scholarly journals Microfluidic device enabled quantitative time-lapse microscopic-photography for phenotyping vegetative and reproductive phases in Fusarium virguliforme, which is pathogenic to soybean

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Jill Marshall ◽  
Xuan Qiao ◽  
Jordan Baumbach ◽  
Jingyu Xie ◽  
Liang Dong ◽  
...  
Keyword(s):  
Microfluidic Device ◽  
Time Lapse ◽  
Fusarium Virguliforme ◽  
Reproductive Phases

scholarly journals DeLTA: Automated cell segmentation, tracking, and lineage reconstruction using deep learning

2019 ◽  
Author(s):  
Jean-Baptiste Lugagne ◽  
Haonan Lin ◽  
Mary J. Dunlop
Keyword(s):  
Gene Expression ◽  
Machine Learning ◽  
Image Analysis ◽  
Deep Learning ◽  
Single Cell ◽  
Real Time ◽  
Microfluidic Device ◽  
Time Lapse ◽  
Cell Segmentation ◽  
Analysis Pipeline

AbstractMicroscopy image analysis is a major bottleneck in quantification of single-cell microscopy data, typically requiring human supervision and curation, which limit both accuracy and throughput. To address this, we developed a deep learning-based image analysis pipeline that performs segmentation, tracking, and lineage reconstruction. Our analysis focuses on time-lapse movies of Escherichia coli cells trapped in a “mother machine” microfluidic device, a scalable platform for long-term single-cell analysis that is widely used in the field. While deep learning has been applied to cell segmentation problems before, our approach is fundamentally innovative in that it also uses machine learning to perform cell tracking and lineage reconstruction. With this framework we are able to get high fidelity results (1% error rate), without human supervision. Further, the algorithm is fast, with complete analysis of a typical frame containing ∼150 cells taking <700msec. The framework is not constrained to a particular experimental set up and has the potential to generalize to time-lapse images of other organisms or different experimental configurations. These advances open the door to a myriad of applications including real-time tracking of gene expression and high throughput analysis of strain libraries at single-cell resolution.Author SummaryAutomated microscopy experiments can generate massive data sets, allowing for detailed analysis of cell physiology and properties such as gene expression. In particular, dynamic measurements of gene expression with time-lapse microscopy have proved invaluable for understanding how gene regulatory networks operate. However, image analysis remains a key bottleneck in the analysis pipeline, typically requiring human supervision and a posteriori processing. Recently, machine learning-based approaches have ushered in a new era of rapid, unsupervised image analysis. In this work, we use and repurpose the U-Net deep learning algorithm to develop an image processing pipeline that can not only accurately identify the location of cells in an image, but also track them over time as they grow and divide. As an application, we focus on multi-hour time-lapse movies of bacteria growing in a microfluidic device. Our algorithm is accurate and fast, with error rates near 1% and requiring less than a second to analyze a typical movie frame. This increase in speed and fidelity has the potential to open new experimental avenues, e.g. where images are analyzed on-the-fly so that experimental conditions can be updated in real time.

scholarly journals An automated microfluidic device for time-lapse imaging of mouse embryonic stem cells

2019 ◽  
Vol 13 (5) ◽  
pp. 054102
Author(s):  
Adam F. Laing ◽  
Venkat Tirumala ◽  
Evan Hegarty ◽  
Sudip Mondal ◽  
Peisen Zhao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Microfluidic Device ◽  
Embryonic Stem ◽  
Time Lapse ◽  
Mouse Embryonic Stem Cells ◽  
Time Lapse Imaging

Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

1978 ◽  
Vol 36 (2) ◽  
pp. 650-651
Author(s):  
Raul I. Garcia ◽  
Evelyn A. Flynn ◽  
George Szabo
Keyword(s):  
Direct Injection ◽  
Skin Pigmentation ◽  
Freeze Fracture ◽  
Pigment Granule ◽  
Time Lapse ◽  
Ultrastructural Level ◽  
Lanthanum Tracer ◽  
A Cell

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.

High-voltage Electron Microscopy of astroglial cell whole mounts

1986 ◽  
Vol 44 ◽  
pp. 316-317
Author(s):  
J.N. Turner ◽  
W.G. Shain ◽  
V. Madelian ◽  
R.A. Grassucci ◽  
D.L. Forman
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Time Lapse ◽  
Astroglial Cells ◽  
High Voltage Electron Microscopy ◽  
Rat Glioma ◽  
Voltage Electron ◽  
High Voltage Electron ◽  
Nervous System Function ◽  
Beta Adrenergic Agonist

Homogeneous cultures of astroglial cells have proved useful for studying biochemical, pharmacological, and toxicological responses of astrocytes to effectors of central nervous system function. LRM 55 astroglial cells, which were derived from a rat glioma and maintained in continuous culture, exhibit a number of astrocyte properties (1-3). Stimulation of LRM 55s and astrocytes in primary cell cultures with the beta-adrenergic agonist isoproterenol results in rapid changes of morphology. Studies with time lapse video light microscopy (VLM) and high-voltage electron microscopy (HVEM) have been correlated to changes in intracellular levels of c-AMP. This report emphasizes the HVEM results.

Measurement of spontaneous neuronal membrane activity by time lapse confocal microscopy

1995 ◽  
Vol 53 ◽  
pp. 972-973
Author(s):  
R H. Selinfreund ◽  
A. H. Cornell-Bell
Keyword(s):  
Membrane Potential ◽  
Confocal Microscopy ◽  
Patch Clamp ◽  
Electrical Activity ◽  
Time Lapse ◽  
Neuronal Firing ◽  
Neuronal Membrane ◽  
Pituitary Cells ◽  
Patch Clamp Recording ◽  
Spontaneous Electrical Activity

Cellular electrophysiological properties are normally monitored by standard patch clamp techniques . The combination of membrane potential dyes with time-lapse laser confocal microscopy provides a more direct, least destructive rapid method for monitoring changes in neuronal electrical activity. Using membrane potential dyes we found that spontaneous action potential firing can be detected using time-lapse confocal microscopy. Initially, patch clamp recording techniques were used to verify spontaneous electrical activity in GH4\C1 pituitary cells. It was found that serum depleted cells had reduced spontaneous electrical activity. Brief exposure to the serum derived growth factor, IGF-1, reconstituted electrical activity. We have examined the possibility of developing a rapid fluorescent assay to measure neuronal activity using membrane potential dyes. This neuronal regeneration assay has been adapted to run on a confocal microscope. Quantitative fluorescence is then used to measure a compounds ability to regenerate neuronal firing.The membrane potential dye di-8-ANEPPS was selected for these experiments. Di-8- ANEPPS is internalized slowly, has a high signal to noise ratio (40:1), has a linear fluorescent response to change in voltage.

Five-Dimensional Microscopy using Widefield-Deconvolution: Practical Considerations and Biological Applications

1996 ◽  
Vol 54 ◽  
pp. 268-269
Author(s):  
W.F. Marshall ◽  
K. Oegema ◽  
J. Nunnari ◽  
A.F. Straight ◽  
D.A. Agard ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
High Speed ◽  
Laser Scanning ◽  
Ccd Camera ◽  
Image Plane ◽  
Time Lapse ◽  
Laser Scanning Confocal Microscopy ◽  
Three Dimensions ◽  
Excitation Light ◽  
Cooled Ccd

The ability to image cells in three dimensions has brought about a revolution in biological microscopy, enabling many questions to be asked which would be inaccessible without this capability. There are currently two major methods of three dimensional microscopy: laser-scanning confocal microscopy and widefield-deconvolution microscopy. The method of widefield-deconvolution uses a cooled CCD to acquire images from a standard widefield microscope, and then computationally removes out of focus blur. Using such a scheme, it is easy to acquire time-lapse 3D images of living cells without killing them, and to do so for multiple wavelengths (using computer-controlled filter wheels). Thus, it is now not only feasible, but routine, to perform five dimensional microscopy (three spatial dimensions, plus time, plus wavelength).Widefield-deconvolution has several advantages over confocal microscopy. The two main advantages are high speed of acquisition (because there is no scanning, a single optical section is acquired at a time by using a cooled CCD camera) and the use of low excitation light levels Excitation intensity can be much lower than in a confocal microscope for three reasons: 1) longer exposures can be taken since the entire 512x512 image plane is acquired in parallel, so that dwell time is not an issue, 2) the higher quantum efficiently of a CCD detect over those typically used in confocal microscopy (although this is expected to change due to advances in confocal detector technology), and 3) because no pinhole is used to reject light, a much larger fraction of the emitted light is collected. Thus we can typically acquire images with thousands of photons per pixel using a mercury lamp, instead of a laser, for illumination. The use of low excitation light is critical for living samples, and also reduces bleaching. The high speed of widefield microscopy is also essential for time-lapse 3D microscopy, since one must acquire images quickly enough to resolve interesting events.

Cleavage of Human Embryos: Options and Diversity

Acta Naturae ◽  
2016 ◽  
Vol 8 (3) ◽  
pp. 88-96
Author(s):  
Yu. K. Doronin ◽  
I. V. Senechkin ◽  
L. V. Hilkevich ◽  
M. A. Kurcer
Keyword(s):  
Time Lapse ◽  
Reproductive Technologies ◽  
Human Embryos ◽  
Imaging Data ◽  
Noninvasive Methods ◽  
Topographic Features ◽  
Time Parameters ◽  
Embryo Cleavage ◽  
Time Lapse Imaging ◽  
Developmental Dynamics

In order to estimate the diversity of embryo cleavage relatives to embryo progress (blastocyst formation), time-lapse imaging data of preimplantation human embryo development were used. This retrospective study is focused on the topographic features and time parameters of the cleavages, with particular emphasis on the lengths of cleavage cycles and the genealogy of blastomeres in 2- to 8-cell human embryos. We have found that all 4-cell human embryos have four developmental variants that are based on the sequence of appearance and orientation of cleavage planes during embryo cleavage from 2 to 4 blastomeres. Each variant of cleavage shows a strong correlation with further developmental dynamics of the embryos (different cleavage cycle characteristics as well as lengths of blastomere cycles). An analysis of the sequence of human blastomere divisions allowed us to postulate that the effects of zygotic determinants are eliminated as a result of cleavage, and that, thereafter, blastomeres acquire the ability of own syntheses, regulation, polarization, formation of functional contacts, and, finally, of specific differentiation. This data on the early development of human embryos obtained using noninvasive methods complements and extend our understanding of the embryogenesis of eutherian mammals and may be applied in the practice of reproductive technologies.

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