Important factors determining the nanoscale tracking precision of dynamic microtubule ends

Tracking dynamic microtubule ends in fluorescence microscopy movies provides insight into the statistical properties of microtubule dynamics and is vital for further analysis that requires knowledge of the trajectories of the microtubule ends. Here we analyse the performance of a previously developed automated microtubule end tracking routine; this has been optimized for comparatively low signal‐to‐noise image sequences that are characteristic of microscopy movies of dynamic microtubules growing in vitro. Sequences of simulated microtubule images were generated assuming a variety of different experimental conditions. The simulated movies were then tracked and the tracking errors were characterized. We found that the growth characteristics of the microtubules within realistic ranges had a negligible effect on the tracking precision. The fluorophore labelling density, the pixel size of the images, and the exposure times were found to be important parameters limiting the tracking precision which could be explained using concepts of single molecule localization microscopy. The signal‐to‐noise ratio was found to be a good single predictor of the tracking precision: typical experimental signal‐to‐noise ratios lead to tracking precisions in the range of tens of nanometres, making the tracking program described here a useful tool for dynamic microtubule end tracking with close to molecular precision.     

Image mean square displacement to study the lateral mobility of Angiotensin II type 1 and Endothelin 1 type A receptors on living cells

The lateral mobility of membrane receptors provides insights into the molecular interactions of protein binding and the complex dynamic plasma membrane. The image mean square displacement (iMSD) analysis is a method used to extract qualitative and quantitative information of the protein diffusion law and infers how diffusion dynamic processes may change when the cellular environment is modified. The aim of the study was to describe the membrane diffusing properties of two G‐protein‐coupled receptors namely Angiotensin II type 1 (AT₁) and Endothelin 1 type A (ET_A) receptors and their corresponding receptor–ligand complexes in living cells using total internal reflection fluorescent microscopy and iMSD analysis. This study showed that both AT₁ and ET_A receptors displayed a mix of three modes of diffusion: free, confined, and partially confined. The confined mode was the predominant at the plasma membrane of living cells and was not affected by ligand binding. However, the local diffusivity and the confinement zone of AT₁ receptors were reduced by the binding of its antagonist losartan, and the long‐range diffusion with the local diffusivity coefficient of ET_A receptors was reduced upon exposure to its antagonist BQ123. To the best of our knowledge, this is the first study addressing the protein diffusion laws of these two receptors on living cells using total internal reflection fluorescence microscopy and iMSD.     

A novel multitarget tracking algorithm for Myosin VI protein molecules on actin filaments in TIRFM sequences

We propose a novel multitarget tracking framework for Myosin VI protein molecules in total internal reflection fluorescence microscopy sequences which integrates an extended Hungarian algorithm with an interacting multiple model filter. The extended Hungarian algorithm, which is a linear assignment problem based method, helps to solve measurement assignment and spot association problems commonly encountered when dealing with multiple targets, although a two‐motion model interacting multiple model filter increases the tracking accuracy by modelling the nonlinear dynamics of Myosin VI protein molecules on actin filaments. The evaluation of our tracking framework is conducted on both real and synthetic total internal reflection fluorescence microscopy sequences. The results show that the framework achieves higher tracking accuracies compared to the state‐of‐the‐art tracking methods, especially for sequences with high spot density.     

Single molecule fluorescence imaging of the photoinduced conversion and bleaching behavior of the fluorescent protein Kaede

Photoconversion and photobleaching behavior of the fluorescent protein Kaede immobilized in polyacrylamide gel matrix at room temperature was studied by single molecule wide-field fluorescence microscopy. Photobleaching kinetics of Kaede molecules upon excitation at 488 nm showed slight heterogeneity, suggesting the presence of different protein conformations and/or the distribution of local environments in the gel matrix. Statistical analysis of intensity trajectories of single molecules revealed four major types of fluorescence dynamics behavior upon short illumination by a violet light pulse (405 nm). In particular, two types of photoswitching behavior were observed: the green-to-red photoconversion (4% of Kaede molecules) and the photoactivation of green fluorescence without emission of red fluorescence (13%). Two other major groups show neither photoconversion nor red emission and demonstrate photoinduced partial deactivation (43%) and partial revival (30%) of green fluorescence. The significantly lower green-to-red conversion ratio as compared with bulk measurements in aqueous solution might be induced by the immobilization of the protein molecules within a polyacrylamide gel. Contrary to Ando et al. (Proc Natl Acad Sci 2002;99:12651-12656), we found a significant increase in green fluorescence emission upon illumination with 405-nm light, which is typical for GFP and related proteins.     

Terminal protein-induced stretching of bacteriophage φ29 DNA

Stretching of DNA molecules helps to resolve detail during the fluorescence microscopy of both single DNA molecules and single DNA–protein complexes. To make stretching occur, intricate procedures of specimen preparation and manipulation have been developed in previous studies. By contrast, the present study demonstrates that conventional procedures of specimen preparation cause DNA stretching to occur, if the specimen is the double‐stranded DNA genome of bacteriophage φ29. Necessary for this stretching is a protein covalently bound at both 5′ termini of φ29 DNA molecules. Some DNA molecules are attached to a cover glass only at the two ends. Others are attached at one end only with the other end free in solution. The extent of stretching varies from ～50% overstretched to ～50% understretched. The understretched DNA molecules are internally mobile to a variable extent. In addition to stretching, some φ29 DNA molecules also undergo assembly to form both linear and branched concatemers observed by single‐molecule fluorescence microscopy. The assembly also requires the terminal protein. The stretched DNA molecules are potentially useful for observing DNA biochemistry at the single molecule level.     

Massively parallel approximate Bayesian computation for estimating nanoparticle diffusion coefficients,sizes and concentrations using confocal laser scanning microscopy

We implement a massively parallel population Monte Carlo approximate Bayesian computation (PMC‐ABC) method for estimating diffusion coefficients, sizes and concentrations of diffusing nanoparticles in liquid suspension using confocal laser scanning microscopy and particle tracking. The method is based on the joint probability distribution of diffusion coefficients and the time spent by a particle inside a detection region where particles are tracked. We present freely available central processing unit (CPU) and graphics processing unit (GPU) versions of the analysis software, and we apply the method to characterize mono‐ and bidisperse samples of fluorescent polystyrene beads.     

Dual-colour microscopy of single fluorophores bound to myosin interacting with fluorescently labelled actin using anti-Stokes fluorescence

We have refined prismless total internal reflection fluorescence microscopy with extremely low background to visualize single fluorophores attached to protein molecules interacting with a filamentous biopolymer labelled with different colour fluorophores. By using Stokes and anti-Stokes fluorescence, two different colour fluorescences from two different colour fluorophores excited with a single wavelength laser can be observed simultaneously. This microscopy was applied to visualize motor proteins, actin and myosin molecules. Single myosin molecules labelled with a tetramethylrhodamine-5-iodoacetamide interacting with a BODIPY FL-labelled actin filament, a filamentous polymer of actin molecules, were observed clearly and simultaneously in aqueous solution. Individual hydrolysis reactions of Cy3-labelled ATP by single myosin molecules and sliding of a BODIPY FL-labelled actin filament along the myosin molecules could also be observed simultaneously. Thus, this technique is useful for observing single molecular processes of proteins interacting with a biological macromolecule such as an actin filament and a DNA.     

Direct visualization of the dynamics of membrane-anchor proteins in living cells

Dynamic properties of proteins have crucial roles in understanding protein function and molecular mechanism within cells. In this paper, we combined total internal reflection fluorescence microscopy with oblique illumination fluorescence microscopy to observe directly the movement and localization of membrane‐anchored green fluorescence proteins in living cells. Total internal reflect illumination allowed the observation of proteins in the cell membrane of living cells since the penetrate depth could be adjusted to about 80 nm, and oblique illumination allowed the observation of proteins both in the cytoplasm and apical membrane, which made this combination a promising tool to investigate the dynamics of proteins through the whole cell. Not only individual protein molecule tracks have been analyzed quantitatively but also cumulative probability distribution function analysis of ensemble trajectories has been done to reveal the mobility of proteins. Finally, single particle tracking has acted as a compensation for single molecule tracking. All the results exhibited green fluorescence protein dynamics within cytoplasm, on the membrane and from cytoplasm to plasma membrane.     

Simultaneous two-photon fluorescence correlation spectroscopy and lifetime imaging of dye molecules in submicrometer fluidic structures

Fluorescence correlation spectroscopy (FCS) is a very sensitive technique that can be used, e.g., for the measurement of low concentrations and for the investigation of transport of fluorescent molecules. Fluorescence lifetime imaging (FLIM) provides spatially resolved information about molecular fluorescence lifetimes reflecting the interactions of the molecules with their environment. We have applied simultaneous two-photon FCS and FLIM to probe the behavior of fluorescent molecules diffusing in submicrometer silicon oxide channels. Our measurements reveal differences in fluorescence lifetimes compared to bulk solution that result from the effects of confinement and the presence of interfaces. Confinement also affects diffusional characteristics of fluorophores as reflected in fluorescence autocorrelation functions. These possible consequences of both spatial confinement and the presence of interfaces between media with different refractive indices on the diffusion and fluorescence lifetime of molecules in nanostructures are discussed in general.