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Anthony, Stephen Michael

Publications and source records attributed to Anthony, Stephen Michael.

18 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Concurrent evaluation of autophagy induction and Burkholderia infection at the single cell level

Autophagy is a natural, regulated cellular process that "cleans up" cellular debris by degrading and recycling dysfunctional proteins. There is a high potential impact of exploiting the benefits of autophagy to complement existing treatments, but little has been done to date on bacterial pathogens of defense concern such as Burkholderia pseudomallei, a highly virulent Select Agent pathogen that is intrinsically resistant to most classes of antibiotics. Assessment of autophagy in the context of infection typically requires use of multiple technologies in combination (e.g., Western analysis paired with microscopy or flow cytometry) as applied to heterogeneous populations of cells. To address this, we have developed a dual target reporter cell line (RAW264.7 LC3-BFP:mPlum, GFP-RelA) that enables concurrent visualization of infection and autophagy induction. We assessed the effect of clinically approved small molecule inducers of autophagy on infection by Burkholderia thailendensis, a closely related but less virulent surrogate for B. pseudomallei. The reporter cells were first infected with a B. thailendensis strain that constitutively expresses GFP, then treated with one of four known autophagy inducers (rapamycin, niclosamide, bromhexine HC1, or flubendazole) for 4 hours. Confocal fluorescence imaging was used to quantify autophagy stimulation at the single cell level. Autophagy maturation was observed as a decrease in BFP LC3 puncta with a concurrent increase in mPlum LC3 puncta. B. thailendensis infection was assessed by monitoring translocation of GFP-RelA (an NFkB subunit) into the nucleus and through quantitating the intracellular bacterial presence in single cells. Preliminary results indicate that bromhexine HC1 and niclosamide may hinder B. thailendensis' ability to replicate intracellularly and reduce overall bacterial survival.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Distinguishing one from many using super-resolution compressive sensing

We present that distinguishing whether a signal corresponds to a single source or a limited number of highly overlapping point spread functions (PSFs) is a ubiquitous problem across all imaging scales, whether detecting receptor-ligand interactions in cells or detecting binary stars. Super-resolution imaging based upon compressed sensing exploits the relative sparseness of the point sources to successfully resolve sources which may be separated by much less than the Rayleigh criterion. However, as a solution to an underdetermined system of linear equations, compressive sensing requires the imposition of constraints which may not always be valid. One typical constraint is that the PSF is known. However, the PSF of the actual optical system may reflect aberrations not present in the theoretical ideal optical system. Even when the optics are well characterized, the actual PSF may reflect factors such as non-uniform emission of the point source (e.g. fluorophore dipole emission). As such, the actual PSF may differ from the PSF used as a constraint. Similarly, multiple different regularization constraints have been suggested including the l 1 -norm, l 0 -norm, and generalized Gaussian Markov random fields (GGMRFs), each of which imposes a different constraint. Other important factors include the signal-to-noise ratio of the point sources and whether the point sources vary in intensity. In this work, we explore how these factors influence super-resolution image recovery robustness, determining the sensitivity and specificity. In conclusion, we determine an approach that is more robust to the types of PSF errors present in actual optical systems.

42 ENGINEERING↗
DOE OSTI2017

Single-Janus Rod Tracking Reveals the “Rock-and-Roll” of Endosomes in Living Cells

Endosomes in cells are known to move directionally along microtubules, but their rotational dynamics have rarely been investigated. Even less is known, specifically, about the rotation of nonspherical endosomes. Here we report a single-Janus rod rotational tracking study to reveal the rich rotational dynamics of rod-shaped endosomes in living cells. The rotational reporters were Janus rods that display patches of different fluorescent colors on opposite sides along their long axes. When the Janus rods are wrapped tightly inside endosomes, their shape and optical anisotropy allow the simultaneous measurements of all three rotational angles (in-plane, out-of-plane, and longitudinal) and the translational motion of single endosomes with high spatiotemporal resolutions. We demonstrate that endosomes undergo in-plane rotation and rolling during intracellular transport and that such rotational dynamics are driven by rapid microtubule fluctuations. We reveal for the first time the “rock-and-roll” of endosomes in living cells and how the intracellular environment modifies such rotational dynamics. As a result, this study demonstrates a unique application of Janus particles as imaging probes in the elucidation of fundamental biological questions.

59 BASIC BIOLOGICAL SCIENCES↗