Time-lapse monitoring of TLR2 ligand internalization with newly developed fluorescent probes

Just two decades ago, life scientists studied biological structure, developmental anatomy and intracellular processes by describing individual snapshots of kinetic events. Today, with so much bioscience research focusing on dynamic processes that occur on the molecular, cellular and whole organ level, it is important to record events as they happen, over seconds, minutes or hours, in living cells. Photographs and camera lucida drawings of fixed, stained cells have given way to live cell imaging using fluorescent probes, warming trays to promote cell viability and cinemicrography as a method of recording events.

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Lanthanum-Doped Hydroxyapatite Nanoparticles as Biocompatible Fluorescent Probes for Cellular Internalization and Biolabeling

Science of Advanced Materials ◽

10.1166/sam.2014.1717 ◽

2014 ◽

Vol 6 (2) ◽

pp. 312-319 ◽

Cited By ~ 16

Author(s):

Sushma Jadalannagari ◽

Ketaki Deshmukh ◽

Anita Kamra Verma ◽

Richa Vohra Kowshik ◽

Sutapa Roy Meenal Ramanan

Keyword(s):

Fluorescent Probes ◽

Cellular Internalization ◽

Hydroxyapatite Nanoparticles

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Time-lapse imaging of cell death in cell culture and whole living organisms using turn-on deep-red fluorescent probes

Journal of Materials Chemistry B ◽

10.1039/c8tb01495g ◽

2018 ◽

Vol 6 (30) ◽

pp. 4963-4971 ◽

Cited By ~ 8

Author(s):

Tia S. Jarvis ◽

Felicia M. Roland ◽

Kyle M. Dubiak ◽

Paul W. Huber ◽

Bradley D. Smith

Keyword(s):

Cell Culture ◽

Cell Death ◽

Fluorescence Imaging ◽

Fluorescent Probes ◽

Time Lapse ◽

Frog Embryo ◽

Non Invasive ◽

Turn On ◽

Living Organisms ◽

Time Lapse Imaging

Targeted solvatochromic probe enables non-invasive, time-lapse fluorescence imaging of cell death in cell culture and living frog embryo.

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Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100084120 ◽

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.

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High-voltage Electron Microscopy of astroglial cell whole mounts

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100143201 ◽

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.

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Measurement of spontaneous neuronal membrane activity by time lapse confocal microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100141238 ◽

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.

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100163800 ◽

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.

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Confocal microscopy of the cytoskeleton in living plant cells following microinjection of fluorescent probes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100146497 ◽

1993 ◽

Vol 51 ◽

pp. 130-131

Author(s):

Ann Cleary

Keyword(s):

Cell Division ◽

Fluorescent Probes ◽

Actin Filaments ◽

Intracellular Transport ◽

Cell Structure ◽

Plant Cells ◽

Cell Plate ◽

Cell Cortex ◽

Living Plant ◽

Rhodamine Phalloidin

Microinjection of fluorescent probes into living plant cells reveals new aspects of cell structure and function. Microtubules and actin filaments are dynamic components of the cytoskeleton and are involved in cell growth, division and intracellular transport. To date, cytoskeletal probes used in microinjection studies have included rhodamine-phalloidin for labelling actin filaments and fluorescently labelled animal tubulin for incorporation into microtubules. From a recent study of Tradescantia stamen hair cells it appears that actin may have a role in defining the plane of cell division. Unlike microtubules, actin is present in the cell cortex and delimits the division site throughout mitosis. Herein, I shall describe actin, its arrangement and putative role in cell plate placement, in another material, living cells of Tradescantia leaf epidermis.The epidermis is peeled from the abaxial surface of young leaves usually without disruption to cytoplasmic streaming or cell division. The peel is stuck to the base of a well slide using 0.1% polyethylenimine and bathed in a solution of 1% mannitol +/− 1 mM probenecid.

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Live Confocal Analysis of Fertilization and Early Development

Microscopy and Microanalysis ◽

10.1017/s1431927600031135 ◽

2001 ◽

Vol 7 (S2) ◽

pp. 1012-1013

Author(s):

Uyen Tram ◽

William Sullivan

Keyword(s):

Drosophila Melanogaster ◽

Embryonic Development ◽

Real Time ◽

Early Development ◽

Fluorescent Probes ◽

Primary Defect ◽

Fluorescent Analysis ◽

Dynamic Event ◽

Enormous Amount ◽

Fluorescent Studies

Embryonic development is a dynamic event and is best studied in live animals in real time. Much of our knowledge of the early events of embryogenesis, however, comes from immunofluourescent analysis of fixed embryos. While these studies provide an enormous amount of information about the organization of different structures during development, they can give only a static glimpse of a very dynamic event. More recently real-time fluorescent studies of living embryos have become much more routine and have given new insights to how different structures and organelles (chromosomes, centrosomes, cytoskeleton, etc.) are coordinately regulated. This is in large part due to the development of commercially available fluorescent probes, GFP technology, and newly developed sensitive fluorescent microscopes. For example, live confocal fluorescent analysis proved essential in determining the primary defect in mutations that disrupt early nuclear divisions in Drosophila melanogaster. For organisms in which GPF transgenics is not available, fluorescent probes that label DNA, microtubules, and actin are available for microinjection.

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Cleavage of Human Embryos: Options and Diversity

Acta Naturae ◽

10.32607/20758251-2016-8-3-88-96 ◽

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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