Term Live Cell Imaging Research Articles

Lysosome-targeting pH indicator based on peri-fused naphthalene monoimide with superior stability for long term live cell imaging.

Lysosomes, the acidic degradation compartments of eukaryotic cells, play an essential role in many physiological processes. Their dysfunction is associated with a number of diseases, which are often related to an altered localization or luminal pH. Thus, the in-depth characterization of lysosomes within the intact eukaryotic cell is of utmost interest. For microscopic evaluation of lysosomal distribution and acidity, a number of labels have been developed, but many showed poor organelle specificity or rapid clearing from lysosomes, rendering them unsuitable for long-term observations. Here, we describe the synthesis and spectroscopic properties of a novel small molecule marker for lysosomes based on naphthalene monoimide with reversible, pH-dependent spectral shifts in both the absorption and the emission spectrum and acidity-associated changes in fluorescence lifetime. The dye can be excited either with single- or two-photon excitation and appears to be very stably associated with lysosomes for several days. We used this chromophore to detect chemically-induced changes of lysosomal pH in HeLa cells by ratiometric and FLIM imaging.

Three - Dimensional Culture For Continuous Long Term Live Cell Imaging

Three - Dimensional Culture For Continuous Long Term Live Cell Imaging

Holo-UNet: hologram-to-hologram neural network restoration for high fidelity low light quantitative phase imaging of live cells.

Intensity shot noise in digital holograms distorts the quality of the phase images after phase retrieval, limiting the usefulness of quantitative phase microscopy (QPM) systems in long term live cell imaging. In this paper, we devise a hologram-to-hologram neural network, Holo-UNet, that restores high quality digital holograms under high shot noise conditions (sub-mW/cm2 intensities) at high acquisition rates (sub-milliseconds). In comparison to current phase recovery methods, Holo-UNet denoises the recorded hologram, and so prevents shot noise from propagating through the phase retrieval step that in turn adversely affects phase and intensity images. Holo-UNet was tested on 2 independent QPM systems without any adjustment to the hardware setting. In both cases, Holo-UNet outperformed existing phase recovery and block-matching techniques by ∼ 1.8 folds in phase fidelity as measured by SSIM. Holo-UNet is immediately applicable to a wide range of other high-speed interferometric phase imaging techniques. The network paves the way towards the expansion of high-speed low light QPM biological imaging with minimal dependence on hardware constraints.

Long-term adult human brain slice cultures as a model system to study human CNS circuitry and disease

Most of our knowledge on human CNS circuitry and related disorders originates from model organisms. How well such data translate to the human CNS remains largely to be determined. Human brain slice cultures derived from neurosurgical resections may offer novel avenues to approach this translational gap. We now demonstrate robust preservation of the complex neuronal cytoarchitecture and electrophysiological properties of human pyramidal neurons in long-term brain slice cultures. Further experiments delineate the optimal conditions for efficient viral transduction of cultures, enabling 'high throughput' fluorescence-mediated 3D reconstruction of genetically targeted neurons at comparable quality to state-of-the-art biocytin fillings, and demonstrate feasibility of long term live cell imaging of human cells in vitro. This model system has implications toward a broad spectrum of translational studies, regarding the validation of data obtained in non-human model systems, for therapeutic screening and genetic dissection of human CNS circuitry.

Abstract 4835: A robust, non-destructive image analysis method for the quantitation and characterization of patient derived organoids

Abstract Patient-derived organoids are an emerging 3D model system that more closely recapitulate the organ functionality of the tissue of origin compared to traditional 2D cell lines. Further advantages to the organoid system include tunability i.e. adding additional cell types, adjusting matrix stiffness, and establishing nutrient gradients, in a physiologically relevant setting, as well as the ability to quickly scale up from a small initial sample. We have established an actively expanding biobank of primary and metastatic colorectal cancer (CRC) tumor organoids under normoxic (21% O2) and physioxic (5% O2) conditions, from a diverse patient population. Concurrently, we isolated and cultured matched cancer-associated fibroblasts (CAFs). With these samples we are able to generate data from a cohort that mimics the CRC population at large, including different ethnic groups, and mutational status. Here we detail a quantitative imaging platform that captures 3D morphometric information in addition to traditional live/dead readouts. Organoids from each patient are heterogeneous in size, shape, symmetry, and various other phenotypic features. Furthermore, the perturbation of microenviromental factors i.e. drugs, CAFs, etc. cause phenotypic changes over time. Common population-based viability assays used in drug screens, such as ATP or MTT, may be inappropriate to capture the complexities of drug response since they are often single end point measurements of an entire population of cells, and do not account for phenotypic variations. Short term quantitative live cell imaging incorporates phenotypic information, and can extend the lifetime of samples, yet still require manipulating samples by using dyes. However, these dyes are phototoxic, making them less than ideal for long term live cell imaging. By restricting image acquisition to brightfield we are able to minimize manipulation of patient samples, leaving them intact and viable. The additional benefit of imaging organoids in 3D enables us to get spatial information not available from assays with a single readout. Using our non-destructive imaging technique we can track 3D morphometric changes in the same organoid population over time. By using a flexible analysis method, and unbiased machine learning algorithms to determine relevant features we can account for these differences yet still get comparable data. Our organoid repository combined with long term image analysis and machine learning techniques provide a robust platform that is flexible enough to handle the heterogeneity seen across the patient population. This platform could be used to advance personalized medicine allowing clinicians to quickly and more accurately determine the appropriate treatment for a patient by screening their tumor prior to determining a course of treatment. Citation Format: Erin Spiller, Roy Lau, Colin Flinders, Shannon Mumenthaler. A robust, non-destructive image analysis method for the quantitation and characterization of patient derived organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4835. doi:10.1158/1538-7445.AM2017-4835

Phase modulation nanoscopy: a simple approach to enhanced optical resolution.

A new modular super-resolution technique called Phase Modulation Nanoscopy (PhMoNa) has been developed in order to break the optical diffraction barrier in Confocal Laser Scanning Microscopy (LSCM). This technique is based on using spatially modulated illumination intensity, whilst harnessing the fluorophore's non-linear emission response. It allows experimental resolution in both lateral and axial domains to be improved by at least a factor of 2. The work is in its initial phase, but by using a custom built Electro Optical Modulator (EOM) in conjunction with functionalised Ln(III) complexes as probes, a sub-diffraction resolution of ∼60 nm was achieved of selected cellular organelles in long term live cell imaging experiments.

THU0511 Macrophage Growth by Polyploidization in Chronic Inflammation

BackgroundHow macrophages grow at sites of chronic inflammation is not well understood. In rheumatoid arthritis and several granulomatous autoimmune diseases, such as sarcoidosis, giant cell arteriitis and granulomatosis with polyangiitis,...

Monitoring the dynamics of primary T cell activation and differentiation using long term live cell imaging in microwell arrays

Methods that allow monitoring of individual cells over time, using live cell imaging, are essential for studying dynamical cellular processes in heterogeneous cell populations such as primary T lymphocytes. However, applying single cell time-lapse microscopy to study activation and differentiation of these cells was limited due to a number of reasons. First, primary naïve T cells are non-adherent and become highly motile upon activation through their antigen receptor. Second, CD4(+) T cell differentiation is a relatively slow process which takes 3-4 days. As a result, long-term dynamic monitoring of individual cells during the course of activation and differentiation is challenging as cells rapidly escape out of the microscope field of view. Here we present and characterize a platform which enables capture and growth of primary T lymphocytes with minimal perturbation, allowing for long-term monitoring of cell activation and differentiation. We use standard cell culture plates combined with PDMS based arrays containing thousands of deep microwells in which primary CD4(+) T cells are trapped and activated by antigen coated microbeads. We demonstrate that this system allows for live cell imaging of individual T cells for up to 72 h, providing quantitative data on cell proliferation and death times. In addition, we continuously monitor dynamics of gene expression in those cells, of either intracellular proteins using cells from transgenic mice expressing fluorescent reporter proteins, or cell surface proteins using fluorescently labeled antibodies. Finally, we show how intercellular interactions between different cell types can be investigated using our device. This system provides a new platform in which dynamical processes and intercellular interactions within heterogeneous populations of primary T cells can be studied at the single cell level.

A modified agar pad method for mycobacterial live-cell imaging

BackgroundTwo general approaches to prokaryotic live-cell imaging have been employed to date, growing bacteria on thin agar pads or growing bacteria in micro-channels. The methods using agar pads 'sandwich' the cells between the agar pad on the bottom and a glass cover slip on top, before sealing the cover slip. The advantages of this technique are that it is simple and relatively inexpensive to set up. However, once the cover slip is sealed, the environmental conditions cannot be manipulated. Furthermore, desiccation of the agar pad, and the growth of cells in a sealed environment where the oxygen concentration will be in gradual decline, may not permit longer term studies such as those required for the slower growing mycobacteria.FindingsWe report here a modified agar pad method where the cells are sandwiched between a cover slip on the bottom and an agar pad on top of the cover slip (rather than the reverse) and the cells viewed from below using an inverted microscope. This critical modification overcomes some of the current limitations with agar pad methods and was used to produce time-lapse images and movies of cell growth for Mycobacterium smegmatis and Mycobacterium bovis BCG.ConclusionsThis method offers improvement on the current agar pad methods in that long term live cell imaging studies can be performed and modification of the media during the experiment is permitted.