Fluocell for Ratiometric and High-Throughput Live-Cell Image Visualization and Quantitation.
ABSTRACT: Spatiotemporal regulation of molecular activities dictates cellular function and fate. Investigation of dynamic molecular activities in live cells often requires the visualization and quantitation of fluorescent ratio image sequences with subcellular resolution and in high throughput. Hence, there is a great need for convenient software tools specifically designed with these capabilities. Here we describe a well-characterized open-source software package, Fluocell, customized to visualize pixelwise ratiometric images and calculate ratio time courses with subcellular resolution and in high throughput. Fluocell also provides group statistics and kinetic analysis functions for the quantified time courses, as well as 3D structure and function visualization for ratio images. The application of Fluocell is demonstrated by the ratiometric analysis of intensity images for several single-chain Förster (or fluorescence) resonance energy transfer (FRET)-based biosensors, allowing efficient quantification of dynamic molecular activities in a heterogeneous population of single live cells. Our analysis revealed distinct activation kinetics of Fyn kinase in the cytosolic and membrane compartments, and visualized a 4D spatiotemporal distribution of epigenetic signals in mitotic cells. Therefore, Fluocell provides an integrated environment for ratiometric live-cell image visualization and analysis, which generates high-quality single-cell dynamic data and allows the quantitative machine-learning of biophysical and biochemical computational models for molecular regulations in cells and tissues.
Project description:Ras and Rho small GTPases are critical for numerous cellular processes including cell division, migration, and intercellular communication. Despite extensive efforts to visualize the spatiotemporal activity of these proteins, achieving the sensitivity and dynamic range necessary for in vivo application has been challenging. Here, we present highly sensitive intensiometric small GTPase biosensors visualizing the activity of multiple small GTPases in single cells in vivo. Red-shifted sensors combined with blue light-controllable optogenetic modules achieved simultaneous monitoring and manipulation of protein activities in a highly spatiotemporal manner. Our biosensors revealed spatial dynamics of Cdc42 and Ras activities upon structural plasticity of single dendritic spines, as well as a broad range of subcellular Ras activities in the brains of freely behaving mice. Thus, these intensiometric small GTPase sensors enable the spatiotemporal dissection of complex protein signaling networks in live animals.
Project description:In most cells, transcriptionally inactive heterochromatin is preferentially localized in the nuclear periphery and transcriptionally active euchromatin is localized in the nuclear interior. Different cell types display characteristic chromatin distribution patterns, which change dramatically during cell differentiation, proliferation, senescence and different pathological conditions. Chromatin organization has been extensively studied on a cell population level, but there is a need to understand dynamic reorganization of chromatin at the single cell level, especially in live cells. We have developed a novel image analysis tool that we term Fluorescence Ratiometric Imaging of Chromatin (FRIC) to quantitatively monitor dynamic spatiotemporal distribution of euchromatin and total chromatin in live cells. A vector (pTandemH) assures stoichiometrically constant expression of the histone variants Histone 3.3 and Histone 2B, fused to EGFP and mCherry, respectively. Quantitative ratiometric (H3.3/H2B) imaging displayed a concentrated distribution of heterochromatin in the periphery of U2OS cell nuclei. As proof of concept, peripheral heterochromatin responded to experimental manipulation of histone acetylation. We also found that peripheral heterochromatin depended on the levels of the inner nuclear membrane protein Samp1, suggesting an important role in promoting peripheral heterochromatin. Taken together, FRIC is a powerful and robust new tool to study dynamic chromatin redistribution in live cells.
Project description:Recently, super-resolution microscopy methods such as stochastic optical reconstruction microscopy (STORM) have enabled visualization of subcellular structures below the optical resolution limit. Due to the poor temporal resolution, however, these methods have mostly been used to image fixed cells or dynamic processes that evolve on slow time-scales. In particular, fast dynamic processes and their relationship to the underlying ultrastructure or nanoscale protein organization cannot be discerned. To overcome this limitation, we have recently developed a correlative and sequential imaging method that combines live-cell and super-resolution microscopy. This approach adds dynamic background to ultrastructural images providing a new dimension to the interpretation of super-resolution data. However, currently, it suffers from the need to carry out tedious steps of sample preparation manually. To alleviate this problem, we implemented a simple and versatile microfluidic platform that streamlines the sample preparation steps in between live-cell and super-resolution imaging. The platform is based on a microfluidic chip with parallel, miniaturized imaging chambers and an automated fluid-injection device, which delivers a precise amount of a specified reagent to the selected imaging chamber at a specific time within the experiment. We demonstrate that this system can be used for live-cell imaging, automated fixation, and immunostaining of adherent mammalian cells in situ followed by STORM imaging. We further demonstrate an application by correlating mitochondrial dynamics, morphology, and nanoscale mitochondrial protein distribution in live and super-resolution images.
Project description:Genetically-encoded biosensors based on fluorescence resonance energy transfer (FRET) have been widely applied to study the spatiotemporal regulation of molecular activity in live cells with high resolution. The efficient and accurate quantification of the large amount of imaging data from these single-cell FRET measurements demands robust and automated data analysis. However, the nonlinear movement of live cells presents tremendous challenge for this task. Based on image registration of the single-cell movement, we have developed automated image analysis methods to track and quantify the FRET signals within user-defined subcellular regions. In addition, the subcellular pixels were classified according to their associated FRET signals and the dynamics of the clusters analyzed. The results revealed that the EGF-induced reduction of RhoA activity in migratory HeLa cells is significantly less than that in stationary cells. Furthermore, the RhoA activity is polarized in the migratory cells, with the gradient of polarity oriented toward the opposite direction of cell migration. In contrast, there is a lack of consistent preference in RhoA polarity among stationary cells. Therefore, our image analysis methods can provide powerful tools for high-throughput and systematic investigation of the spatiotemporal molecular activities in regulating functions of live cells with their shapes and positions continuously changing in time.
Project description:Translation is under tight spatial and temporal controls to ensure protein production in the right time and place in cells. Methods that allow real-time, high-resolution visualization of translation in live cells are essential for understanding the spatiotemporal dynamics of translation regulation. Based on multivalent fluorescence amplification of the nascent polypeptide signal, we develop a method to image translation on individual mRNA molecules in real time in live cells, allowing direct visualization of translation events at the translation sites. Using this approach, we monitor transient changes of translation dynamics in responses to environmental stresses, capture distinct mobilities of individual polysomes in different subcellular compartments, and detect 3' UTR-dependent local translation and active transport of polysomes in dendrites of primary neurons.
Project description:High-resolution spatiotemporal imaging of histidine in single living mammalian cells faces technical challenges. Here, we developed a series of ratiometric, highly responsive, and single fluorescent protein-based histidine sensors of wide dynamic range. We used these sensors to quantify subcellular free-histidine concentrations in glucose-deprived cells and glucose-fed cells. Results showed that cytosolic free-histidine concentration was higher and more sensitive to the environment than free histidine in the mitochondria. Moreover, histidine was readily transported across the plasma membrane and mitochondrial inner membrane, which had almost similar transport rates and transport constants, and histidine transport was not influenced by cellular metabolic state. These sensors are potential tools for tracking histidine dynamics inside subcellular organelles, and they will open an avenue to explore complex histidine signaling.
Project description:Dynamic reorganization of photosystems I and II is suggested to occur in chloroplast thylakoid membranes to maintain the efficiency of photosynthesis under fluctuating light conditions. To directly observe the process in action, live-cell imaging techniques are necessary. Using live-cell imaging, we have shown that the fine thylakoid structures in the moss Physcomitrella patens are flexible in time. However, the spatiotemporal resolution of a conventional confocal microscopy limits more precise visualization of entire thylakoid structures and understanding of the structural dynamics. Here, we discuss the issues related to observing chlorophyll fluorescence at multiple spatiotemporal scales in vivo and in vitro.
Project description:Genetically encoded biosensors based on FRET have enabled the visualization of signaling events in live cells with high spatiotemporal resolution. However, the limited sensitivity of these biosensors has hindered their broad application in biological studies. We have paired enhanced CFP (ECFP) with YPet, a variant of YFP. This ECFP/YPet FRET pair markedly enhanced the sensitivity of biosensors (several folds enhancement without the need of tailored optimization for each individual biosensor) for a variety of signaling molecules, including tyrosine kinase Src, small GTPase Rac, calcium, and a membrane-bound matrix metalloproteinase MT1-MMP. The application of these improved biosensors revealed that the activations of Src and Rac by PDGF displayed distinct subcellular patterns during directional cell migration on micropatterned surface. The activity of Rac is highly polarized and concentrated at the leading edge, whereas Src activity is relatively uniform. These FRET biosensors also led to the discovery that Src and Rac mutually regulate each other. Our findings indicate that molecules within the same signaling feedback loop can be differentially regulated at different subcellular locations. In summary, ECFP/YPet may serve as a general FRET pair for the development of highly sensitive biosensors to allow the determination of molecular hierarchies at subcellular locations in live cells.
Project description:Glycans are attractive targets for molecular imaging but have been inaccessible because of their incompatibility with genetically encoded reporters. We demonstrated the noninvasive imaging of glycans in live developing zebrafish, using a chemical reporter strategy. Zebrafish embryos were treated with an unnatural sugar to metabolically label their cell-surface glycans with azides. Subsequently, the embryos were reacted with fluorophore conjugates by means of copper-free click chemistry, enabling the visualization of glycans in vivo at subcellular resolution during development. At 60 hours after fertilization, we observed an increase in de novo glycan biosynthesis in the jaw region, pectoral fins, and olfactory organs. Using a multicolor detection strategy, we performed a spatiotemporal analysis of glycan expression and trafficking and identified patterns that would be undetectable with conventional molecular imaging approaches.
Project description:Carboxylesterases (CEs) are widely distributed enzymes in the human body that catalyze hydrolysis of various endogenous and exogenous substrates. They are directly linked to hepatic drug metabolisms and steatosis, and their regulations are important issues in pharmacological and clinical applications. In this work, we have developed an emission ratiometric two-photon probe (SE1) for quantitatively detecting CE in situ. This probe is based on a translation of intramolecular charge transfer character upon reaction with CE. It shows a sensitive blue-to-yellow emission change in response to human CE activity, easy loading into cells, insensitivity to pH and other metabolites including ROS and RNS, high photostability, and low cytotoxicity. Using live hepatocytes and liver tissues, we found that ratiometric two-photon microscopic imaging with SE1 is an effective tool for monitoring CE activities at the subcellular level in live tissues. This probe will find useful applications in biomedical research, including studies of hepatic steatosis and drug developments.