Michael A. Thompson

Three-dimensional single-molecule fluorescence imaging beyond the diffraction limit using a double-helix point spread function

We demonstrate single-molecule fluorescence imaging beyond the optical diffraction limit in 3 dimensions with a wide-field microscope that exhibits a double-helix point spread function (DH-PSF). The DH-PSF design features high and uniform Fisher information and has 2 dominant lobes in the image plane whose angular orientation rotates with the axial (z) position of the emitter. Single fluorescent molecules in a thick polymer sample are localized in single 500-ms acquisitions with 10- to 20-nm precision over a large depth of field (2 μm) by finding the center of the 2 DH-PSF lobes. By using a photoactivatable fluorophore, repeated imaging of sparse subsets with a DH-PSF microscope provides superresolution imaging of high concentrations of molecules in all 3 dimensions. The combination of optical PSF design and digital postprocessing with photoactivatable fluorophores opens up avenues for improving 3D imaging resolution beyond the Rayleigh diffraction limit.

BubR1 insufficiency causes early onset of aging-associated phenotypes and infertility in mice.

Faithful segregation of replicated chromosomes is essential for maintenance of genetic stability and seems to be monitored by several mitotic checkpoints. Various components of these checkpoints have been identified in mammals, but their physiological relevance is largely unknown. Here we show that mutant mice with low levels of the spindle assembly checkpoint protein BubR1 develop progressive aneuploidy along with a variety of progeroid features, including short lifespan, cachectic dwarfism, lordokyphosis, cataracts, loss of subcutaneous fat and impaired wound healing. Graded reduction of BubR1 expression in mouse embryonic fibroblasts causes increased aneuploidy and senescence. Male and female mutant mice have defects in meiotic chromosome segregation and are infertile. Natural aging of wild-type mice is marked by decreased expression of BubR1 in multiple tissues, including testis and ovary. These results suggest a role for BubR1 in regulating aging and infertility.

Effect of Nanoparticle Surface Charge at the Plasma Membrane and Beyond

Herein, we demonstrate that the surface charge of gold nanoparticles (AuNPs) plays a critical role in modulating membrane potential of different malignant and nonmalignant cell types and subsequent downstream intracellular events. The findings presented here describe a novel mechanism for cell-nanoparticle interactions and AuNP uptake: modulation of membrane potential and its effect on intracellular events. These studies will help understand the biology of cell-nanoparticle interactions and facilitate the engineering of nanoparticles for specific intracellular targets.

Deep sequencing of the small RNA transcriptome of normal and malignant human B cells identifies hundreds of novel microRNAs.

A role for microRNA (miRNA) has been recognized in nearly every biologic system examined thus far. A complete delineation of their role must be preceded by the identification of all miRNAs present in any system. We elucidated the complete small RNA transcriptome of normal and malignant B cells through deep sequencing of 31 normal and malignant human B-cell samples that comprise the spectrum of B-cell differentiation and common malignant phenotypes. We identified the expression of 333 known miRNAs, which is more than twice the number previously recognized in any tissue type. We further identified the expression of 286 candidate novel miRNAs in normal and malignant B cells. These miRNAs were validated at a high rate (92%) using quantitative polymerase chain reaction, and we demonstrated their application in the distinction of clinically relevant subgroups of lymphoma. We further demonstrated that a novel miRNA cluster, previously annotated as a hypothetical gene LOC100130622, contains 6 novel miRNAs that regulate the transforming growth factor-β pathway. Thus, our work suggests that more than a third of the miRNAs present in most cellular types are currently unknown and that these miRNAs may regulate important cellular functions.

Localizing and Tracking Single Nanoscale Emitters in Three Dimensions with High Spatiotemporal Resolution Using a Double-Helix Point Spread Function

Three-dimensional nanoscale localization and tracking of dim single emitters can be obtained with a widefield fluorescence microscope exhibiting a double-helix point spread function (DH-PSF). We describe in detail how the localization precision quantitatively depends upon the number of photons detected and the z position of the nanoscale emitter, thereby showing a approximately 10 nm localization capability along x, y, and z in the limit of weak emitters. Experimental measurements are compared to Fisher information calculations of the ultimate localization precision inherent in the DH-PSF. The DH-PSF, for the first time, is used to track single quantum dots in aqueous solution and a quantum dot-labeled structure inside a living cell in three dimensions.

Evidence of systemic Th2 driven chronic inflammation in patients with metastatic melanoma

Purpose: Immunotherapeutic modalities are commonly used for treatment of patients with melanoma. The therapeutic success in preclinical models has not yielded the expected clinical results. To understand this discrepancy, we attempted to define immune homeostasis of 209 patients with melanoma across stages of disease relative to normal controls. Experimental Design: Peripheral blood mononuclear cells (PBMC) and plasma were collected from patients and healthy donors. PBMC were analyzed for frequencies of natural killer, dendritic, and T cells and their functional status. Matched plasma samples were analyzed for the concentrations of 27 cytokines, chemokines, and growth factors. RNA was isolated from 24 metastatic melanoma tumor biopsies and profiled by microarray analysis. Results: The frequency of natural killer, T, and dendritic cells in patients does not significantly change across stages of melanoma. However, plasma concentrations of Th2 cytokines [interleukin (IL)-4, IL-5, IL-10, and IL-13] in tumor-bearing patients were significantly higher than those with resected melanoma. Expression array analysis of metastatic melanoma revealed that the malignant melanocytes were not the source of the Th2 cytokines but did highly up-regulate vascular endothelial growth factor (VEGF) transcripts, consistent with plasma VEGF concentrations. In vitro VEGF exposure of normal PBMC lead to repolarization from Th1 to Th2 emulating the state of metastatic melanoma. Conclusions: Patients with metastatic melanoma exist in a state of Th2-mediated “chronic inflammation” as a result of at least VEGF overproduction by malignant tumors. These data support prior observations regarding the effect of VEGF on immune cell function and suggests consideration of VEGF inhibitors in future cancer immunotherapy clinical studies in metastatic melanoma.

Three-dimensional tracking of single mRNA particles in Saccharomyces cerevisiae using a double-helix point spread function.

Optical imaging of single biomolecules and complexes in living cells provides a useful window into cellular processes. However, the three-dimensional dynamics of most important biomolecules in living cells remains essentially uncharacterized. The precise subcellular localization of mRNA-protein complexes plays a critical role in the spatial and temporal control of gene expression, and a full understanding of the control of gene expression requires precise characterization of mRNA transport dynamics beyond the optical diffraction limit. In this paper, we describe three-dimensional tracking of single mRNA particles with 25-nm precision in the x and y dimensions and 50-nm precision in the z dimension in live budding yeast cells using a microscope with a double-helix point spread function. Two statistical methods to detect intermittently confined and directed transport were used to quantify the three-dimensional trajectories of mRNA for the first time, using ARG3 mRNA as a model. Measurements and analysis show that the dynamics of ARG3 mRNA molecules are mostly diffusive, although periods of non-Brownian confinement and directed transport are observed. The quantitative methods detailed in this paper can be broadly applied to the study of mRNA localization and the dynamics of diverse other biomolecules in a wide variety of cell types.

Phase II Trial of Intravenous Administration of Reolysin® (Reovirus Serotype-3-dearing Strain) in Patients with Metastatic Melanoma

Reovirus, a replication competent RNA virus, has preclinical activity against melanoma lines and xenografts. We conducted a phase II trial of reovirus in metastatic melanoma patients. Patients received 3 × 10(10) TCID50 on days 1-5 of each 28 day cycle, administered intravenously. Twenty-one eligible patients were enrolled. Treatment was well tolerated without any dose reductions having to be implemented. Post-treatment biopsy samples were obtained in 15 patients, 13/15 contained adequate tumor for correlative analysis. In two patients, productive reoviral replication (viral antigen coexpression with tubulin) was demonstrated, despite increase in neutralizing antibody titers. There were no objective responses although 75-90% tumor necrosis, consistent with treatment effect, was observed in one patient who had metastatic lesions surgically removed. Median time to progression and survival were 45 days (range 13-96 days) and 165 days (range 15 days-15.8 months) respectively. In conclusion, reovirus treatment was well tolerated in metastatic melanoma patients; viral replication was demonstrated in biopsy samples. Based on preclinical data showing synergy with taxane and platinum compounds, a phase II combination trial in metastatic melanoma patients is ongoing.Reovirus, a replication competent RNA virus, has preclinical activity against melanoma lines and xenografts. We conducted a phase II trial of reovirus in metastatic melanoma patients. Patients received 3 × 1010 TCID50 on days 1-5 of each 28 day cycle, administered intravenously. Twenty-one eligible patients were enrolled. Treatment was well tolerated without any dose reductions having to be implemented. Post-treatment biopsy samples were obtained in 15 patients, 13/15 contained adequate tumor for correlative analysis. In two patients, productive reoviral replication (viral antigen coexpression with tubulin) was demonstrated, despite increase in neutralizing antibody titers. There were no objective responses although 75-90% tumor necrosis, consistent with treatment effect, was observed in one patient who had metastatic lesions surgically removed. Median time to progression and survival were 45 days (range 13-96 days) and 165 days (range 15 days-15.8 months) respectively. In conclusion, reovirus treatment was well tolerated in metastatic melanoma patients; viral replication was demonstrated in biopsy samples. Based on preclinical data showing synergy with taxane and platinum compounds, a phase II combination trial in metastatic melanoma patients is ongoing.

Three-dimensional localization precision of the double-helix point spread function versus astigmatism and biplane.

Wide-field microscopy with a double-helix point spread function (DH-PSF) provides three-dimensional (3D) position information beyond the optical diffraction limit. We compare the theoretical localization precision for an unbiased estimator of the DH-PSF to that for 3D localization by astigmatic and biplane imaging using Fisher information analysis including pixelation and varying levels of background. The DH-PSF results in almost constant localization precision in all three dimensions for a 2 μm thick depth of field while astigmatism and biplane improve the axial localization precision over smaller axial ranges. For high signal-to-background ratio, the DH-PSF on average achieves better localization precision.

Cigarette smoke-induced mitochondrial fragmentation and dysfunction in human airway smooth muscle

The balance between mitochondrial fission and fusion is crucial for mitochondria to perform its normal cellular functions. We hypothesized that cigarette smoke (CS) disrupts this balance and enhances mitochondrial dysfunction in the airway. In nonasthmatic human airway smooth muscle (ASM) cells, CS extract (CSE) induced mitochondrial fragmentation and damages their networked morphology in a concentration-dependent fashion, via increased expression of mitochondrial fission protein dynamin-related protein 1 (Drp1) and decreased fusion protein mitofusin (Mfn) 2. CSE effects on Drp1 vs. Mfn2 and mitochondrial network morphology involved reactive oxygen species (ROS), activation of extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), protein kinase C (PKC) and proteasome pathways, as well as transcriptional regulation via factors such as NF-κB and nuclear erythroid 2-related factor 2. Inhibiting Drp1 prevented CSE effects on mitochondrial networks and ROS generation, whereas blocking Mfn2 had the opposite, detrimental effect. In ASM from asmatic patients, mitochondria exhibited substantial morphological defects at baseline and showed increased Drp1 but decreased Mfn2 expression, with exacerbating effects of CSE. Overall, these results highlight the importance of mitochondrial networks and their regulation in the context of cellular changes induced by insults such as inflammation (as in asthma) or CS. Altered mitochondrial fission/fusion proteins have a further potential to influence parameters such as ROS and cell proliferation and apoptosis relevant to airway diseases.