure in the material.  New continuous fibre reinforced composite (CFRC) materials based on ceramics are very sensitive to defects or anomalies in their microstructure, such as misalignment of fibres.  However, they are capable of withstanding temperatures in excess of 1500 degrees Centigrade, which offers the opportunity to use them in jet engines or nuclear power plants to help generate energy more efficiently.  Therefore, it is worthwhile investigating effective methods of inspecting their microstructure which we can do either destructively by repetitively polishing away the surface of a sample and viewing it in a microscope, or non-destructively using x-ray tomography.  In both cases, the result is hundreds of ‘images’ containing millions of data values from which it is challenging to extract useful information.  In our work, we have used a little lateral thinking, to show how digital image correlation, usually used to track deformation of structures using multiple images collected over time [see ‘256 shades of grey‘ on January 22nd, 2014] , can be used to track fibres through the multiple images of the layers of the microstructure.  The result is the sort of ‘stick’ diagram in the image showing the orientation of fibres through the sample.  We have demonstrated that our new algorithm was more reliable and 30 times faster than its nearest rival.

The image shows, at the top, a typical stack of images from the microscope of a ceramic matrix composite; and, at the bottom, a plot of 3d profiles of the fibres tracked using the DIC-based method with the fibres orientated nominally at ±45° from the sectioning (x-y) plane shown in red and green colours.

Source:

Amjad K, Christian WJR, Dvurecenska K, Chapman MG, Uchic MD, Przybyla CP & Patterson EA, Computationally efficient method of tracking fibres in composite materials using digital image correlation, Composites Part A, 129:105683, 2020.

 

Size matters

Most of us have a sub-conscious understanding of the forces that control the interaction of objects in the size scale in which we exist, i.e. from millimetres through to metres.  In this size scale gravitational and inertial forces dominate the interactions of bodies.  However, at the size scale that we cannot see, even when we use an optical microscope, the forces that the dominate the behaviour of objects interacting with one another are different.  There was a hint of this change in behaviour observed in our studies of the diffusion of nanoparticles [see ‘Slow moving nanoparticles‘ on December 13th, 2017], when we found that the movement of nanoparticles less than 100 nanometres in diameter was independent of their size.  Last month we published another article in one of the Nature journals, Scientific Reports, on ‘The influence of inter-particle forces on diffusion at the nanoscale‘, in which we have demonstrated by experiment that Van der Waals forces and electrostatic forces are the dominant forces at the nanoscale.  These forces control the diffusion of nanoparticles as well as surface adhesion, friction and colloid stability.  This finding is significant because the ionic strength of the medium in which the particles are moving will influence the strength of these forces and hence the behaviour of the nanopartices.  Since biological fluids contain ions, this will be important in understanding and predicting the behaviour of nanoparticles in biological applications where they might be used for drug delivery, or have a toxicological impact, depending on their composition.

Van der Waals forces are weak attractive forces between uncharged molecules that are distance dependent.  They are named after a Dutch physicist, Johannes Diderik van der Waals (1837-1923).  Electrostatic forces occur between charged particles or molecules and are usually repulsive with the result that van der Waals and electrostatic forces can balance each other, or not depending on the circumstances.

Sources:

Giorgi F, Coglitore D, Curran JM, Gilliland D, Macko P, Whelan M, Worth A & Patterson EA, The influence of inter-particle forces on diffusion at the nanoscale, Scientific Reports, 9:12689, 2019.

Coglitore D, Edwardson SP, Macko P, Patterson EA, Whelan MP, Transition from fractional to classical Stokes-Einstein behaviour in simple fluids, Royal Society Open Science, 4:170507, 2017. doi: .

Patterson EA & Whelan MP, Tracking nanoparticles in an optical microscope using caustics. Nanotechnology, 19 (10): 105502, 2009.

Image: from Giorgi et al 2019, figure 1 showing a photograph of a caustic (top) generated by a 50 nm gold nanoparticle in water taken with the optical microscope adjusted for Kohler illumination and closing the condenser field aperture to its minimum following method of Patterson and Whelan with its 2d random walk over a period of 3 seconds superimposed and a plot of the same walk (bottom).

Assessing nanoparticle populations in historic nuclear waste

Together with colleagues at the JRC Ispra, my research group has shown that the motion of small nanoparticles at low concentrations is independent of their size, density and material [1], [see ‘Slow moving nanoparticles‘ on December 13th, 2017].  This means that commercially-available instruments for evaluating the size and number of nanoparticles in a solution will give erroneous results under certain conditions.  In a proposed PhD project, we are planning to extend our work to develop an instrument with capability to automatically identify and size nanoparticles, in the range from 1 to 150 nanometres, using the three-dimensional optical signature, or caustic, which particles generate in an optical microscope, that can be several orders of magnitude larger than the particle [2],  [see ‘Toxic nanoparticles?‘ on November 13th, 2013].  The motivation for the work is the need to characterise particles present in solution in legacy ponds at Sellafield.  Legacy ponds at the Sellafield site have been used to store historic radioactive waste for decades and progress is being made in reducing the risks associated with these facilities [3].  Over time, there has been a deterioration in the condition of the ponds and their contents that has resulted in particles being present in solution in the ponds.  It is important to characterise these particles in order to facilitate reductions in the risks associated with the ponds.  We plan to use our existing facilities at the University of Liverpool to develop a novel instrument using simple solutions probably with gold nanoparticles and then to progress to non-radioactive simulants of the pond solutions.  The long-term goal will be to transition the technology to the Sellafield site perhaps with an intermediate step involving a demonstration of  the technology on pond solutions using the facilities of the National Nuclear Laboratory.

The PhD project is fully-funded for UK and EU citizens as part of a Centre for Doctoral Training and will involve a year of specialist training followed by three years of research.  For more information following this link.

References:

[1] Coglitore, D., Edwardson, S.P., Macko, P., Patterson, E.A., & Whelan, M.P., Transition from fractional to classical Stokes-Einstein behaviour in simple fluids, Royal Society Open Science, 4:170507, 2017.

[2] Patterson, E.A., Whelan, P., 2008, ‘Optical signatures of small nanoparticles in a conventional microscopeSmall, 4(10):1703-1706.

[3] Comptroller and Auditor General, The Nuclear Decommissioning Authority: progress with reducing risk at Sellafield, National Audit Office, HC 1126, Session 2017-19, 20 June 2018.