Automated Microtubule Tracking and Analysis (original) (raw)
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MTrack: Automated Detection, Tracking, and Analysis of Dynamic Microtubules
Scientific Reports
Microtubules are polar, dynamic filaments fundamental to many cellular processes. In vitro reconstitution approaches with purified tubulin are essential to elucidate different aspects of microtubule behavior. To date, deriving data from fluorescence microscopy images by manually creating and analyzing kymographs is still commonplace. Here, we present Mtrack, implemented as a plug-in for the open-source platform Fiji, which automatically identifies and tracks dynamic microtubules with sub-pixel resolution using advanced objection recognition. Mtrack provides automatic data interpretation yielding relevant parameters of microtubule dynamic instability together with population statistics. the application of our software produces unbiased and comparable quantitative datasets in a fully automated fashion. this helps the experimentalist to achieve higher reproducibility at higher throughput on a user-friendly platform. We use simulated data and real data to benchmark our algorithm and show that it reliably detects, tracks, and analyzes dynamic microtubules and achieves sub-pixel precision even at low signal-to-noise ratios. Microtubules are dynamic filaments essential for many cellular processes such as intracellular transport, cell motility and chromosome segregation. They assemble from dimeric αβ-tubulin subunits that polymerize in a head-to-tail fashion into polar filaments 1 (Fig. 1). Microtubules show a behavior termed dynamic instability, which can be empirically described by four parameters: (1) the polymerization velocity at which microtubules grow (vg), (2) the depolymerization velocity at which microtubules shrink (vs), (3) the catastrophe frequency at which microtubules switch from growth to shrinkage (fc), and (4) the rescue frequency at which microtubules switch from shrinkage to growth (fs) 2. This dynamic behavior is intrinsic to microtubules. In a cellular context, however, the dynamic properties of microtubules are modulated by motors and accessory proteins known as microtubule associated proteins (MAPs) 3-7. In most cases, the cellular context is too complex to study a single protein's contribution to microtubule dynamics. Therefore, biochemical activities of individual proteins have primarily been characterized in vitro using purified components and total-internal reflection fluorescence (TIRF) microscopy 8-17. Furthermore, microtubule dynamics are strongly affected by a set of drugs routinely used to treat diseases such as cancer 18 and malaria 19. Owing to their clinical relevance, it is a viable need to understand the exact regulation of microtubule dynamics by a given drug and thereby elucidate the underlying molecular mechanisms. Given the growing interest in biochemical reconstitution systems 3,4,20 , automation of data analysis will unveil the full potential of the experimental approaches as described above. Quantitatively deriving dynamic microtubule parameters from fluorescence microscopy images by manually creating and analyzing kymographs (spatial position over time) is still common practice 21. This limits the collection of statistically significant amounts of data. Moreover, manual analysis can bias data collection and introduce variability. Thus, methods have been developed that allow microtubule detection and/or tracking 22-27. However, to date, there is no fully automated workflow that provides detection and tracking of microtubules followed by automated data analysis and statistics collection. Here, we present the software MTrack, which detects, tracks, measures, and analyses the behavior of fluorescently labeled microtubules imaged by TIRF microscopy
plusTipTracker: Quantitative image analysis software for the measurement of microtubule dynamics
2011
Here we introduce plusTipTracker, a Matlab-based open source software package that combines automated tracking, data analysis, and visualization tools for movies of fluorescently-labeled microtubule (MT) plus end binding proteins (+TIPs). Although +TIPs mark only phases of MT growth, the plusTipTracker software allows inference of additional MT dynamics, including phases of pause and shrinkage, by linking collinear, sequential growth tracks. The algorithm underlying the reconstruction of full MT trajectories relies on the spatially and temporally global tracking framework described in (Jaqaman et al., 2008). Post-processing of track populations yields a wealth of quantitative phenotypic information about MT network architecture that can be explored using several visualization modalities and bioinformatics tools included in plusTipTracker. Graphical user interfaces enable novice Matlab users to track thousands of MTs in minutes. In this paper we describe the algorithms used by plusTipTracker and show how the package can be used to study regional differences in the relative proportion of MT subpopulations within a single cell. The strategy of grouping +TIP growth tracks for the analysis of MT dynamics has been introduced before (Matov et al., 2010). The numerical methods and analytical functionality incorporated in plusTipTracker substantially advance this previous work in terms of flexibility and robustness. To illustrate the enhanced performance of the new software we thus compare computer-assembled +TIP-marked trajectories to manually-traced MT trajectories from the same movie used in (Matov et al., 2010).
Traffic, 2017
High throughput confocal imaging poses challenges in the computational image analysis of complex subcellular structures such as the microtubule cytoskeleton. Here, we developed CellArchitect, an automated image analysis tool that quantifies changes to subcellular patterns illustrated by microtubule markers in plants. We screened microtubule-targeted herbicides and demonstrate that high throughput confocal imaging with integrated image This article is protected by copyright. All rights reserved. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as
Cell Motility and The Cytoskeleton, 1988
The ability to tag biological molecules fluorescently and to detect their distribution in living cells has promoted the study of cytoplasmic organization in general and microtubule dynamics in particular. The techniques that we have selected and developed allowed the determination of spatial and temporal changes of the microtubule network in living fibroblasts at the level of individual microtubules. We have employed two general approaches for determining pattern changes: direct video microscopy and photobleaching and subsequent observation. Direct observation of fluorescent microtubules by high-definition video microscopy provided good spatial resolution at several time points, but was limited to the less congested and thinner periphery of the cell. This approach was made possible by a relatively bright, photostable reporter, xrhodamine-tubulin, and showed that microtubules underwent rounds of assembly and disassembly from their ends. Bleaching and subsequent observation of lysed cells improved the signal to noise ratio by extracting soluble chromophore and permitted observations in congested areas, but was limited to a single time interval. This approach demonstrated that microtubule domains were replaced one by one and that turnover was most rapid at the cell periphery. Antibodies specific for nonbleached chromophore can be used to enhance the signal to noise ratio further or to extend spatial resolution by the use of immunoelectron microscopy. Direct video microscopy and photo-bleaching are two approaches to the study of dynamics that have complementary strengths and wide application to the biology of living cells.
Motion tracking of the outer tips of microtubules
Medical Image Analysis, 2008
Microtubules play numerous critical roles in a cell such as providing structural tracks for the anchoring and movement of vesicles and chromosomes. Also, the assembly of microtubules coordinates cell division and migration. Abnormal function of the assembly is involved in cancer. To date the study of the microtubule assembly dynamics has been done by visual inspection or manually. In this work we have developed a method to automatically track microtubule tips so as to enable high throughput quantitative studies. Our approach first estimates the region where a tip is expected to lie. In that region a tip feature is computed for all time and used to form the tip trajectory. Last, we evaluate our method with phantom data as well as real sequences of fluorescently tagged living cells.
Detection and Tracking of Astral Microtubules in Fluorescence Microscopy Images
2018 25th IEEE International Conference on Image Processing (ICIP), 2018
In this paper we explore detection and tracking of astral microtubules, a sub-population of microtubules which only exists during and immediately before mitosis and aids in the spindle orientation by connecting it to the cell cortex. Its analysis can be useful to determine the presence of certain diseases, such as brain pathologies and cancer. The proposed algorithm focuses on overcoming the problems regarding fluorescence microscopy images and microtubule behaviour by using various image processing techniques and is then compared with three existing algorithms, tested on consistent sets of images.
A new directionality tool for assessing microtubule pattern alterations
Cytoskeleton, 2014
The cytoskeleton (microtubules, actin and intermediate filaments) has a cell type-specific spatial organization that is essential and reflects cell health. We are interested in understanding how changes in the organization of microtubules contribute to muscle diseases such as Duchenne muscular dystrophy (DMD). The grid-like immunofluorescence microtubule pattern of fast-twitch muscle fibers lends itself well to visual assessment. The more complicated pattern of other fibers does not. Furthermore, visual assessment is not quantitative. Therefore we have developed a robust software program for detecting and quantitating microtubule directionality. Such a tool was necessary because existing methods focus mainly on local image features and are not well suited for microtubules. Our tool, TeDT, is based on the Haralick texture method and takes into account both local and global features with more weight on the latter. The results are expressed in a graphic form responsive to subtle variations in microtubule distribution, while a numerical score allows quantitation of directionality. Furthermore, the results are not affected by imaging conditions or post-imaging procedures. TeDT successfully assesses test images and microtubules in fast-twitch fibers of wild-type and mdx mice (a model for DMD); TeDT also identifies and quantitates microtubule directionality in slow-twitch fibers, in the fibers of young animals, and in other mouse models which could not be assessed visually. TeDT might also contribute to directionality assessments of other cytoskeletal components.
Increased visualization of microtubules by an improved fixation procedure
Journal of Histochemistry & Cytochemistry, 1977
We have found that when a buffer utilized for in vitro polymerization of microtubules, i.e., 1 mM guanosine triphosphate, 1 mM MgSO4, 2 mM ethylene glycol bis(beta-aminoethyl ether)-N, N'-tetraacetic acid 100 mM piperazine-N,N'-bis(2-ethanesulfonic acid), pH 6.9 polymerization mix, was used in the glutaraldehyde prefixation regimen instead of classical fixative buffers, i.e., isotonic cacodylate or phosphate buffer, the following features were observed in thin-sections of the cytoplasm of interphase HeLa cells: (a) a greater than 2-fold increase in total microtubule contour length, (b) a 2-fold increase in a number of microtubules greater than or equal to 1 mu long, (c) an enhanced association of microtubules with cytoplasmic organelles, and (d) an increased clustering of 100 A filaments located in a perinuclear region of the cell. Furthermore, we found that after we incubated purified chick brain microtubules on a Sephadex G-25 column pre-equilibrated with polymerization mi...