Mark D. Holton

Statistical analysis of nanoparticle dosing in a dynamic cellular system

The delivery of nanoparticles into cells is important in therapeutic applications and in nanotoxicology. Nanoparticles are generally targeted to receptors on the surfaces of cells and internalized into endosomes by endocytosis, but the kinetics of the process and the way in which cell division redistributes the particles remain unclear. Here we show that the chance of success or failure of nanoparticle uptake and inheritance is random. Statistical analysis of nanoparticle-loaded endosomes indicates that particle capture is described by an over-dispersed Poisson probability distribution that is consistent with heterogeneous adsorption and internalization. Partitioning of nanoparticles in cell division is random and asymmetric, following a binomial distribution with mean probability of 0.52-0.72. These results show that cellular targeting of nanoparticles is inherently imprecise due to the randomness of nature at the molecular scale, and the statistical framework offers a way to predict nanoparticle dosage for therapy and for the study of nanotoxins.

Quantitative characterization of nanoparticle agglomeration within biological media

Quantitative analysis of nanoparticle dispersion state within biological media is essential to understanding cellular uptake and the roles of diffusion, sedimentation, and endocytosis in determining nanoparticle dose. The dispersion of polymer-coated CdTe/ZnS quantum dots in water and cell growth medium with and without fetal bovine serum was analyzed by transmission electron microscopy (TEM) and dynamic light scattering (DLS) techniques. Characterization by TEM of samples prepared by plunge freezing the blotted solutions into liquid ethane was sensitive to the dispersion state of the quantum dots and enabled measurement of agglomerate size distributions even in the presence of serum proteins where DLS failed. In addition, TEM showed a reduced packing fraction of quantum dots per agglomerate when dispersed in biological media and serum compared to just water, highlighting the effect of interactions between the media, serum proteins, and the quantum dots. The identification of a heterogeneous distribution of quantum dots and quantum dot agglomerates in cell growth medium and serum by TEM will enable correlation with the previously reported optical metrology of in vitro cellular uptake of this quantum dot dispersion. In this paper, we present a comparative study of TEM and DLS and show that plunge-freeze TEM provides a robust assessment of nanoparticle agglomeration state.

Quantification of Nanoparticle Dose and Vesicular Inheritance in Proliferating Cells

Assessing dose in nanoparticle–cell interactions is inherently difficult due to a complex multiplicity of possible mechanisms and metrics controlling particle uptake. The fundamental unit of nanoparticle dose is the number of particles internalized per cell; we show that this can be obtained for large cell populations that internalize fluorescent nanoparticles by endocytosis, through calibration of cytometry measurements to transmission electron microscopy data. Low-throughput, high-resolution electron imaging of quantum dots in U-2 OS cells is quantified and correlated with high-throughput, low-resolution optical imaging of the nanoparticle-loaded cells. From the correlated data, we obtain probability distribution functions of vesicles per cell and nanoparticles per vesicle. Sampling of these distributions and comparison to fluorescence intensity histograms from flow cytometry provide the calibration factor required to transform the cytometry metric to total particle dose per cell, the mean value of which is 2.4 million. Use of the probability distribution functions to analyze particle partitioning during cell division indicates that, while vesicle inheritance is near symmetric, highly variable vesicle loading leads to a highly asymmetric particle dose within the daughter cells.

A transfer function approach to measuring cell inheritance

BackgroundThe inheritance of cellular material between parent and daughter cells during mitosis is highly influential in defining the properties of the cell and therefore the population lineage. This is of particular relevance when studying cell population evolution to assess the impact of a disease or the perturbation due to a drug treatment. The usual technique to investigate inheritance is to use time-lapse microscopy with an appropriate biological marker, however, this is time consuming and the number of inheritance events captured are too low to be statistically meaningful.ResultsHere we demonstrate the use of a high throughput fluorescence measurement technique e.g. flow cytometry, to measure the fluorescence from quantum dot markers which can be used to target particular cellular sites. By relating, the fluorescence intensity measured between two time intervals to a transfer function we are able to deconvolve the inheritance of cellular material during mitosis. To demonstrate our methodology we use CdTe/ZnS quantum dots to measure the ratio of endosomes inherited by the two daughter cells during mitosis in the U2-OS, human osteoscarcoma cell line. The ratio determined is in excellent agreement with results obtained previously using a more complex and computational intensive bespoke stochastic model.ConclusionsThe use of a transfer function approach allows us to utilise high throughput measurement of large cell populations to derive statistically relevant measurements of the inheritance of cellular material. This approach can be used to measure the inheritance of organelles, proteins etc. and also particles introduced to cells for drug delivery.

Stroboscopic fluorescence lifetime imaging

We report a fluorescence lifetime imaging technique that uses the time integrated response to a periodic optical excitation, eliminating the need for time resolution in detection. A Dirac pulse train of variable period is used to probe the frequency response of the total fluorescence per pulse leading to a frequency roll-off that is dependent on the relaxation rate of the fluorophores. The technique is validated by demonstrating wide-field, realtime, lifetime imaging of the endocytosis of inorganic quantum dots by a cancer cell line. Surface charging of the dots in the intra-cellular environment produces a switch in the fluorescence lifetime from approximately 40 ns to < 10 ns. A temporal resolution of half the excitation period is possible which in this instance is 15 ns. This stroboscopic technique offers lifetime based imaging at video rates with standard CCD cameras and has application in probing millisecond cell dynamics and in high throughput imaging assays.

Imaging concentric modulations in transverse modes of a vertical-cavity surface emitting laser using a scanning near-field optical microscope

Concentric standing waves have been spatially imaged in the near-field regime within the optical aperture of a vertical-cavity surface emitting laser (VCSEL) using a scanning near-field optical microscope. Using the microscope’s near-field collection mode and subsequent detection via a spectrometer it has been possible to design an experiment to determine the spatial location of multiple lasing modes in addition to concentric standing waves. At low injection current above threshold the standing waves influence and modulate the optical emission from multiple transverse modes. These results are discussed in relation to cavity and aperture effects, and pattern formation in VCSELs. Surface defects arising in the aperture are also seen to affect the optical output and are briefly discussed.

Mitigating the squash effect: sloths breathe easily upside down

Sloths are mammals renowned for spending a large proportion of time hanging inverted. In this position, the weight of the abdominal contents is expected to act on the lungs and increase the energetic costs of inspiration. Here, we show that three-fingered sloths Bradypus variegatus possess unique fibrinous adhesions that anchor the abdominal organs, particularly the liver and glandular stomach, to the lower ribs. The key locations of these adhesions, close to the diaphragm, prevent the weight of the abdominal contents from acting on the lungs when the sloth is inverted. Using ventilation rate and body orientation data collected from captive and wild sloths, we use an energetics-based model to estimate that these small adhesions could reduce the energy expenditure of a sloth at any time it is fully inverted by almost 13%. Given body angle preferences for individual sloths in our study over time, this equates to mean energy saving of 0.8–1.5% across individuals (with individual values ranging between 0.01 and 8.6%) per day. Given the sloths reduced metabolic rate compared with other mammals and extremely low energy diet, these seemingly innocuous adhesions are likely to be important in the animals energy budget and survival.

TEM analysis of nanoparticle dispersions with application towards the quantification of in vitro cellular uptake

TEM analysis of nanoparticle dispersions in solution has previously been limited, in the main, to the measurement of primary particle size due to the drying effects that occur during sample preparation. We show that solutions prepared for TEM by plunge freezing specimen grids coated with a blotted solution is a sensitive and potentially representative route to measuring a nanoparticle dispersion. We have started to benchmark the technique against the more commonly used dynamic light scattering method and show that the TEM route is potentially more sensitive route to counting individual nanoparticles in a dispersion that also contains nanoparticle agglomerates. Accurate measurement of nanoparticle dispersion in biological solutions could be a key step in the application of nanoparticles in medicine.

Fluorescence lifetime based, contrast imaging using variable period excitation pulse trains

The development of an experimental setup capable of contrasting fluorescent materials by their recombinative lifetimes in an imaging mode is discussed. Such materials might include molecular dyes and QDs. The system is comprised of a standard upright microscope fitted with an imaging CCD, and a white light laser that illuminates a circular region within the field of view with variable period excitation pulse trains. Different fluorescent species within this region absorb the laser light and fluoresce with a recombination lifetime dependent on material composition and local environment. Species with differing fluorescent lifetimes can be distinguished in an imaging mode by their contrasting intensity response to the pulse train at the range of different pulse frequencies. The technique is discussed and applied to samples containing both CdTe (705 nm) and CdSe (611 nm) QDs, showing contrast between long (70-100 ns) and (relatively) short (25-35 ns) lifetime within an image.