Application of Zernike Phase-Contrast Electron Microscopy and Electron Tomography for Frozen Hydrated Complex Biological Specimens

Compared with images of negatively stained single particle specimens, those obtained by cryo-electron microscopy have the following new features: (a) higher “signal” variability due to a higher variability of particle orientation; (b) reduced signal/noise ratio (S/N); (c) virtual absence of low-spatial-frequency information related to elastic scattering, due to the properties of the phase contrast transfer function (PCTF); and (d) reduced resolution due to the efforts of the microscopist to boost the PCTF at low spatial frequencies, in his attempt to obtain recognizable particle images.

Download Full-text

Imaging sub-micron objects with light microscopy: Visualization of the T-4 bacteriophage

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100156158 ◽

1989 ◽

Vol 47 ◽

pp. 834-835

Author(s):

Malcolm Brown ◽

Reynolds M. Delgado ◽

Michael J. Fink

Keyword(s):

Electron Microscopy ◽

Light Microscopy ◽

Focal Plane ◽

Phase Contrast ◽

Dark Field ◽

E Coli ◽

Polystyrene Beads ◽

Television Camera ◽

Transmission Electron ◽

Universal Microscope

While light microscopy has been used to image sub-micron objects, numerous problems with diffraction-limitations often preclude extraction of useful information. Using conventional dark-field and phase contrast light microscopy coupled with image processing, we have studied the following objects: (a) polystyrene beads (88nm, 264nm, and 557mn); (b) frustules of the diatom, Pleurosigma angulatum, and the T-4 bacteriophage attached to its host, E. coli or free in the medium. Equivalent images of the same areas of polystyrene beads and T-4 bacteriophages were produced using transmission electron microscopy.For light microscopy, we used a Zeiss universal microscope. For phase contrast observations a 100X Neofluar objective (N.A.=1.3) was applied. With dark-field, a 100X planachromat objective (N.A.=1.25) in combination with an ultra-condenser (N.A.=1.25) was employed. An intermediate magnifier (Optivar) was available to conveniently give magnification settings of 1.25, 1.6, and 2.0. The image was projected onto the back focal plane of a film or television camera with a Carl Zeiss Jena 18X Compens ocular.

Download Full-text

Capturing Enveloped Viruses on Affinity Grids for Downstream Cryo-Electron Microscopy Applications

Microscopy and Microanalysis ◽

10.1017/s1431927613013937 ◽

2013 ◽

Vol 20 (1) ◽

pp. 164-174 ◽

Cited By ~ 10

Author(s):

Gabriella Kiss ◽

Xuemin Chen ◽

Melinda A. Brindley ◽

Patricia Campbell ◽

Claudio L. Afonso ◽

...

Keyword(s):

Electron Microscopy ◽

Measles Virus ◽

Influenza A ◽

Electron Tomography ◽

Three Dimensional ◽

Nitrilotriacetic Acid ◽

Dimensional Structure ◽

Enveloped Viruses ◽

Cryo Electron Microscopy ◽

Human Immunodeficiency Virus Virus

AbstractElectron microscopy (EM), cryo-electron microscopy (cryo-EM), and cryo-electron tomography (cryo-ET) are essential techniques used for characterizing basic virus morphology and determining the three-dimensional structure of viruses. Enveloped viruses, which contain an outer lipoprotein coat, constitute the largest group of pathogenic viruses to humans. The purification of enveloped viruses from cell culture presents certain challenges. Specifically, the inclusion of host-membrane-derived vesicles, the complete destruction of the viruses, and the disruption of the internal architecture of individual virus particles. Here, we present a strategy for capturing enveloped viruses on affinity grids (AG) for use in both conventional EM and cryo-EM/ET applications. We examined the utility of AG for the selective capture of human immunodeficiency virus virus-like particles, influenza A, and measles virus. We applied nickel-nitrilotriacetic acid lipid layers in combination with molecular adaptors to selectively adhere the viruses to the AG surface. This further development of the AG method may prove essential for the gentle and selective purification of enveloped viruses directly onto EM grids for ultrastructural analyses.

Download Full-text

Lattice Micropatterning of Electron Microscopy Grids for Improved Cellular Cryo-Electron Tomography Throughput

Biophysical Journal ◽

10.1016/j.bpj.2020.11.1218 ◽

2021 ◽

Vol 120 (3) ◽

pp. 173a

Author(s):

Leeya Engel ◽

Claudia G. Vasquez ◽

Elizabeth A. Montabana ◽

Belle M. Sow ◽

Marcin P. Walkiewicz ◽

...

Keyword(s):

Electron Microscopy ◽

Electron Tomography ◽

Cryo Electron Tomography

Download Full-text

THE SUBMICROSCOPIC ORGANIZATION OF AXON MATERIAL ISOLATED FROM MYELIN NERVE FIBERS

Journal of Experimental Medicine ◽

10.1084/jem.98.3.269 ◽

1953 ◽

Vol 98 (3) ◽

pp. 269-276 ◽

Cited By ~ 20

Author(s):

E. De Robertis ◽

C. M. Franchi

Keyword(s):

Electron Microscopy ◽

Electron Microscope ◽

Phase Contrast ◽

Nerve Fibers ◽

Myelinated Nerve ◽

Myelinated Nerve Fibers ◽

Dense Granules ◽

Small Clusters ◽

Membranous Material ◽

The Way

A technique has been developed for the extrusion of axon material from myelinated nerve fibers. This material is then compressed and prepared for observation with the electron microscope. All the stages of preparation and purification of the axon material can be checked microscopically and in the present paper they are illustrated with phase contrast photomicrographs. Observation with the electron microscope of the compressed axons showed the presence of the following components: granules, fibrils, and a membranous material. Only the larger granules could be seen with the ordinary microscope. A considerable number of dense granules were observed. Of these the largest resemble typical mitochondria of 250 mµ by 900 mµ. In addition rows or small clusters of dense granules ranging in diameter from 250 to 90 mµ were present. In several specimens fragments of a membrane 120 to 140 A thick and intimately connected with the axon were found. The entire axon appeared to be constituted of a large bundle of parallel tightly packed fibrils among which the granules are interspersed. The fibrils are of indefinite length and generally smooth. They are rather labile structures, less resistant in the rat than in the toad nerve. They varied between 100 and 400 A in diameter and in some cases disintegrated into very fine filaments (less than 100 A thick). The significance is discussed of the submicroscopic structures revealed by electron microscopy of the material prepared in the way described.

Download Full-text

HIGH RESOLUTION ELECTRON MICROSCOPE STUDY OF CdTe-Cd0.6Mn0.4 Te SUPERLATTICES

MRS Proceedings ◽

10.1557/proc-56-235 ◽

1985 ◽

Vol 56 ◽

Cited By ~ 1

Author(s):

C. CHOI ◽

N. OTSUKA ◽

L. A. KOLODZIEJSKI ◽

R. L. GUNSHOR-a

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Phase Contrast ◽

Electron Microscope Study ◽

Lattice Mismatch ◽

Thick Layer ◽

High Resolution Electron Microscopy ◽

Misfit Dislocations ◽

Resolution Electron ◽

High Resolution Electron Microscope

AbstractStructures of CdTe-Cd0.6Mn0.4Te superlattices which are caused by the lattice mismatch between suterlattice layers have been studied by high resolution electron microscopy (HREM). In thin-layer superlattices, the crystal lattice in each layeris elastically distorted, resulting in the change of the crystal symmetry from cubic to rhombohedral. The presence of the small rhombohedral distrotion has been confirmed through a phase contrast effect in HREM images. In a thick-layer superlattice, the lattice mismatch is accommodated by dissociated misfit dislocations. Burgers vectors of partial misfit dislocations have been identified from the shift of lattice fringes in HREM images.

Download Full-text

Efficient linear phase contrast in scanning transmission electron microscopy with matched illumination and detector interferometry

Nature Communications ◽

10.1038/ncomms10719 ◽

2016 ◽

Vol 7 (1) ◽

Cited By ~ 60

Author(s):

Colin Ophus ◽

Jim Ciston ◽

Jordan Pierce ◽

Tyler R. Harvey ◽

Jordan Chess ◽

...

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Scanning Transmission Electron Microscopy ◽

Phase Contrast ◽

Linear Phase ◽

Scanning Transmission Electron ◽

Transmission Electron ◽

Scanning Transmission

Download Full-text

Phase-contrast and electron-microscopic observations on a membranous complex in primary spermatocytes of the mouse

Journal of Cell Science ◽

10.1242/jcs.18.1.1 ◽

1975 ◽

Vol 18 (1) ◽

pp. 1-17

Author(s):

A. Pleshkewych ◽

L. Levine

Keyword(s):

Electron Microscopy ◽

Endoplasmic Reticulum ◽

Phase Contrast ◽

Cultured Cells ◽

Light And Electron Microscopy ◽

Cytoplasmic Inclusion ◽

Electron Microscopic ◽

Secondary Spermatocyte ◽

Living Mouse ◽

Circular Contour

A prominent cytoplasmic inclusion present in living mouse primary spermatocytes has been observed by both light and electron microscopy. It began to form at prometaphase and continued to increase in thickness and length as the cells developed. By metaphase it was a distinct sausage-shaped boundary that enclosed a portion of the cytoplasm between the spindle and the cell membrane. At the end of metaphase, the inclusion reached its maximum length. At telophase, it was divided between the daughter secondaries. The inclusion persisted as a circular contour in the interphase secondary spermatocyte. Electron microscopy of the same cultured cells that were previously observed with light microscopy revealed that the inclusion was a distinctive formation of membranes. It consisted of agranular cisternae and vesicles, and was therefore a membranous complex. Many of the smaller vesicles in the membranous complex resembled those found in the spindle. The cisternae in the membranous complex were identical to the cisternal endoplasmic reticulum of interphase primary spermatocytes. Nevertheless, the organization of vesicles and cisternae into the membranous complex was unique for the primaries in division stages, since such an organization was not present in their interphase stages.

Download Full-text

Coelomomyces canadense stat. et comb. nov. (Blastocladiales: Coelomomycetaceae) from Psectrocladius sp. G (Diptera: Chironomidae)

Canadian Journal of Botany ◽

10.1139/b78-276 ◽

1978 ◽

Vol 56 (18) ◽

pp. 2303-2306 ◽

Cited By ~ 1

Author(s):

Richard A. Nolan

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Phase Contrast ◽

Field Phase ◽

Species Status ◽

Bright Field ◽

Latin Diagnosis ◽

Taxonomic Criterion ◽

Sporangial Wall ◽

Scanning Electron

Resistant sporangia of Coelomomyces chironomi var. canadense Weiser and McCauley were examined by bright-field, phase-contrast, and scanning electron microscopy (SEM). The use of SEM facilitated the observation of previously undescribed complex furrows in the sporangial wall. The taxonomic criterion for varietal status is discussed, and the variety is elevated to species status. Coelomomyces canadense (Weiser and McCauley) Nolan stat. et comb. nov. is described with an emended Latin diagnosis.

Download Full-text