Automated tracking of gene expression in individual cells and cell compartments.
ABSTRACT: Many intracellular signal transduction processes involve the reversible translocation from the cytoplasm to the nucleus of transcription factors. The advent of fluorescently tagged protein derivatives has revolutionized cell biology, such that it is now possible to follow the location of such protein molecules in individual cells in real time. However, the quantitative analysis of the location of such proteins in microscopic images is very time consuming. We describe CellTracker, a software tool designed for the automated measurement of the cellular location and intensity of fluorescently tagged proteins. CellTracker runs in the MS Windows environment, is freely available (at http://www.dbkgroup.org/celltracker/), and combines automated cell tracking methods with powerful image-processing algorithms that are optimized for these applications. When tested in an application involving the nuclear transcription factor NF-kappaB, CellTracker is competitive in accuracy with the manual human analysis of such images but is more than 20 times faster, even on a small task where human fatigue is not an issue. This will lead to substantial benefits for time-lapse-based high-content screening.
Project description:Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics(1,2). One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization(1). Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs(1-3). Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker(4), following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones(5). This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types(6-8).
Project description:The genetic expression of cloned fluorescent proteins coupled to time-lapse fluorescence microscopy has opened the door to the direct visualization of a wide range of molecular interactions in living cells. In particular, the dynamic translocation of proteins can now be explored in real time at the single-cell level. Here we propose a reliable, easy-to-implement, quantitative image processing method to assess protein translocation in living cells based on the computation of spatial variance maps of time-lapse images. The method is first illustrated and validated on simulated images of a fluorescently-labeled protein translocating from mitochondria to cytoplasm, and then applied to experimental data obtained with fluorescently-labeled hexokinase 2 in different cell types imaged by regular or confocal microscopy. The method was found to be robust with respect to cell morphology changes and mitochondrial dynamics (fusion, fission, movement) during the time-lapse imaging. Its ease of implementation should facilitate its application to a broad spectrum of time-lapse imaging studies.
Project description:Cellulose nanofibers (CNFs) have great potential to be a layer in packaging materials because of their good barrier properties. When paper is coated with CNFs, they are difficult to distinguish from the base sheet. This issue creates challenges when trying to determine where CNFs migrate relative to the paper fibers during coating and drying. A three- dimensional analysis is possible by using confocal laser scanning microscopy (CLSM) if CNFs can be tagged with fluorescently active groups. In this study, CNFs were fluorescently tagged through adsorption of fluorescent dyes such as fluorescein isothiocyanate (FITC) and thioflavin by mixing with CNFs in their native suspension followed by purification. The adsorbed dye remained attached during typical coating procedures, low pH values, and high ionic strengths, but not for high pH and in contact with acetone. CNFs were also covalently tagged with FITC following methods reported in the literature as a comparison to already established methods for tagging cellulose nanocrystals (CNCs). Images of never dried samples indicated that covalently tagging CNFs altered the state of the fines dispersion, while dye adsorption did not. Coatings of the adsorbed dye tagged CNFs on paper were successfully imaged by CLSM since the concentration of dye in the water phase was low enough to provide a good contrast between regions of CNFs and paper. With this method, the location and potential migration of CNFs coated on paper were successfully determined for the first time to the best of our knowledge. CNF based coatings with solids larger than 2.8% were found to have a distinct layer of CNFs at the paper surface with little CNFs penetrating into the paper structure, but lower solids result in significant penetration into the paper.
Project description:Assessing the cytoplasmic uptake of fluorescently-tagged drugs in heterogeneous cell types currently involves time-consuming manual segmentation of confocal microscopy images. We developed a set of methods that incorporate map algebra techniques to facilitate and expedite image segmentation, particularly of the parenchyma of intermediate cells in the stria vascularis of the inner ear. Map algebra is used to apply a convolution kernel to pixel neighborhoods to create a masking image to select pixels in the original image for further operations. Here, we describe the utility of integrated intensity-based, percentile-based, and local autocorrelation-based methods to automate segmentation of images into putative morphological regions for pixel intensity analysis. Integrated intensity-based methods are variants of watershed segmentation tools that determine morphological boundaries from rates of change in integrated pixel intensity. Percentile- and local autocorrelation-based methods evolved out of the process of developing map algebra- and integrated intensity-based tools. We identified several simplifications that are surprisingly effective for image segmentation and pixel intensity analysis. These methods were empirically validated on three levels: first, the algorithms were developed based on iterations of inspected results; second, algorithms were tested for various types of robustness; and third, developed algorithms were validated against results from manually-segmented images. We conclude the key to automated segmentation is supervision of output data.
Project description:In patients with cancer receiving potentially cardio-toxic chemotherapy, measurements of left ventricular (LV) circumferential or longitudinal strain are often used clinically to identify myocardial dysfunction. Using a new software algorithm, we sought to determine in individuals receiving treatment for cancer the association between automated assessments of LV mean mid-wall circumferential strain and conventional measures of LV ejection fraction (EF) both obtained from cardiovascular magnetic resonance (CMR) cine balanced steady-state free-precession (bSSFP) white-blood acquisitions.Before and 3 months after initiating treatment with potentially cardio-toxic chemotherapy, 72 individuals (aged 54 ± 14 years with breast cancer [39%], lymphoma [49%], or sarcoma [12%]) underwent serial CMR cine bSSFP assessments of LV volumes and EF, and mean mid-wall circumferential strain determined from these same cine images as well as from additional tagged CMR images. On the cine images, assessments of strain were obtained using the newly developed deformation-based segmentation algorithm. Assessments of LV volumes/EF from the cine images and strain from tagged CMR were accomplished using commercially available software. All measures were analyzed in a blinded fashion independent of one another.Acceptable measures for the automated assessments of mean mid-wall circumferential strain from the cine images were obtained in 142 of 144 visits (98.6%) with an overall analysis time averaging 6:47 ± 1:06 min. The results from these automated measures averaged -18.8 ± 2.9 at baseline and -17.6 ± 3.1 at 3 months (p = 0.001). Left ventricular EF declined slightly from 65 ± 7% at baseline to 62 ± 7% at 3 months (p = 0.0002). The correlation between strain from cine imaging and LVEF was r = -0.61 (p < 0.0001). In addition, the 3-month changes in LV strain and LVEF were correlated (r = -0.49; p < 0.0001). The correlation between cine and tagged derived assessments of strain was r = 0.23; p = 0.01.Automated measures of LV mean mid-wall circumferential strain can be obtained in 6¾ minutes from cine bSSFP LV short-axis images (used concurrently to assess LV volumes and EF) in 98.6% of patients receiving treatment for cancer with potentially cardio-toxic chemotherapy. These cine derived measures of circumferential strain correlate with early subclinical declines in LVEF.
Project description:Previously we described a method to estimate the average number of virus genomes expressed in an infected cell. By analyzing the color spectrum of cells infected with a mixture of isogenic pseudorabies virus (PRV) recombinants expressing three fluorophores, we estimated that fewer than seven incoming genomes are expressed, replicated, and packaged into progeny per cell. In this report, we expand this work and describe experiments demonstrating the generality of the method, as well as providing more insight into herpesvirus replication. We used three isogenic PRV recombinants, each expressing a fluorescently tagged VP26 fusion protein (VP26 is a capsid protein) under the viral VP26 late promoter. We calculated a similar finite limit on the number of expressed viral genomes, indicating that this method is independent of the promoter used to transcribe the fluorophore genes, the time of expression of the fluorophore (early versus late), and the insertion site of the fluorophore gene in the PRV genome (UL versus US). Importantly, these VP26 fusion proteins are distributed equally in punctate virion assembly structures in each nucleus, which improves the signal-to-noise ratio when determining the color spectrum of each cell. To understand how the small number of genomes are distributed among the replication compartments, we used a two-color fluorescent in situ hybridization assay. Most viral replication compartments in the nucleus occupy unique nuclear territories, implying that they arose from single genomes. Our experiments suggest a correlation between the small number of expressed viral genomes and the limited number of replication compartments.
Project description:Nitric oxide (NO) plays an essential role within the immune system since it is involved in the break-down of infectious agents such as viruses and bacteria. The ability to measure the presence of NO in the intracellular environment would provide a greater understanding of the pathophysiological mechanism of this important molecule. Here we report the detection of NO from the intracellular phagolysosome using a fluorescently tagged metalloprotein-gold nanoparticle conjugate. The metalloprotein cytochrome c, fluorescently tagged with an Alexa Fluor dye, was self-assembled onto gold nanoparticles to produce a NO specific nanobiosensor. Upon binding of NO, the cytochrome c protein changes conformation which induces an increase of fluorescence intensity of the tagged protein proportional to the NO concentration. The nanobiosensor was sensitive to NO in a reversible and selective manner, and exhibited a linear response at NO concentrations between 1 and 300 μM. In RAW264.7γ NO- macrophage cells, the nanobiosensor was used to detect the presence of NO that had been endogenously generated upon stimulation of the cells with interferon-γ and lipopolysaccharide, or spontaneously released following treatment of the cells with a NO donor. Significantly, the nanobiosensor was shown to be taken up by the macrophages within phagolysosomes, i.e., the precise location where the NO, together with other species, destroys bacterial infection. The nanobiosensor measured, for the first time, increasing concentrations of NO produced during combined stimulation and phagocytosis of Escherichia coli bacteria from within localised intracellular phagolysosomes, a key part of the immune system.
Project description:BACKGROUND:As sequence data sets used for the investigation of pathogen transmission patterns increase in size, automated tools and standardized methods for cluster analysis have become necessary. We have developed an automated Cluster Picker which identifies monophyletic clades meeting user-input criteria for bootstrap support and maximum genetic distance within large phylogenetic trees. A second tool, the Cluster Matcher, automates the process of linking genetic data to epidemiological or clinical data, and matches clusters between runs of the Cluster Picker. RESULTS:We explore the effect of different bootstrap and genetic distance thresholds on clusters identified in a data set of publicly available HIV sequences, and compare these results to those of a previously published tool for cluster identification. To demonstrate their utility, we then use the Cluster Picker and Cluster Matcher together to investigate how clusters in the data set changed over time. We find that clusters containing sequences from more than one UK location at the first time point (multiple origin) were significantly more likely to grow than those representing only a single location. CONCLUSIONS:The Cluster Picker and Cluster Matcher can rapidly process phylogenetic trees containing tens of thousands of sequences. Together these tools will facilitate comparisons of pathogen transmission dynamics between studies and countries.
Project description:Femtosecond laser nanosurgery has been widely accepted as an axonal injury model, enabling nerve regeneration studies in the small model organism, Caenorhabditis elegans. To overcome the time limitations of manual worm handling techniques, automation and new immobilization technologies must be adopted to improve throughput in these studies. While new microfluidic immobilization techniques have been developed that promise to reduce the time required for axotomies, there is a need for automated procedures to minimize the required amount of human intervention and accelerate the axotomy processes crucial for high-throughput. Here, we report a fully automated microfluidic platform for performing laser axotomies of fluorescently tagged neurons in living Caenorhabditis elegans. The presented automation process reduces the time required to perform axotomies within individual worms to ?17 s/worm, at least one order of magnitude faster than manual approaches. The full automation is achieved with a unique chip design and an operation sequence that is fully computer controlled and synchronized with efficient and accurate image processing algorithms. The microfluidic device includes a T-shaped architecture and three-dimensional microfluidic interconnects to serially transport, position, and immobilize worms. The image processing algorithms can identify and precisely position axons targeted for ablation. There were no statistically significant differences observed in reconnection probabilities between axotomies carried out with the automated system and those performed manually with anesthetics. The overall success rate of automated axotomies was 67.4±3.2% of the cases (236/350) at an average processing rate of 17.0±2.4 s. This fully automated platform establishes a promising methodology for prospective genome-wide screening of nerve regeneration in C. elegans in a truly high-throughput manner.