Three-dimensional localization of immunogold markers using two tilted electron microscope recordings

Starink, J. P. P.; Humbel, Bruno M.; Verkleij, A.J.

doi:10.1016/s0006-3495(95)80399-1

Cited by 7 publications

(9 citation statements)

References 45 publications

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“…As known from any ultra‐small gold conjugate subjected to SE, the here generated NANOGOLD‐silver particles varied in size and shape (eg, Figures B and A), including aggregates of several NANOGOLD particles within the very same silver shell . For merely qualitative TEM‐analyses in 2D particle heterogeneity seems rather an esthetic than fundamental problem.…”

Section: Resultsmentioning

confidence: 85%

“…On the other hand becomes the entire surface of organelles or the cytoskeleton (potentially) accessible to immuno‐reagents, thanks to the partial extraction resulting from membrane perforation. Such samples are, therefore, appropriate for 3D reconstruction of label distribution in the context of organelle morphology . However, a serious limitation of pre‐embedding labeling is its virtual incompatibility with rapid freezing methods for EM (henceforth referred to as cryofixation).…”

Section: Introductionmentioning

confidence: 99%

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Combining high‐pressure freezing with pre‐embedding immunogold electron microscopy and tomography

Hess

Vogel

Yordanov

et al. 2018

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Immunogold labeling of permeabilized whole-mount cells or thin-sectioned material is widely used for the subcellular localization of biomolecules at the high spatial resolution of electron microscopy (EM). Those approaches are well compatible with either 3-dimensional (3D) reconstruction of organelle morphology and antigen distribution or with rapid cryofixation-but not easily with both at once. We describe here a specimen preparation and labeling protocol for animal cell cultures, which represents a novel blend of specifically adapted versions of established techniques. It combines the virtues of reliably preserved organelle ultrastructure, as trapped by rapid freezing within milliseconds followed by freeze-substitution and specimen rehydration, with the advantages of robust labeling of intracellular constituents in 3D through means of pre-embedding NANOGOLD-silver immunocytochemistry. So obtained thin and semi-thick epoxy resin sections are suitable for transmission EM imaging, as well as tomographic reconstruction and modeling of labeling patterns in the 3D cellular context.

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Section: Resultsmentioning

confidence: 85%

Section: Introductionmentioning

confidence: 99%

Combining high‐pressure freezing with pre‐embedding immunogold electron microscopy and tomography

Hess

Vogel

Yordanov

et al. 2018

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“…Such superpositional effects are typical for stained structures in 50-100-nm thick resin or cryosections. Statistical methods (Kemler and Schwarz, 1989), tilting sections (stereology: Slot and Geuze, 1983;van Bergen en Henegouwen and Leunissen, 1986;Stierhof et al, 1991b;Kaufmann et al, 1994), or 3-D reconstruction using EM tomography (Starink et al, 1995) can improve spatial resolution.…”

Section: Labeling Precisionmentioning

confidence: 99%

Preparation, use, and enlargement of ultrasmall gold particles in immunoelectron microscopy

Baschong

Stierhof

1998

Microsc. Res. Tech.

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“…The algorithm was to generate a model particle with random cluster locations on the surface, rotate it in space by three random rotation angles, displace the cluster positions randomly within a sphere of radius ''cluster-noise,'' record projection coordinates including a random ''EM-noise'' displacement, rotate the particle by an additional 45°about a known tilt axis, record a second set of projection coordinates including another random ''EM-noise'' displacement, and finally calculate x, y, and z coordinates for each cluster based on these two sets of projection coordinates. We assumed in the simulation that corresponding particles and clusters in the tilted images were previously identified as well as the direction and magnitude of tilt, noting that algorithms for these tasks are routine and well established (10,27,28). The accuracy of the procedure then depends mostly on the uniformity of cluster positions with respect to the particle (cluster-noise), on the precision to which clusters can be located in the micrograph (EM-noise), and on the number of particles averaged.…”

Section: Simulationsmentioning

confidence: 99%

Single-particle selection and alignment with heavy atom cluster-antibody conjugates

Jensen

Kornberg

1998

Proc. Natl. Acad. Sci. U.S.A.

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A method is proposed for selecting and aligning images of single biological particles to obtain highresolution structural information by cryoelectron microscopy. The particles will be labeled with multiple heavy atom clusters to permit the precise determination of particle locations and relative orientations even when imaged close to focus with a low electron dose, conditions optimal for recording highresolution detail. Heavy atom clusters should also allow selection of images free from many kinds of defects, including specimen movement and particle inhomogeneity. Heavy atom clusters may be introduced in a general way by the construction of ''adaptor'' molecules based on single-chain Fv antibody fragments, consisting of a constant framework region engineered for optimal cluster binding and a variable antigen binding region selected for a specific target. The success of the method depends on the mobility of the heavy atom cluster on the particle, on the precision to which clusters can be located in an image, and on the sufficiency of cluster projections alone to orient and select particles for averaging. The necessary computational algorithms were developed and implemented in simulations that address the feasibility of the method.Electron microscopy of biological specimens is limited in resolution by beam-induced specimen damage because single organic molecules are destroyed by electron irradiation sufficient to reveal structural details. In addition to damaging the specimen, electrons, even at low doses, impair the quality of electron imaging by causing localized heating, specimen movement, and specimen charging. These difficulties can be overcome by image averaging and special data collection techniques (1-3). The possibility of structure determination to atomic resolution has been indicated (4) and in some cases attained (2,5,6).Image averaging to improve the signal-to-noise ratio of low-dose electron micrographs has been accomplished in the past by diffraction from ordered arrays of molecules or by computational methods of aligning the images of single particles. Development of the diffraction approach exploited naturally occurring ordered arrays, such as virus particles, muscle fibers, and two-dimensional (2-D) crystals of membrane proteins (7,8). A general method of forming 2-D crystals was devised to bring a wide range of proteins within reach of the approach (9). The necessity of forming a crystalline specimen nonetheless remains an impediment. It prevents the study of a great many biological objects, including partially irregular or inhomogeneous molecules and molecular complexes. The very large multiprotein complexes of most biological interest are especially prone to these limitations.Escape from the requirement for crystals by computational alignment of single particles relies on the detection of image details to determine the relative orientations of the particles and permit image averaging (10). The very paucity of detail in a low-dose electron micrograph that necessitates averaging unavoida...

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Three-dimensional localization of immunogold markers using two tilted electron microscope recordings

Cited by 7 publications

References 45 publications

Combining high‐pressure freezing with pre‐embedding immunogold electron microscopy and tomography

Combining high‐pressure freezing with pre‐embedding immunogold electron microscopy and tomography

Preparation, use, and enlargement of ultrasmall gold particles in immunoelectron microscopy

Single-particle selection and alignment with heavy atom cluster-antibody conjugates

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