SUMMARY: Analysis of intra- and extracellular dynamic like vesicles transport involves particle tracking algorithms. The design of a particle tracking pipeline is a routine but tedious task. Therefore, particle dynamics analysis is often performed by combining several pieces of software (filtering, detection, tracking, etc.) requiring many manual operations, and thus leading to poorly reproducible results. Given the new segmentation tools based on deep learning, modularity and interoperability between software have become essential in particle tracking algorithms. A good synergy between a particle detector and a tracker is of paramount importance. In addition, a user-friendly interface to control the quality of estimated trajectories is necessary. To address these issues, we developed STracking, a Python library that allows combining algorithms into standardized particle tracking pipelines. AVAILABILITY AND IMPLEMENTATION: STracking is available as a Python library using 'pip install' and the source code is publicly available on GitHub (https://github.com/sylvainprigent/stracking). A graphical interface is available using two napari plugins: napari-stracking and napari-tracks-reader. These napari plugins can be installed via the napari plugins menu or using 'pip install'. The napari plugin source codes are available on GitHub (https://github.com/sylvainprigent/napari-tracks-reader, https://github.com/sylvainprigent/napari-stracking). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

BACKGROUND: Characterizing membrane dynamics is a key issue to understand cell exchanges with the extra-cellular medium. Total internal reflection fluorescence microscopy (TIRFM) is well suited to focus on the late steps of exocytosis at the plasma membrane. However, it is still a challenging task to quantify (lateral) diffusion and estimate local dynamics of proteins. RESULTS: A new model was introduced to represent the behavior of cargo transmembrane proteins during the vesicle fusion to the plasma membrane at the end of the exocytosis process. Two biophysical parameters, the diffusion coefficient and the release rate parameter, are automatically estimated from TIRFM image sequences, to account for both the lateral diffusion of molecules at the membrane and the continuous release of the proteins from the vesicle to the plasma membrane. Quantitative evaluation on 300 realistic computer-generated image sequences demonstrated the efficiency and accuracy of the method. The application of our method on 16 real TIRFM image sequences additionally revealed differences in the dynamic behavior of Transferrin Receptor (TfR) and Langerin proteins. CONCLUSION: An automated method has been designed to simultaneously estimate the diffusion coefficient and the release rate for each individual vesicle fusion event at the plasma membrane in TIRFM image sequences. It can be exploited for further deciphering cell membrane dynamics.

Accurately detecting subcellular particles in fluorescence microscopy is of primary interest for further quantitative analysis such as counting, tracking, or classification. Our primary goal is to segment vesicles likely to share nearly the same size in fluorescence microscopy images. Our method termed adaptive thresholding of Laplacian of Gaussian (LoG) images with autoselected scale (ATLAS) automatically selects the optimal scale corresponding to the most frequent spot size in the image. Four criteria are proposed and compared to determine the optimal scale in a scale-space framework. Then, the segmentation stage amounts to thresholding the LoG of the intensity image. In contrast to other methods, the threshold is locally adapted given a probability of false alarm (PFA) specified by the user for the whole set of images to be processed. The local threshold is automatically derived from the PFA value and local image statistics estimated in a window whose size is not a critical parameter. We also propose a new data set for benchmarking, consisting of six collections of one hundred images each, which exploits backgrounds extracted from real microscopy images. We have carried out an extensive comparative evaluation on several data sets with ground-truth, which demonstrates that ATLAS outperforms existing methods. ATLAS does not need any fine parameter tuning and requires very low computation time. Convincing results are also reported on real total internal reflection fluorescence microscopy images.

Image analysis applied to fluorescence live cell microscopy has become a key tool in molecular biology since it enables to characterize biological processes in space and time at the subcellular level. In fluorescence microscopy imaging, the moving tagged structures of interest, such as vesicles, appear as bright spots over a static or nonstatic background. In this paper, we consider the problem of vesicle segmentation and time-varying background estimation at the cellular scale. The main idea is to formulate the joint segmentation-estimation problem in the general conditional random field framework. Furthermore, segmentation of vesicles and background estimation are alternatively performed by energy minimization using a min cut-max flow algorithm. The proposed approach relies on a detection measure computed from intensity contrasts between neighboring blocks in fluorescence microscopy images. This approach permits analysis of either 2D + time or 3D + time data. We demonstrate the performance of the so-called C-CRAFT through an experimental comparison with the state-of-the-art methods in fluorescence video-microscopy. We also use this method to characterize the spatial and temporal distribution of Rab6 transport carriers at the cell periphery for two different specific adhesion geometries.

We present a nonparametric regression method for denoising 3-D image sequences acquired via fluorescence microscopy. The proposed method exploits the redundancy of the 3-D+time information to improve the signal-to-noise ratio of images corrupted by Poisson-Gaussian noise. A variance stabilization transform is first applied to the image-data to remove the dependence between the mean and variance of intensity values. This preprocessing requires the knowledge of parameters related to the acquisition system, also estimated in our approach. In a second step, we propose an original statistical patch-based framework for noise reduction and preservation of space-time discontinuities. In our study, discontinuities are related to small moving spots with high velocity observed in fluorescence video-microscopy. The idea is to minimize an objective nonlocal energy functional involving spatio-temporal image patches. The minimizer has a simple form and is defined as the weighted average of input data taken in spatially-varying neighborhoods. The size of each neighborhood is optimized to improve the performance of the pointwise estimator. The performance of the algorithm (which requires no motion estimation) is then evaluated on both synthetic and real image sequences using qualitative and quantitative criteria.