Cryo-negative staining reduces electron-beam sensitivity of vitrified biological particles

Carlo, Sacha De; El-Bez, C.; Álvarez-Rúa, Carmen; Borge, Javier; Dubochet, Jacques

doi:10.1016/s1047-8477(02)00035-7

Cited by 58 publications

(57 citation statements)

References 37 publications

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“…Thus, the use of frozen-hydrated sample preparation is necessary, but this approach is limited by the sample radiation damage, especially when tomographic image recording is performed on standard low-voltage electron microscopes (120 KV, LaB 6 ). In this context, it was of interest to evaluate the use of new preparation techniques that protect samples against the radiation damage such as cryo-negative staining (De Carlo et al, 2002). We have used this approach to generate sixteen independent tomographic volumes.…”

Section: Discussionmentioning

confidence: 99%

“…Indeed, cryo-electron tomography present two major drawbacks: the low contrast of recorded images and the sample radiation damage. In the present work we have tested, on T4 bacteriophage samples, the use of a new preparation technique, cryo-negative staining (Adrian et al, 1998), which reduces the radiation damage while preserving a high signal-to-noise ratio (De Carlo et al, 2002). Our results demonstrate that the combination of cryo-negative staining in tomography with standard cryo-microscopy and single particle analysis results in a methodological approach that could be useful in the study of biological structures ranging in the T4 bacteriophage size.…”

Section: Introductionmentioning

confidence: 99%

“…Nevertheless, the use in tomography of these preparation techniques is limited by the low signal-to-noise-ratio of the recorded images and by the sample radiation damage. In this context, cryo-negative staining (Adrian et al, 1998) has recently been demonstrated to be effective in protecting samples from irradiation while allowing a contrast enhancement of the images (De Carlo et al, 2002). However, cryonegative staining has never been tested in cryo-tomography.…”

Section: Introductionmentioning

confidence: 99%

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Use of cryo‐negative staining in tomographic reconstruction of biological objects: application to T4 bacteriophage

Messaoudi

Boudier

Lechaire³

et al. 2003

Biology of the Cell

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Recent advances in electron microscopy and image analysis techniques have resulted in the development of tomography, which makes possible the study of structures neither accessible to X-ray crystallography nor nuclear magnetic resonance. However, the use of tomography to study biological structures, ranging from 100 to 500 nm, requires developments in sample preparation and image analysis. Indeed, cryo-electron tomography present two major drawbacks: the low contrast of recorded images and the sample radiation damage. In the present work we have tested, on T4 bacteriophage samples, the use of a new preparation technique, cryo-negative staining (Adrian et al., 1998), which reduces the radiation damage while preserving a high signal-to-noise ratio (De Carlo et al., 2002). Our results demonstrate that the combination of cryo-negative staining in tomography with standard cryo-microscopy and single particle analysis results in a methodological approach that could be useful in the study of biological structures ranging in the T4 bacteriophage size.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Use of cryo‐negative staining in tomographic reconstruction of biological objects: application to T4 bacteriophage

Messaoudi

Boudier

Lechaire³

et al. 2003

Biology of the Cell

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“…Cryo-TEM of macromolecules is now becoming routine and has the advantage that specimens are observed without chemical fixation, staining or coating whilst minimising radiation damage. Problems may arise due to the inherently low contrast of biological macromolecules, and although new techniques combine negative stains with cryofixation to increase contrast, stain-based artifacts remain an issue [2,17].…”

Section: Advances In Sample Preparation Techniquesmentioning

confidence: 99%

Towards native-state imaging in biological context in the electron microscope

2009

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Modern cell biology is reliant on light and fluorescence microscopy for analysis of cells, tissues and protein localisation. However, these powerful techniques are ultimately limited in resolution by the wavelength of light. Electron microscopes offer much greater resolution due to the shorter effective wavelength of electrons, allowing direct imaging of sub-cellular architecture. The harsh environment of the electron microscope chamber and the properties of the electron beam have led to complex chemical and mechanical preparation techniques, which distance biological samples from their native state and complicate data interpretation. Here we describe recent advances in sample preparation and instrumentation, which push the boundaries of high-resolution imaging. Cryopreparation, cryoelectron microscopy and environmental scanning electron microscopy strive to image samples in near native state. Advances in correlative microscopy and markers enable high-resolution localisation of proteins. Innovation in microscope design has pushed the boundaries of resolution to atomic scale, whilst automatic acquisition of high-resolution electron microscopy data through large volumes is finally able to place ultrastructure in biological context.

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“…We had at our disposal a set of experimental GroEL images acquired by De Carlo et al [41] and a first estimation of their orientational and translational parameters. This first assignment has been obtained after having performed one iteration of PM for refinement of a starting model (the zero-iteration model), as explained in Ref.…”

Section: Validation Without Ground-truth Alignmentmentioning

confidence: 99%

Spline-based image-to-volume registration for three-dimensional electron microscopy

et al. 2005

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This paper presents an algorithm based on a continuous framework for a posteriori angular and translational assignment in three-dimensional electron microscopy (3DEM) of single particles. Our algorithm can be used advantageously to refine the assignment of standard quantized-parameter methods by registering the images to a reference 3D particle model. We achieve the registration by employing a gradient-based iterative minimization of a least-squares measure of dissimilarity between an image and a projection of the volume in the Fourier transform (FT) domain. We compute the FT of the projection using the central-slice theorem (CST). To compute the gradient accurately, we take advantage of a cubic B-spline model of the data in the frequency domain. To improve the robustness of the algorithm, we weight the cost function in the FT domain and apply a ''mixed'' strategy for the assignment based on the minimum value of the cost function at registration for several different initializations. We validate our algorithm in a fully controlled simulation environment. We show that the mixed strategy improves the assignment accuracy; on our data, the quality of the angular and translational assignment was better than 2 voxel (i.e., 6.54 Å ). We also test the performance of our algorithm on real EM data. We conclude that our algorithm outperforms a standard projection-matching refinement in terms of both consistency of 3D reconstructions and speed. r

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Cryo-negative staining reduces electron-beam sensitivity of vitrified biological particles

Cited by 58 publications

References 37 publications

Use of cryo‐negative staining in tomographic reconstruction of biological objects: application to T4 bacteriophage

Use of cryo‐negative staining in tomographic reconstruction of biological objects: application to T4 bacteriophage

Towards native-state imaging in biological context in the electron microscope

Spline-based image-to-volume registration for three-dimensional electron microscopy

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