Video‐enhanced contrast, differential interference contrast (AVEC‐DIC) microscopy: A new method capable of analyzing microtubule‐related motility in the reticulopodial network of allogromia laticollaris

Allen, Robert D.; Allen, Nina S.; Travis, Jeffrey L.

doi:10.1002/cm.970010303

Cited by 411 publications

(147 citation statements)

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“…The movement of axoplasmic organelles on nucleated skeletal muscle actin filaments or endogenous neuronal actin filaments was observed by AVEC-DIC microscopy (Allen et al, 1981Weiss et al, 1989) and video intensified fluorescence microscopy (Weiss et al, 1989). A Zeiss Axiophot microscope (C. Zeiss, Inc., Thornwood, NY) equipped with oil immersion condenser (NA 1.4) and ×100 DIC Plan Neofluar oil objective (NA 1.32) were used.…”

Section: Avec-dic and Fluorescence Microscopymentioning

confidence: 99%

“…A Hamamatsu C2400-07 Newvicon camera was used to acquire DIC images and a Hamamatsu C2400-97 intensified CCD camera system (Hamamatsu Photonics, Inc., Bridgewater, NJ) for fluorescence images. The video signals were first subjected to analog contrast enhancement (Allen et al, 1981Weiss et al, 1989), followed by real-time digital image processing in the following steps: subtraction of an out-of-focus background (mottle pattern), accumulation or averaging of images to increase signal to noise ratio, and finally selection of the desired range of gray levels (Allen and Allen, 1983;Inoué, 1986;Weiss and Maile, 1993). The analog and digital processing of fluorescence and DIC signals was performed in parallel using two ARGUS 10 real time image processors (Hamamatsu Photonics Inc.).…”

Section: Avec-dic and Fluorescence Microscopymentioning

confidence: 99%

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Movement of axoplasmic organelles on actin filaments from skeletal muscle

Kuznetsov

Rivera

Severin

et al. 1994

Cell Motil. Cytoskeleton

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It was recently shown that, in addition to the well‐established microtubule‐dependent mechanism, fast transport of organelles in squid giant axons also occurs in the presence of actin filaments [Kuznetsov et al., 1992, Nature 356:722‐725]. The objectives of this study were to obtain direct evidence of axoplasmic organelle movement on actin filaments and to demonstrate that these organelles are able to move on skeletal muscle actin filaments. Organelles and actin filaments were visualized by video‐enhanced contrast differential interference contrast (AVEC‐DIC) microscopy and by video intensified fluorescence microscopy. Actin filaments, prepared by polymerization of monomeric actin purified from rabbit skeletal muscle, were stabilized with rhodamine‐phalloidin and adsorbed to cover slips. When axoplasm was extruded on these cover slips in the buffer containing cytochalasin B that prevents the formation of endogenous axonal actin filaments, organelles were observed to move at the fast transport rate. Also, axoplasmic organelles were observed to move on bundles of actin filaments that were of sufficient thickness to be detected directly by AVEC‐DIC microscopy. The range of average velocities of movement on the muscle actin filaments was not statistically different from that on axonal filaments. The level of motile activity (number of organelles moving/min/field) on the exogenous filaments was less than on endogenous filaments probably due to the entanglement of filaments on the cover slip surface. We also found that calmodulin (CaM) increased the level of motile activity of organelles on actin filaments. In addition, CaM stimulated the movement of elongated membranous organelles that appeared to be tubular elements of smooth endoplasmic reticulum or extensions of prelysosomes. These studies provide the first direct evidence that organelles from higher animal cells such as neurons move on biochemically defined actin filaments. © 1994 Wiley‐Liss, Inc.

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Section: Avec-dic and Fluorescence Microscopymentioning

confidence: 99%

Section: Avec-dic and Fluorescence Microscopymentioning

confidence: 99%

Movement of axoplasmic organelles on actin filaments from skeletal muscle

Kuznetsov

Rivera

Severin

et al. 1994

Cell Motil. Cytoskeleton

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“…The development of optical and video methods for visualizing individual MTs in cell-free systems (2,29) has led to the discovery and characterization of several motor enzymes that move over a MT surface: kinesin, which generally moves over MTs in vitro toward their plus ends (86)(87)(88)(89), and cytoplasmic dynein, which usually moves in the opposite direction (37,64, and reviewed in reference 45) . More recently, however, members of the kinesin family have been found that move in the same direction as dynein (42,93, and reviewed in reference 11), and a dynein-like protein seems capable of moving in either direction (81) .…”

Section: The Generation Ofmitotic Forcesmentioning

confidence: 99%

Mitotic motors.

McIntosh

Pfarr

1991

The Journal of Cell Biology

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T HE complex but well-controlled motions of chromosomes during mitosis have long evoked the view that the mitotic spindle contains enzymes capable oftransducing chemical energy into mechanical work, but the nature ofthese "motor" molecules has been elusive . During the last 40 years, three ideas have dominated the field : (a) microtubules (MT)' attach to chromosomes and move them by the addition and removal of tubulin; (b) mitotic forces are produced by mechanochemical enzymes, probably ATPases, that interact with MTs; and (c) mitotic forces are generated by muscle proteins. Possibility c now seems remote, but recent data from studies on the dynamics of spindle MTs, on the genes and gene products that are required for normal chromosome motion, and on the time-dependent changes in spindle structure reveal that both possibilities a and b are pertinent for explaining mitotic movements. While it is too soon to say just how chromosomes are moved during cell division, there is now enough information to identify key phenomena and the kinds of proteins involved . This paper is a sketch of the motile events that occur during mitosis and a discussion of the ways that MTs and their associated motor enzymes may cause these motions . Mitotic Motions and Forces MotionsThe essential features of chromosome motion are well known (Fig. 1). The interactions between MTs and chromosomes begin at the transition from prophase to prometaphase; in higher eukaryotes this occurs when the nuclear envelope disperses, while in many lower eukaryotes spindle proteins assemble in the nucleus . MTs grow out from the centrosomes (spindle poles) and interact with the chromosomes and with each other. Initial chromosome movements are usually directed toward the nearby pole and are often comparatively fast (ca. 25 pm/min). One ofthe two sister kinetochores is the principal site at which the force for this motion is generated (71). Eventually sister kinetochores attach to MTs growing from opposite poles, and each chromosome shows a net motion toward the spindle equator. During this "congression" to the metaphase plate, which is usually the sum of many movements toward and away from each pole, the rate ofchromosome motion decreases . Anaphase begins with the splitting of the centromeres, and sister chromatids move apart at 0.5-2 jAm/min . The decrease in distance be-1 . Abbreviations used in this paper : MT, microtubule .

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“…Video-enhanced DIC allows enchantment of contrast while removing unwanted background signal (such as fixed image noise due to dust particles, non-uniform illumination or other imperfections in the optical system) by subtraction of a reference image with no specimen 5 . Also S. Inoué and R.D.…”

Section: Introductionmentioning

confidence: 99%

Orientation-independent differential interference contrast microscopy

Shribak¹,

Inoué²

2008

Collected Works of Shinya Inoué

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The image in a regular DIC microscope reflects the orientation of the prism shear direction and the optical path gradients in a phase specimen. If the shear direction lies parallel to the specimen boundary no contrast is generated. Also a bias retardance is generally introduced, which creates a gray background and reduces image contrast. Here we describe the theoretical foundation for a new DIC technique, which records phase gradients independently of their orientation and with the digitally generated gradient magnitude image as well as the optical path distribution image free from the gray background. Separate images can show the magnitude distribution of the optical path gradients and of the azimuths, or the two images can be combined into one picture e.g., with the brightness representing magnitudes and color showing azimuths respectively.For experimental verification of the proposed technique we investigated various specimens such as glass rods embedded in Permount, Siemens star nano-fabricated in 90-nm thick silicon oxide layer, Bovine pulmonary artery endothelial cell, etc, using regular DIC optics on a microscope equipped with a precision rotating stage. Several images were recorded with the specimen oriented in different directions, but with the prism bias unchanged, followed by digital alignment and processing of the images. The results demonstrate that the proposed DIC technique can successfully image and measure phase gradients of transparent specimens, independent of the 2 directions of the gradient. The orientation-independent DIC data obtained can also be used to compute the quantitative distribution of specimen phase or to generate enhanced, regular DIC images with any desired shear direction.We are currently developing a new device using special DIC prisms, which allows the bias and shear directions to be switched rapidly without the need to mechanically rotate the specimen or the prism (US Patent Application 2005-0152030). With the new system an orientation independent DIC image should be obtained in a fraction of a second. A detailed description of the new system will be given in a future publication.

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Video‐enhanced contrast, differential interference contrast (AVEC‐DIC) microscopy: A new method capable of analyzing microtubule‐related motility in the reticulopodial network of allogromia laticollaris

Cited by 411 publications

References 12 publications

Movement of axoplasmic organelles on actin filaments from skeletal muscle

Movement of axoplasmic organelles on actin filaments from skeletal muscle

Mitotic motors.

Orientation-independent differential interference contrast microscopy

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