Nonexponential statistics of fluorescence photobleaching

In this paper, I consider theoretical models of the decay via photobleaching of a sample of surface-immobilized fluorescent molecules excited by a spatially varying laser intensity profile. I show that, with mild restrictions on the photobleaching mechanism, the fluorescence decay measured in a nonuniform excitation profile is always nonexponential. Under the same conditions, the fluorescence decay can always be approximated by a discrete sum of exponentials. A particular example is given in which a homogeneous population of fluorophores with a single (intensity-dependent) photobleaching lifetime, when illuminated by a Gaussian laser, exhibits power law fluorescence decay at long times. These results indicate that the observation of multiple exponentials in single molecule or ensemble photobleaching lifetime measurements can arise solely as an artifact of a spatially varying laser profile and is not necessarily indicative of heterogeneity in molecular internal states, conformation, or local environment

Tracking-FCS: Fluorescence correlation spectroscopy of individual particles

We exploit recent advances in single-particle tracking to perform fluorescence correlation spectroscopy on individual fluorescent particles, in contrast to traditional methods that build up statistics over a sequence of many measurements. By rapidly scanning the focus of an excitation laser in a circular pattern, demodulating the measured fluorescence, and feeding these results back to a piezoelectric translation stage, we track the Brownian motion of fluorescent polymer microspheres in aqueous solution in the plane transverse to the laser axis. We discuss the estimation of particle diffusion statistics from closed-loop position measurements, and we present a generalized theory of fluorescence correlation spectroscopy for the case that the motion of a single fluorescent particle is actively tracked by a time-dependent laser intensity. We model the motion of a tracked particle using Ornstein-Uhlenbeck statistics, using a general theory that contains a umber of existing results as specific cases. We find good agreement between our theory and experimental results, and discuss possible future applications of these techniques to passive, single-shot, single-molecule fluorescence measurements with many orders of magnitude in time resolution

Photon statistics and dynamics of Fluorescence Resonance Energy Transfer

We report high time-resolution measurements of photon statistics from pairs of dye molecules coupled by fluorescence resonance energy transfer (FRET). In addition to quantum-optical photon antibunching, we observe photon bunching on a timescale of several nanoseconds. We show by numerical simulation that configuration fluctuations in the coupled fluorophore system could account for minor deviations of our data from predictions of basic Forster theory. With further characterization we believe that FRET photon statistics could provide a unique tool for studying DNA mechanics on timescales from 10^-9 to 10^-3 s.Comment: 4 pages, 4 figures, submitted to Physical Review Letter

Feedback localization of freely diffusing fluorescent particles near the optical shot-noise limit

We report near-optimal tracking of freely diffusing fluorescent particles in a quasi-two-dimensional geometry via photon counting and real-time feedback. We present a quantitative statistical model of our feedback network and find excellent agreement with the experiment. We monitor the motion of a single fluorescent particle with a sensitivity of 15 nm/sqrt Hz while collecting fewer than 5000 fluorescence photons/s. Fluorescent microspheres (diffusion coefficient 1.3 μm^2/s) are tracked with a root-mean-square tracking error of 170 nm, within a factor of 2 of the theoretical limit set by photon counting shot noise

Expand+Functional selection and systematic analysis of intronic splicing elements identify active sequence motifs and associated splicing factors

Despite the critical role of pre-mRNA splicing in generating proteomic diversity and regulating gene expression, the sequence composition and function of intronic splicing regulatory elements (ISREs) have not been well elucidated. Here, we employed a high-throughput in vivo Screening PLatform for Intronic Control Elements (SPLICE) to identify 125 unique ISRE sequences from a random nucleotide library in human cells. Bioinformatic analyses reveal consensus motifs that resemble splicing regulatory elements and binding sites for characterized splicing factors and that are enriched in the introns of naturally occurring spliced genes, supporting their biological relevance. In vivo characterization, including an RNAi silencing study, demonstrate that ISRE sequences can exhibit combinatorial regulatory activity and that multiple trans-acting factors are involved in the regulatory effect of a single ISRE. Our work provides an initial examination into the sequence characteristics and function of ISREs, providing an important contribution to the splicing code

Narrow-line magneto-optical cooling and trapping of strongly magnetic atoms

Laser cooling on weak transitions is a useful technique for reaching ultracold temperatures in atoms with multiple valence electrons. However, for strongly magnetic atoms a conventional narrow-line magneto-optical trap (MOT) is destabilized by competition between optical and magnetic forces. We overcome this difficulty in Er by developing an unusual narrow-line MOT that balances optical and magnetic forces using laser light tuned to the blue side of a narrow (8 kHz) transition. The trap population is spin-polarized with temperatures reaching below 2 microkelvin. Our results constitute an alternative method for laser cooling on weak transitions, applicable to rare-earth-metal and metastable alkaline earth elements.Comment: To appear in Phys. Rev. Lett. 4 pages, 5 figure

Conservation of context-dependent splicing activity in distant Muscleblind homologs

The Muscleblind (MBL) protein family is a deeply conserved family of RNA binding proteins that regulate alternative splicing, alternative polyadenylation, RNA stability and RNA localization. Their inactivation due to sequestration by expanded CUG repeats causes symptoms in the neuromuscular disease myotonic dystrophy. MBL zinc fingers are the most highly conserved portion of these proteins, and directly interact with RNA. We identified putative MBL homologs in Ciona intestinalis and Trichoplax adhaerens, and investigated their ability, as well as that of MBL homologs from human/mouse, fly and worm, to regulate alternative splicing. We found that all homologs can regulate alternative splicing in mouse cells, with some regulating over 100 events. The cis-elements through which each homolog exerts its splicing activities are likely to be highly similar to mammalian Muscleblind-like proteins (MBNLs), as suggested by motif analyses and the ability of expanded CUG repeats to inactivate homolog-mediated splicing. While regulation of specific target exons by MBL/MBNL has not been broadly conserved across these species, genes enriched for MBL/MBNL binding sites in their introns may play roles in cell adhesion, ion transport and axon guidance, among other biological pathways, suggesting a specific, conserved role for these proteins across a broad range of metazoan species.National Institutes of Health (U.S.) (DP5 OD017865

MBNL1 binds GC motifs embedded in pyrimidines to regulate alternative splicing

Muscleblind-like 1 (MBNL1) regulates alternative splicing and is a key player in the disease mechanism of myotonic dystrophy (DM). In DM, MBNL1 becomes sequestered to expanded CUG/CCUG repeat RNAs resulting in splicing defects, which lead to disease symptoms. In order to understand MBNL1’s role in both the disease mechanism of DM and alternative splicing regulation, we sought to identify its RNA-binding motif. A doped SELEX was performed on a known MBNL1-binding site. After five rounds of SELEX, MBNL1 selected pyrimidine-rich RNAs containing YGCY motifs. Insertion of multiple YGCY motifs into a normally MBNL1-independent splicing reporter was sufficient to promote regulation by MBNL1. MBNL1 was also shown to regulate the splicing of exon 22 in the ATP2A1 pre-mRNA, an exon mis-spliced in DM, via YGCY motifs. A search for YGCY motifs in 24 pre-mRNA transcripts that are mis-spliced in DM1 patients revealed an interesting pattern relative to the regulated exon. The intronic regions upstream of exons that are excluded in normal tissues relative to DM1, are enriched in YGCY motifs. Meanwhile, the intronic regions downstream of exons that are included in normal tissues relative to DM1, are enriched in YGCY motifs