Fast Optimized Cluster Algorithm for Localizations (FOCAL): a spatial cluster analysis for super-resolved microscopy.

MOTIVATION: Single-molecule localization microscopy (SMLM) microscopy provides images of cellular structure at a resolution an order of magnitude below what can be achieved by conventional diffraction limited techniques. The concomitantly larger data sets generated by SMLM require increasingly efficient image analysis software. Density based clustering algorithms, with the most ubiquitous being DBSCAN, are commonly used to quantitatively assess sub-cellular assemblies. DBSCAN, however, is slow, scaling with the number of localizations like O(n log (n)) at best, and it's performance is highly dependent upon a subjectively selected choice of parameters. RESULTS: We have developed a grid-based clustering algorithm FOCAL, which explicitly accounts for several dominant artifacts arising in SMLM image reconstructions. FOCAL is fast and efficient, scaling like O(n), and only has one set parameter. We assess DBSCAN and FOCAL on experimental dSTORM data of clusters of eukaryotic RNAP II and PALM data of the bacterial protein H-NS, then provide a detailed comparison via simulation. FOCAL performs comparable and often superior to DBSCAN while yielding a significantly faster analysis. Additionally, FOCAL provides a novel method for filtering out of focus clusters from complex SMLM images. AVAILABILITY AND IMPLEMENTATION: The data and code are available at: http://www.utm.utoronto.ca/milsteinlab/resources/Software/FOCAL/ CONTACT: josh.milstein@utoronto.ca SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

On the role of DNA biomechanics in the regulation of gene expression.

DNA is traditionally seen as a linear sequence of instructions for cellular functions that are expressed through biochemical processes. Cellular DNA, however, is also organized as a complex hierarchical structure with a mosaic of mechanical features, and a growing body of evidence is now emerging to imply that these mechanical features are connected to genetic function. Mechanical tension, for instance, which must be felt by DNA within the heavily constrained and continually fluctuating cellular environment, can affect a number of regulatory processes implicating a role for biomechanics in gene expression complementary to that of biochemical regulation. In this article, we review evidence for such mechanical pathways of genetic regulation.

Bead size effects on protein-mediated DNA looping in tethered-particle motion experiments.

Tethered particle motion (TPM) has become an important tool for single-molecule studies of biomolecules; however, concerns remain that the method may alter the dynamics of the biophysical process under study. We investigate the effect of the attached microsphere on an illustrative biological example: the formation and breakdown of protein-mediated DNA loops in the lac repressor system. By comparing data from a conventional TPM experiment with 800 nm polystyrene beads and dark-field TPM using 50 nm Au nanoparticles, we found that the lifetimes of the looped and unlooped states are only weakly modified, less than two-fold, by the presence of the large bead. This is consistent with our expectation of weak excluded-volume effects and hydrodynamic surface interactions from the cover glass and microsphere.

Protein-mediated DNA loop formation and breakdown in a fluctuating environment.

Living cells provide a fluctuating, out-of-equilibrium environment in which genes must coordinate cellular function. DNA looping, which is a common means of regulating transcription, is very much a stochastic process; the loops arise from the thermal motion of the DNA and other fluctuations of the cellular environment. We present single-molecule measurements of DNA loop formation and breakdown when an artificial fluctuating force, applied to mimic a fluctuating cellular environment, is imposed on the DNA. We show that loop formation is greatly enhanced in the presence of noise of only a fraction of k_{B}T, yet find that hypothetical regulatory schemes that employ mechanical tension in the DNA-as a sensitive switch to control transcription-can be surprisingly robust due to a fortuitous cancellation of noise effects.

Femtonewton entropic forces can control the formation of protein-mediated DNA loops.

We show that minuscule entropic forces, on the order of 100 fN, can prevent the formation of DNA loops-a ubiquitous means of regulating the expression of genes. We observe a tenfold decrease in the rate of LacI-mediated DNA loop formation when a tension of 200 fN is applied to the substrate DNA, biasing the thermal fluctuations that drive loop formation and breakdown events. Conversely, once looped, the DNA-protein complex is insensitive to applied force. Our measurements are in excellent agreement with a simple polymer model of loop formation in DNA, and show that an antiparallel topology is the preferred LacI-DNA loop conformation for a generic loop-forming construct.

Signatures of resonance superfluidity in a quantum Fermi gas.

We predict a direct and observable signature of the superfluid phase in a quantum Fermi gas, in a temperature regime already accessible in current experiments. We apply the theory of resonance superfluidity to a gas confined in a harmonic potential and demonstrate that a significant increase in density will be observed in the vicinity of the trap center.