Salvatore Lanzavecchia

Electron-tomographic analysis of intraflagellar transport particle trains in situ

Ultrastructural study of Chlamydomonas cilia shows that anterograde IFT particles form trains that are long and narrow, while retrograde IFT form short, compact particle trains.

Conical Electron Tomography of a Chemical Synapse: Polyhedral Cages Dock Vesicles to the Active Zone

In this study, we tested the hypothesis that the structure of the active zone of chemical synapses has remained uncertain because of limitations of conventional electron microscopy. To resolve these limitations, we reconstructed chemical synapses of rat neocortex, the archetypical “average” synapse, by conical electron tomography, a method that exhibits an isotropic in plane resolution of ∼3 nm and eliminates the need to impose symmetry or use averaging methods to increase signal-to-noise ratios. Analysis of 17 reconstructions by semiautomatic density segmentation indicated that the active zone was constructed of a variable number of distinct “synaptic units” comprising a polyhedral cage and a corona of approximately seven vesicles. The polyhedral cages measured ∼60 nm in diameter, with a density of ∼44/μm2 and were associated with vesicles at the active zone (“first tier”). Vesicles in this first-tier position represented ∼7.5% of the total number of vesicles in the terminal and were contiguous, hemifused (∼4% of total), or fully fused (∼0.5% of total) to the plasma membrane. Our study supports the hypothesis that rat neocortical synapses are constructed of variable numbers of distinct synaptic units that facilitate the docking of vesicles to the active zone and determine the number of vesicles available for immediate release.

The sperm tail of a gall midge and its microtubular arrangement studied by two strategies of image analysis (cecidomyiidae, diptera, insecta)

Abstract Spermatozoa of the gall midge, Asphondylia ruebsaamen, were found to have an axoneme with about 2500 microtubular doublets, each carrying a single dynein arm that corresponds to the outer dynein arm of conventional flagella. The doublets form a tightly wound double spiral, in which doublets of the inner spiral and outer one are joined pairwise. Whereas most axonemes had their doublets in a curved spiral, some axonemes showed large blocks of doublets lying in a rather regular crystalline arrangement. The axoneme hence could appear in two states, spiral and crystalline, with intermediate forms. The doublets and their dynein arms and other connections have been subjected to a computer analysis that aims at calculating an average image from a great number of doublet pairs. Two strategies of image analysis were used. In one method the average doublet pair was obtained from a number of isolated image motifs by correlation methods; this strategy could be applied to both the spiral regions and crystalline ones and showed that there are definite differences in the angle between the two doublets of a pair and in the greater regularity of the dynein arm orientation in the crystalline state. Further, it can be seen that the shape of the two doublets in the pair is the same in the spiral regions and differs somewhat in the crystalline one. In the other strategy a filtration of noisy components was performed from the Fourier transform of periodic arrays portrayed in crystalline regions. This strategy included a determination of distortions in the lattice, and removal of these distortions by image resampling in iterated steps. The average doublet pairs appear much the same in reconstructions made with either strategy. It could be seen that the interspace between the microtubular protofilaments varied somewhat and appeared widened at those places where the dynein arm or other bridges were inserted. The lumen of the microtubules could also be seen to have slender radial strands that are particularly well resolved in the B-tubules of the doublets in the spiral state. The axoneme was seen to have one polypeptide band only in the dynein band region.

Simultaneous alignment of dual-axis tilt series.

We present a strategy for the alignment of dual-axis tomographic series, based on reference points and simultaneous alignment of both series. Each series is first aligned individually, an affine transformation is determined to bring the two series in a unique reference system, and all experimental coordinates are combined in a single system of equations. In case of severe shrinkage, a global and a local refinement of the orientation parameters are performed to correct all minors misalignments. The strategy is illustrated on tomographic experiments performed on sections from plastic-embedded biological samples. The efficiency in correcting the misalignment of gold particles and in improving the quality of the reconstruction is documented both visually and quantitatively. In our approach every region of the tomogram is associated with its own orientation parameters and can be eventually reconstructed with the preferred algorithm. This is convenient in the computation of 3D averages of equivalent structures. A simulation experiment is presented to show that the performances of this approach are superior to those of the method of rotation in direct space.

The variance of icosahedral virus models is a key indicator in the structure determination: a model-free reconstruction of viruses, suitable for refractory particles

A model-free method to determine the three-dimensional structure of icosahedral viruses is described. The novel strategy is based upon the approximate principle that correct virus structures have high variance as do all other well-detailed structures, even wrong ones. The original projections of individual particles are reduced to a radius of 25 pixels and are used to compute single particle reconstruction models by assigning them 1800 different Euler triads. The variance of the models obtained from all projections is stored in maps and a decimation process is carried out. In a first stage, thresholds are adopted for the variance values, and in a second stage, carried out by correspondence analysis and classification, 30 clusters of models are sorted out. The clusters are refined to yield models contained in boxes of 64(3) voxels. The refined models with highest variance and closest similarity represent the correct solution. Once enlarged, these models can be used to align all available projections in their original scale in a customary projection-matching process. The method has proved successful in determining the structures of poliovirus, of the empty and filled capsids of L-A virus, and of a modified capsid of hepatitis B virus.

Electron tomography in conical tilt geometry. The accuracy of a direct Fourier method (DFM) and the suppression of non-tomographic noise

Abstract SPARK is a direct Fourier method (DFM) to reconstruct 3D structures from projections observed in the electron microscope. It is suitable for both single axis tilting and random conical tilting. The present article reports accuracy tests performed in reconstructing phantom structures from analytical projections in conical tilt geometry, both noise-free and corrupted with noise. Statistical reliability indices, similar to those used in X-ray crystallography, are proposed and reported for all tests. Phase agreement tests are discussed in connection with the possibility of determining the internal consistency of data without dividing projections into two sets and are presented for the reconstructions of phantoms and of a haematic pigment. The mathematical formulation of the 3D Fourier transform of an ordered stack of projections is illustrated. With conical tilt geometry, the transform is almost zero within an elliptic cone. This property is exploited to suppress “non-tomographic noise” (NTN) and to recover missing projections in a POCS-like implementation. The suppression of non-tomographic noise, which is possible even if reconstructions are done by back-projection methods, allows one to push low-pass filtering to a higher frequency and to achieve improved resolution.

A two exposures technique of electron tomography from projections with random orientations and a quasi-Boolean angular reconstitution

Abstract The reconstruction of three-dimensional structures of macromolecular assemblies from projections with random orientation is commonly based on the detection of lines shared among couples of sinograms. This common line detection may be affected by large errors, even in simulations with noise-free data. A two exposures technique, with use of a symmetric tilt of the specimen, is proposed here. Relatively small tilt angles (±20–25°) alleviate defocus problems. The essential point of the technique is that one knows a priori the common line and the dihedral angle formed by couples of projections of the same particle. Inherent to a two exposures technique is the possibility to determine ab initio the correct handedness of the structure. The orientation of each couple with respect to a reference couple is determined by the simultaneous detection of four common lines, a task performed by a quasi-Boolean algorithm. Since multiple exposures would damage the sample, the reconstruction should be based on the first exposure, use of the second exposure being limited to the orientation process. Tests carried out with projections of asymmetric phantoms corrupted with a substantial amount of noise indicate a good algorithm performance.

SPARK, a kernel of software programs for spatial reconstruction in electron microscopy

SPARK, an acronym for ‘SPAtial Reconstruction Kernel’, is the nucleus of a software library being developed for the three‐dimensional (3‐D) reconstruction of objects observed by the electron microscope. A unifying concept is used: the Fourier transform, known in several central sections, is resampled to obtain a 3‐D Cartesian grid, which is inverted by a fast Fourier transform. This technique is used for both single‐axis tilting (of 2‐D periodic layers or of isolated objects) and for the random conical‐tilt technique. The principles that make it possible to recover Cartesian grids in the two different geometries are illustrated and some preliminary results are reported. SPARK resamples the Cartesian grids with the use of a fast and efficient algorithm of Shannon interpolation developed by the authors.

A strategy for the reconstruction of structures possessing axial symmetry: sectioned axonemes in sperm flagella

The images of complex biological structures seen in the electron microscope, possessing an n‐fold rotational symmetry, can be enhanced by averaging the axially repeating motif in order to improve their signal‐to‐noise ratio; this requires that the slices with n‐fold symmetry do not exhibit distortions relative to one another.

POLCA, a library running in a modern environment, implements a protocol for averaging randomly oriented images

The library POLCA implements the averaging of biological structures whose images are recorded in digital form from electron micrographs. The averaging protocol is based upon a method developed about ten years ago, which allows one to operate on a sequence of objects oriented and displaced at random within their frame; the relative rotations and the displacements of the structures are detected with the use of correlation algorithms and modified to make all objects appear the same, apart from their noisy components. The average image is then obtained by a simple addition and the signal-to-noise ratio is improved by a factor equal to the square root of the number of objects used to calculate the average. With respect to the original implementation of the method, two novel features characterize the library: the first one deals with the functions that are cross-correlated to determine the relative rotations of the structures; the functions used here are the inverse transforms of the amplitude spectra (IAS functions), which give rise to sharp maxima when they are cross-correlated. The second peculiarity is the systematic adoption, in the transformations of coordinates and in other circumstances, of an interpolation technique based upon the Fourier series kernel. POLCA is written in C and runs on a VME machine under the UNIX V/68 operating system. A programming style has been adopted to exploit fully the machine resources.