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Biomedical subjects

Ryan McAllister

Publications and source records attributed to Ryan McAllister.

9 recordsLinked to original sources
PubMed2006

Spinning disk confocal microscopy of live, intraerythrocytic malarial parasites. 1. Quantification of hemozoin development for drug sensitive versus resistant malaria.

We have customized a Nipkow spinning disk confocal microscope (SDCM) to acquire three-dimensional (3D) versus time data for live, intraerythrocytic malarial parasites. Since live parasites wiggle within red blood cells, conventional laser scanning confocal microscopy produces blurred 3D images after reconstruction of z stack data. In contrast, since SDCM data sets at high x, y, and z resolution can be acquired in hundreds of milliseconds, key aspects of live parasite cellular biochemistry can be much better resolved on physiologically meaningful times scales. In this paper, we present the first 3D DIC transmittance "z stack" images of live malarial parasites and use those to quantify hemozoin (Hz) produced within the living parasite digestive vacuole, under physiologic conditions. Using live synchronized cultures and voxel analysis of sharpened DIC z stacks, we present the first quantitative in vivo analysis of the rate of Hz growth for chloroquine sensitive (CQS) versus resistant (CQR) malarial parasites. We present data for laboratory strains, as well as pfcrt transfectants expressing a CQR conferring mutant pfcrt gene. We also analyze the rate of Hz growth in the presence and absence of physiologically relevant doses of chloroquine (CQ) and verapamil (VPL) and thereby present the first in vivo quantification of key predictions from the well-known Fitch hypothesis for CQ pharmacology. In the following paper [Gligorijevic, B., et al. (2006) Biochemistry 45, pp 12411-12423], we acquire fluorescent images of live parasite DV via SDCM and use those to quantify DV volume for CQS versus CQR parasites.

Animals↗
PubMed2006

Spinning disk confocal microscopy of live, intraerythrocytic malarial parasites. 2. Altered vacuolar volume regulation in drug resistant malaria.

In the previous paper [Gligorijevic, B., et al. (2006) Biochemistry 45, pp 12400-12410], we reported on a customized Nipkow spinning disk confocal microscopy (SDCM) system and its initial application to DIC imaging of hemozoin within live, synchronized, intraerythrocytic Plasmodium falciparum malarial parasites. In this paper, we probe the biogenesis as well as the volume and pH regulation of the parasite digestive vacuole (DV), using the fluorescence imaging capabilities of the system. Several previous reports have suggested that mutant PfCRT protein, which causes chloroquine resistance (CQR) in P. falciparum, also causes increased acidification of the DV. Since pH and volume regulation are often linked, we wondered whether DV volume differences might be associated with CQR. Using fast acquisition of SDCM z stacks for synchronized parasites with OGd internalized into the DV, followed by iterative deconvolution using experimental point spread functions, we quantify the volume of the DV for live, intraerythrocytic HB3 (CQS), Dd2 (CQR via drug selection), GCO3 (CQS), and GCO3/C3(Dd2) (CQR via transfection with mutant pfcrt) malarial parasites as they develop within the human red blood cell. We find that relative to both CQS strains, both CQR strains show significantly increased DV volume as the organelle forms upon entry into the trophozoite stage of development and that this persists until the trophozoite-schizont boundary. A more acidic DV pH is found for CQR parasites as soon as the organelle forms and persists throughout the trophozoite stage. We probe DV volume and pH changes upon ATP depletion, hypo- and hypertonic shock, and rapid withdrawal of perfusate chloride. Taken together, these data suggest that the PfCRT mutations that cause CQR also lead to altered DV volume regulation.

Animals↗
PubMed2006

Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope.

We develop an extension of fluorescence correlation spectroscopy (FCS) using a spinning disk confocal microscope. This approach can spatially map diffusion coefficients or flow velocities at up to approximately 10(5) independent locations simultaneously. Commercially available cameras with frame rates of 1000 Hz allow FCS measurements of systems with diffusion coefficients D~10(-7) cm(2)/s or smaller. This speed is adequate to measure small microspheres (200-nm diameter) diffusing in water, or hindered diffusion of macromolecules in complex media (e.g., tumors, cell nuclei, or the extracellular matrix). There have been a number of recent extensions to FCS based on laser scanning microscopy. Spinning disk confocal microscopy, however, has the potential for significantly higher speed at high spatial resolution. We show how to account for a pixel size effect encountered with spinning disk confocal FCS that is not present in standard or scanning FCS, and we introduce a new method to correct for photobleaching. Finally, we apply spinning disk confocal FCS to microspheres diffusing in Type I collagen, which show complex spatially varying diffusion caused by hydrodynamic and steric interactions with the collagen matrix.

Animals↗
PubMed2004

Consistency of nonlinear system response to complex drive signals.

The consistency of a nonlinear system's response to a repeated complex waveform drive signal is an important consideration in classical and quantum systems as diverse as lasers, neuronal networks, and manufacturing plants. We show from a consideration of different characteristic waveforms that there is typically an optimal drive amplitude for the most consistent response; internal noise sources dominate for small amplitude driving while deterministic system nonlinearity reduces consistency for large amplitudes. We test this general concept and its measurement experimentally and numerically on the specific example of a laser system.

Algorithms↗
PubMed2003

Generalized synchronization of chaos in identical systems with hidden degrees of freedom.

We demonstrate generalized synchronization of chaos in a two-mode laser system. The total intensity of the laser output (the sum of the individual mode intensities) is used as the drive signal. This lumped variable transmitted to the identical response system does not generate identical synchronization. Generalized synchronization is observed instead of identical synchronization because of the hidden internal degrees of freedom.

Feedback↗
PubMed2003

Competition between two frequencies for phase synchronization of a chaotic laser.

Competition between two distinct driving frequencies to phase synchronize the intensity dynamics of a chaotic laser has been observed. The phase of the chaotic intensity signal is constructed using the complex analytic signal. Competing frequencies alternately show phase locking and phase slipping. Competition has been quantified by calculating the portion of time the laser phase locks to each of the driving frequencies and their average.

Journal Article↗
PubMed2003

Measuring intense rotation and dissipation in turbulent flows.

Turbulent flows are highly intermittent--for example, they exhibit intense bursts of vorticity and strain. Kolmogorov theory describes such behaviour in the form of energy cascades from large to small spatial and temporal scales, where energy is dissipated as heat. But the causes of high intermittency in turbulence, which show non-gaussian statistics, are not well understood. Such intermittency can be important, for example, for enhancing the mixing of chemicals, by producing sharp drops in local pressure that can induce cavitation (damaging mechanical components and biological organisms), and by causing intense vortices in atmospheric flows. Here we present observations of the three components of velocity and all nine velocity gradients within a small volume, which allow us to determine simultaneously the dissipation (a measure of strain) and enstrophy (a measure of rotational energy) of a turbulent flow. Combining the statistics of all measurements and the evolution of individual bursts, we find that a typical sequence for intense events begins with rapid strain growth, followed by rising vorticity and a final sudden decline in stretching. We suggest two mechanisms which can produce these characteristics, depending whether they are due to the advection of coherent structures through our observed volume or caused locally.

Journal Article↗
PubMed2002

Chaotic function generator: complex dynamics and its control in a loss-modulated Nd:YAG laser.

The complex dynamics resulting from electronic feedback of a laser's intensity are explored and characterized. Distinct stable and chaotic regimes can be elicited from the laser by tuning the bias of the feedback loop. An additional branch of the feedback loop, containing a derivative filter, provides access to new kinds of dynamics, including a more gradual transition to chaos. The whole feedback network together allows the laser dynamics to be selected from among a wide range of chaotic wave forms distinguished by statistical or spectral information. In other words, this laser system can be used as a tunable generator of chaotic functions.

Journal Article↗