3D Deconvolution Microscopy

Biggs, David S. C.

doi:10.1002/0471142956.cy1219s52

Cited by 78 publications

(70 citation statements)

References 14 publications

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“…Computer algorithms are used to compensate the absence of interplane detail and to reconstruct a three-dimensional image from a rendered z-stack [18]. This method is commonly employed to study the dynamics of IS formation in lateral T-cell-APC conjugates [8,19], and can be used to rapidly acquire threedimensional information of the IS morphology and structure.…”

Section: Introductionmentioning

confidence: 99%

High-resolution imaging of the immunological synapse and T-cell receptor microclustering through microfabricated substrates

Biggs

Milone

Santos

et al. 2011

J. R. Soc. Interface.

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T-cell activation via antigen presentation is associated with the formation of a macromolecular membrane assembly termed the immunological synapse (IS). The genesis of the IS and the onset of juxtacrine signalling is characterized by the formation of cell membrane microclusters and the organization of such into segregated microdomains. A central zone rich in T-cell receptor (TCR) -major histocompatibility complex microclusters termed the central supramolecular activation cluster (cSMAC) forms the bullseye of this structure, while the cellular interface surrounding the cSMAC is characterized by regions enriched in adhesion and co-stimulatory molecules. In vitro, the study of dynamic TCR microcluster coalescence and IS genesis in T-cell populations is hampered by cell migration within the culture system and resolution constraints resulting from lateral cell -cell contact. Here, we detail a novel system describing the fabrication of micropit arrays designed to sequester single T-cell -antigen presenting cell (APC) conjugates and promote IS formation in the horizontal imaging plane for high-resolution studies of microcluster dynamics. We subsequently use this system to describe the formation of the cSMAC in T-cell populations and to investigate the morphology of the interfacial APC membrane.

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Section: Introductionmentioning

confidence: 99%

High-resolution imaging of the immunological synapse and T-cell receptor microclustering through microfabricated substrates

Biggs

Milone

Santos

et al. 2011

J. R. Soc. Interface.

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“…The noise model for wide-field microscopy with its larger photon fluxes approaches Gaussian noise, but wide-field images can still be deconvolved using R-L [13]. With an appropriate PSF, this algorithm can thus be applied to multiple modes of microscopy.…”

Section: Deconvolution Resultsmentioning

confidence: 99%

Real-time GPU-based 3D Deconvolution

Bruce¹,

Butte²

2013

Opt. Express

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Abstract:Confocal microscopy is an oft-used technique in biology. Deconvolution of 3D images reduces blurring from out-of-focus light and enables quantitative analyses, but existing software for deconvolution is slow and expensive. We present a parallelized software method that runs within ImageJ and deconvolves 3D images ~100 times faster than conventional software (few seconds per image) by running on a low-cost graphics processor board (GPU). We demonstrate the utility of this software by analyzing microclusters of T cell receptors in the immunological synapse of a CD4 + T cell and dendritic cell. This software provides a lowcost and rapid way to improve the accuracy of 3D microscopic images obtained by any method.

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“…For very light-sensitive sparsely-labeled samples like our electroporated spinal cord slices, therefore, wide-field microscopy performs better than confocal microscopy. When combined with image restoration by deconvolution, which removes out of focus information and improves contrast, wide-field is then particularly good for detection of small, dim objects 11 . While not appropriate for every tissue imaging experiment, this approach has been very effective for our live cell imaging application.…”

Section: Discussionmentioning

confidence: 99%

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

Das

Wilcock

Swedlow

et al. 2012

JoVE

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The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation 1, 2 , we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy ( Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.This assay may be modified to image a range of embryonic tissues 3,4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling 5 ) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development. Video LinkThe video component of this article can be found at https://www.jove.com/video/3920/ Protocol 1. Dish Preparation 1. Dishes used for slice culture are glass-bottomed (with a coverslip as the base) (WillCo dishes). These are placed on lens tissue in a 6 cm tissue culture dish to keep the glass bottom clean. 2. On the day before the experiment, add 2 ml 0.1% poly-L-lysine solution to the glass bottomed dish and incubate for 5min at room temperature to allow the poly-L-lysine to coat the glass bottom. 3. Remove poly-L-lysine solution and rinse three times in deionised water, and then once in 70% ethanol. 4. Leave to dry overnight. Dishes can also be dried by gently heating in a microwave at low power for 30-45 seconds. Embryo Electroporation1. Incubate eggs at 37 °C to Hamburger-Hamilton (HH) stage 10 (~36 hours) (or other desired stage). 2. Before beginning prepare glass needles (we use a Flaming/Brown model p87 microcapillary puller) and break off the tip of the needle using a fine forceps under a dissecting microscope. The end of the needle should be sharp enough to pierce the embryo while not being so narrow that it impedes injection of the DNA solution. 3. Window eggs and place electrodes (5mm apart) on either side of the embryo. 4. Inject DNA (~0.025 -0.5 μg/μl in deionised water colored with a small amount of fast green) into the neural tube. 5. Apply current -12-17 V three times, 50ms pulse length with 950 ms between pulses. 6. We use low concentrations of DNA and low electroporation voltages to achieve mosaic expression so that we can follow individual cells. 7. Cover window in the eggshell with cellotape and make sure it is sealed. 8. Allow embryos to recove...

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3D Deconvolution Microscopy

Cited by 78 publications

References 14 publications

High-resolution imaging of the immunological synapse and T-cell receptor microclustering through microfabricated substrates

High-resolution imaging of the immunological synapse and T-cell receptor microclustering through microfabricated substrates

Real-time GPU-based 3D Deconvolution

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

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