Fluorescence recovery after photobleaching (FRAP) is a widely used tool for estimating mobility parameters of fluorescently tagged molecules in cells. Despite the widespread use of confocal laser scanning microscopes (CLSMs) to perform photobleaching experiments, quantitative data analysis has been limited by lack of appropriate practical models. Here, we present a new approximate FRAP model for use on any standard CLSM. The main novelty of the method is that it takes into account diffusion of highly mobile molecules during the bleach phase. In fact, we show that by the time the first postbleach image is acquired in a CLSM a significant fluorescence recovery of fast-moving molecules has already taken place. The model was tested by generating simulated FRAP recovery curves for a wide range of diffusion coefficients and immobile fractions. The method was further validated by an experimental determination of the diffusion coefficient of fluorescent dextrans and green fluorescent protein. The new FRAP method was used to compare the mobility rates of fluorescent dextrans of 20, 40, 70, and 500 kDa in aqueous solution and in the nucleus of living HeLa cells. Diffusion coefficients were lower in the nucleoplasm, particularly for higher molecular weight dextrans. This is most likely caused by a sterical hindrance effect imposed by nuclear components. Decreasing the temperature from 37 to 22 degrees C reduces the dextran diffusion rates by approximately 30% in aqueous solution but has little effect on mobility in the nucleoplasm. This suggests that spatial constraints to diffusion of dextrans inside the nucleus are insensitive to temperature.

Abstract-This paper presents an extensive analysis of the integral line-of-sight (ILOS) guidance method for path following tasks of underactuated marine vehicles, operating on and below the sea surface. It is shown that due to the embedded integral action, the guidance law makes the vessels follow straight lines by compensating for the drift effect of environmental disturbances such as currents, wind and waves. The ILOS guidance is first applied to a 2D model of surface vessels that includes the underactauted sway dynamics of the vehicle as well as disturbances in the form of constant irrotational ocean currents and constant dynamic, attitude dependent, forces. The actuated dynamics are not taken into account at this point. A Lyapunov closed loop analysis yields explicit bounds on the guidance law gains to guarantee uniform global asymptotic stability (UGAS) and uniform local exponential stability (ULES).The complete kinematic and dynamic closed loop system of the 3D ILOS guidance law is analyzed next, hence extending the analysis to underactuated AUVs for 3D straight-line path following applications in the presence of constant irrotational ocean currents. The actuated surge, pitch and yaw dynamics are included in the analysis where the closed loop system forms a cascade, and the properties of UGAS and ULES are shown. The 3D ILOS control system is a generalization of the 2D ILOS guidance. Finally, results from simulations and experiments are presented to validate and illustrate the theoretical results, where the 2D ILOS guidance is applied to the CART and the LAUV vehicles.

This paper presents the design and control of a multirotor-based aerial manipulator developed for outdoor operation. The multi-rotor has eight rotors and large payload to integrate a 7-degrees of freedom arm and to carry sensors and processing hardware needed for outdoor positioning. The arm can also carry an end-effector and sensors to perform different missions. The paper focuses on the control design and implementation aspects. A stable backstepping-based controller for the multirotor that uses the coupled full dynamic model is proposed, and an admittance controller for the manipulator arm is outlined. Several experimental tests with the aerial manipulator are also presented. In one of the experiments, the performance of the pitch attitude controller is compared to a PID controller. Other experiments of the arm controller following an object with the camera are also presented.

fAter being released from transcription sites, messenger ribonucleoprotein particles (mRNPs) must reach the nuclear pore complexes in order to be translocated to the cytoplasm. Whether the intranuclear movement of mRNPs results largely from Brownian motion or involves molecular motors remains unknown. Here we have used quantitative photobleaching techniques to monitor the intranuclear mobility of protein components of mRNPs tagged with GFP. The results show that the diffusion coefficients of the poly(A)-binding protein II (PABP2) and the export factor TAP are significantly reduced when these proteins are bound to mRNP complexes, as compared with nonbound proteins. The data further show that the mobility of wild-type PABP2 and TAP, but not of a point mutant variant of PABP2 that fails to bind to RNA, is significantly reduced when cells are ATP depleted or incubated at 22°C. Energy depletion has only minor effects on the intranuclear mobility of a 2,000-kD dextran (which corresponds approximately in size to 40S mRNP particles), suggesting that the reduced mobility of PABP2 and TAP is not caused by a general alteration of the nuclear environment. Taken together, the data suggest that the mobility of mRNPs in the living cell nucleus involves a combination of passive diffusion and ATP-dependent processes.

How splicing factors are recruited to nascent transcripts in the nucleus in order to assemble spliceosomes on newly synthesised pre-mRNAs is unknown. To address this question, we compared the intranuclear trafficking kinetics of small nuclear ribonucleoprotein particles (snRNP) and non-snRNP proteins in the presence and absence of splicing activity. Photobleaching experiments clearly show that spliceosomal proteins move continuously throughout the entire nucleus independently of ongoing transcription or splicing. Using quantitative experimental data, a mathematical model was applied for spliceosome assembly and recycling in the nucleus. The model assumes that splicing proteins move by Brownian diffusion and interact stochastically with binding sites located at different subnuclear compartments. Inhibition of splicing, which reduces the number of pre-mRNA binding sites available for spliceosome assembly, was modeled as a decrease in the on-rate binding constant in the nucleoplasm. Simulation of microscopy experiments before and after splicing inhibition yielded results consistent with the experimental observations. Taken together, our data argue against the view that spliceosomal components are stored in nuclear speckles until a signal triggers their recruitment to nascent transcripts. Rather, the results suggest that splicing proteins are constantly diffusing throughout the entire nucleus and collide randomly and transiently with pre-mRNAs.