Tag Archives: nanoparticles

Real-time label-free tracking of bacteriophages interacting with bacteria

(a) Two-dimensional random dynamics (blue line) of a pelp20 bacteriophage monitored for a period of 5 s. Scale bar, 2 µm. (b) A plot of the same dynamics and (c) the mean square displacement (MSD) of the random walk. The MSD of the random walk is represented by square data points, and a linear fit (black line) has been applied to the data (Figure 4 from https://royalsocietypublishing.org/view-large/figure/20098614/rsif.2026.0250.f004.tif)

I was excited last month when our latest research on tracking nano-entities was published by the Journal of the Royal Society Interface. The paper describes the real-time and label-free tracking of bacteriophages, or phages, in an optical microscope using caustics (see right thumbnail). Phages are of interest due to the potential applications in biotechnology and medicine. They selectively infect and replicate within bacteria and play an important role in regulating bacterial populations across many ecosystems. I have written previously about the threat of antimicrobial resistant (AMR) infections and our research on the real-time tracking of individual bacterium that could be responsible for such infections [see ‘Label-free real-time tracking of individual bacterium‘ on January 25th, 2023]. In this newly published paper, we describe tracking phages as they interact with and compromise bacteria (see bottom thumbnail) using the same technique, optical caustics [see ‘Caustics‘ on October 15th, 2024 and application to ‘Nanoparticle motion through heterogeneous hydrogels‘ on November 6th, 2024 and to ‘Corona-induced transition from molecular to particle motion in biological media‘ on December 4th, 2024]. Traditionally, phages have been monitored using fluorescent labelling because their size is nanometric which renders them invisible in a conventional optical microscope. However, chemically attaching labels to nano entities has been shown to influence their dynamics. Hence, this new study represents a significant advance that will accelerate the real-time observation of phage-bacteria interactions which will enable the development of phage-based diagnostics and antimicrobial therapies.

Sources:

Francesco Giorgi, Samuel Chenery, Liberty Duignan, Joanne L. Fothergill, Eann Patterson, Judith M. Curran; Elucidating bacteriophage dynamics and interactions with real-time label-free optical imaging. J R Soc Interface 1 May 2026; 23 (238): 20260250. https://doi.org/10.1098/rsif.2026.0250

Details of E. coli bacteria: (a) not exposed (reprinted from [17]) and (b,c) exposed to a population of EcoLiv25 phages in solution. In (b), the arrow points at the supposed presence of a phage attached to the bacterium’s external membrane, while in (c), the arrows point at the compromised sections of the bacterium’s external membrane as a result of phage infection. Scale bars, 2 µm.

Details of E. coli bacteria: (a) not exposed and (b,c) exposed to a population of EcoLiv25 phages in solution. In (b), the arrow points at the supposed presence of a phage attached to the bacterium’s external membrane, while in (c), the arrows point at the compromised sections of the bacterium’s external membrane as a result of phage infection. Scale bars, 2 µm (Figure 6 from https://royalsocietypublishing.org/view-large/figure/20098622/rsif.2026.0250.f006.tif).

Passive nanorheology measurements

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What do marshmallows, jelly (or Jell-O), cream cheese and Chinese soup dumplings have in common? They are often made with gelatin. Gelatin is derived from the skin and bones of cattle and pigs through the partial hydrolysis of collagen. Gelatin is a physical hydrogel meaning that it consists of a three-dimensional network of polymer molecules in which a large amount of water is absorbed, as much as 90% in gelatin. These polymer molecules are cross-linked by hydrogen bonds, hydrophobic interactions and chain entanglements. External stimuli, such as temperature, can change the level of cross-linking causing the material to transition between its solid, liquid and gel states. This is why jelly sets in the fridge and melts when it’s heated up – the cross-links holding the molecules together break down. This type of responsive behaviour allows the properties of hydrogels to be controlled at the micro and sub-micron scale for a host of applications including tissue engineering, drug delivery, water treatment, wearable technologies, and supercapacitors. However, the design and manufacture of soft hydrogels can be challenging due to the lack of technology for measuring the local properties. Current quantitative techniques for measuring the properties of hydrogels usually focus on bulk properties and provide little data about local variations or real-time responses to external stimuli. My colleagues and I have used gold nanoparticles as probes in hydrogels to map the properties at the microscale of thermosensitive hydrogels undergoing real-time transition from the solid to gel phases [see ‘Passive nanorheological tool to characterise hydrogels’]. This is an extension, or perhaps more accurately an application, of our earlier work on tracking nanoparticles through the vitreous humour of the eye [see ‘Nanoparticle motion-through heterogeneous hydrogels’ on November 6^th, 2024]. The novel technique, which yields passive nanorheological measurements, allows us to evaluate local viscosity, identify time-varying heterogeniety and monitor dynamic phase transitions at the micro through to nano scale. The significant challenges of other techniques, such as weak signals due to high water content and the dynamism of hydrogels, are overcome with a fast, inexpensive and user-friendly technology. Although, even with these advantages, you are unlikely to use it when you are making jelly or roasting marshmallows over the campfire; however, it is really useful for understanding the transport of drugs through biological hydrogels or designing manufacturing processes for artificial tissue.

Reference

Moira Lorenzo Lopez, Victoria R. Kearns, Eann A. Patterson & Judith M. Curran, Passive nanorheological tool to characterise hydrogels, Nanoscale, 2025,17, 15338-15347.

Image: Figure 5 from the above reference showing a hydrogel transitioning to a gel phase as result of an increase in temperature with 100 nm diameter gold nanoparticles with some particles (yellow arrows) at the interface between phases. The image was taken in an inverted optical microscope set up for tracking the nanoparticles.

Corona-induced transition from molecular to particle motion in biological media

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In last month’s post [see ‘Nanoparticle motion through heterogeneous hydrogels’ on November 6^th, 2024], I described our recent work on tracking nanoparticles through a model of the vitreous humour and mentioned it was the first of two articles published in the Nature journal, Scientific Reports. In the second article, we explored the use of caustics in an optical microscope [see ‘Seeing the invisible’ on October 29^th, 2014] to track nanoparticles in biofluids. Nanoparticles are below the resolution of an optical microscope because they are substantially smaller than the wavelength of visible light; hence, they are usually tracked using fluorescent markers or tags attached chemically to the nanoparticles. These tags can influence both the motion of the particles and biological activity so caustics provide a label-free technique that allows particles to be tracked in real-time using a standard optical microscope. In most of our previous research, we have tracked nanoparticles in transparent fluids such as water, glycerol-water mixtures, or the hydrogels described in last month’s post. In our latest work, we have tracked small nanoparticles with diameters from 10 to 100 nm in common cell culture media with different concentrations of serum proteins. These fluids are a ‘soup’ of complex protein molecules that interact with one another and the gold nanoparticles being tracked. We found that the presence of proteins caused a reduction in the rate of diffusion for both positively- and negatively-charged particles and we concluded that the proteins form a corona around each nanoparticle effectively enlarging its diameter. For larger nanoparticles, and those positively-charged, the enlargement appears to cause a transition from molecular motion, in which particle diameter is unimportant, to particle motion where larger particles diffuse more slowly. We first explored this transition from fractional to classical Stokes-Einstein behaviour in simple fluids in 2017 [‘Slow moving nanoparticles‘ on December 13th 2017] and it seems likely to be complicated in these complex fluids. Hence, understanding protein dynamics as well nanoparticle dynamics will be essential to the development of nanotechnologies applicable in biological environments. So, we have lots more work to do!

Sources:

Schleyer G, Patterson EA, Curran JM. Label free tracking to quantify nanoparticle diffusion through biological media. Scientific Reports. 2024 Aug 13;14(1):18822.

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.

Nanoparticle motion through heterogeneous hydrogels

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Over the last couple of years, we have been transitioning a technique, which we developed for tracking the motion of nanoparticles using caustics [see ‘Slow moving nanoparticles‘ on December 13th 2017], from its initial use in exploring mechanics at the nanoscale to applications in nanobiology [See ‘Label-free real-time tracking of individual bacterium‘ on January 25th, 2023] where it has the advantages of functioning in real-time and being label-free (chemical labels can impact motion, protein interactions and cell behaviour). In the summer, we had couple of articles published in consecutive issues of the Nature journal, Scientific Reports which describe our recent work. In the first, we have explored the diffusion of nanoparticles through a synthetic analogue of the vitreous humour in order to support the design of novel therapeutics for retinal diseases. Retinal diseases, such as macular degeneration and diabetic retinopathy, affects millions of people globally and treatment often involves frequent intravitreal injections of anti-vascular endothelium growth factor agents and corticoids. Delivery of the appropriate dose to the retinal cell layer is challenging due to the complex nature of the vitreous and functionalised nanoparticles offer a potential solution. In vivo animal testing is inappropriate because of the ethical concerns and poor representation of human eyes and ex vivo testing of cadaveric eyes is unreliable due to the instability of biomechanical and biochemical properties of the vitreous humour. Hence, we used agar-hyaluronic acid hydrogels as an in vitro model of the vitreous and employed the caustic technique to track the motion of nanoparticles through the hydrogels. The hydrogels had been validated as a representative model of the vitreous humour by other research groups. Our tracking technique revealed that the electric charge on the nanoparticles did not affect their diffusion through the hydrogel; however, both the diameter of the particles and the heterogeneous nature of the gel influenced the diffusion. Nanoparticles with diameters of 200, 100 and 50 nm moved progressively more quickly and over a larger area. The diffusion rates in hydrogels with a high viscosity (about 450 Pa.s) were consistent throughout the gel implying that the gel was homogeneous, while gels with medium (about 40 Pa.s) to low (about 3 Pa.s) viscosity generated diffusion rates that were distributed bi-modally suggesting a heterogeneous gel with zones of low and high density in which the particles moved more or less freely. The heterogeneity of a gel renders a global value for viscosity somewhat meaningless and makes comparisons difficult with the vitreous humour because it is also heterogeneous; however, global values of viscosity for porcine vitreous humour are typically 1 Pa.s. We are continuing this research; however, our published work has demonstrated that the use of caustics in an optical microscope is a reproducible and inexpensive technique for exploring the design of novel nanoscale drug delivery systems for the eye.

Source: Lorenzo Lopez M, Kearns VR, Curran JM, Patterson EA. Diffusion of nanoparticles in heterogeneous hydrogels as vitreous humour in vitro substitutes. Scientific reports. 2024 Jul 29;14(1):1744.

Image: Random track of a nanoparticle superimposed on its image generated in the microscope using a pin-hole and narrowband filter.

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