Quantitative orientation-independent differential interference contrast microscope with fast switching shear direction and bias modulation

Experimental Cell Research

Bruist

et al. 2015

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In order to obtain fine details in 3 dimensions (3D) over time, it is critical for motile biological specimens to be appropriately immobilized. Of the many immobilization options available, the mechanical microcompressor offers many benefits. Our device, previously described, achieves gentle flattening of a cell, allowing us to image finely detailed structures of numerous organelles and physiological processes in living cells. We have imaged protozoa and other small metazoans using differential interference contrast (DIC) microscopy, orientation-independent (OI) DIC, and real-time birefringence imaging using a video-enhanced polychromatic polscope. We also describe an enhancement of our previous design by engineering a new device where the coverslip mount is fashioned onto the top of the base; so the entire apparatus is accessible on top of the stage. The new location allows for easier manipulation of the mount when compressing or releasing a specimen on an inverted microscope. Using this improved design, we imaged immobilized bacteria, yeast, paramecia, and nematode worms and obtained an unprecedented view of cell and specimen details. A variety of microscopic techniques were used to obtain high resolution images of static and dynamic cellular and physiological events.

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A mechanical microcompressor for high resolution imaging of motile specimens

Zinskie

Experimental Cell Research

Bruist

et al. 2015

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“…[1][2][3][4][5][6] The DIC microscope can employ a high numerical aperture (NA) objective and condenser lenses and, therefore, provides good lateral resolution as well as good axial discrimination. The shear distance is usually smaller than the Airy disk radius, 7 and it does not affect the lateral resolution substantially. Very weak features can be seen with good contrast because the image intensity is a sine squared function 8 of the specimen's phase gradient in the direction of the shear.…”

Section: Introductionmentioning

confidence: 99%

Mapping optical path length and image enhancement using quantitative orientation-independent differential interference contrast microscopy

Larkin²,

Biggs

2017

J. Biomed. Opt

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We describe the principles of using orientation-independent differential interference contrast (OI-DIC) microscopy for mapping optical path length (OPL). Computation of the scalar two-dimensional OPL map is based on an experimentally received map of the OPL gradient vector field. Two methods of contrast enhancement for the OPL image, which reveal hardly visible structures and organelles, are presented. The results obtained can be used for reconstruction of a volume image. We have confirmed that a standard research grade light microscope equipped with the OI-DIC and 100 × / 1.3 NA objective lens, which was not specially selected for minimum wavefront and polarization aberrations, provides OPL noise level of ? 0.5 ?? nm and lateral resolution if ? 300 ?? nm at a wavelength of 546 nm. The new technology is the next step in the development of the DIC microscopy. It can replace standard DIC prisms on existing commercial microscope systems without modification. This will allow biological researchers that already have microscopy setups to expand the performance of their systems.

“…The image contrast also depends on the initial phase difference (bias) between the interfering beams. To overcome the limitations of traditional DIC systems, Shribak has developed a quantitative orientation-independent differential interference contrast (OI-DIC) microscope, which allows the bias to be modulated and shear directions to be switched rapidly without mechanically rotating the specimen or the prisms (Fig.6A) (Shribak, 2013). A set of raw DIC images with orthogonal shear directions and different biases is captured within a second.…”

Section: Orientation-independent Differential Interference Contrast Mmentioning

confidence: 99%

Living Cells and Dynamic Molecules Observed with the Polarized Light Microscope: the Legacy of Shinya Inoué

Tani

The Biological Bulletin

Oldenbourg

2016

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In 1948, Shinya Inoué arrived in the United States for graduate studies at Princeton. A year later he came to Woods Hole, starting a long tradition of summer research at MBL, which quickly became Inoué’s scientific home. Primed by his Japanese mentor Katsuma Dan, Inoué followed Dan’s mantra to work with healthy living cells, on a fundamental problem (mitosis), with a unique tool set that he refined for precise and quantitative observations (polarized light microscopy), and a fresh and brilliant mind that was unafraid of challenging current dogma. Building on this potent combination, Inoué contributed landmark observations and concepts in cell biology, including the notion of dynamic fine structures inside living cells in which molecular assemblies such as mitotic spindle fibers exist in a delicate equilibrium with their molecular building blocks suspended in the cytoplasm. In the late 1970s and 80s, Inoué and others at the MBL were instrumental in conceiving of video microscopy, a groundbreaking technique that married light microscopy and electronic imaging and ushered in a revolution in how we know and what we know about living cells and the molecular mechanisms of life. This article recounts some of Inoué’s accomplishments and how his legacy has shaped current activities in polarized light imaging at the MBL.