We have developed a new data collection method and processing framework in full field cryo soft X-ray tomography to computationally extend the depth of field (DOF) of a Fresnel zone plate lens. Structural features of 3D-reconstructed eukaryotic cells that are affected by DOF artifacts in standard reconstruction are now recovered. This approach, based on focal series projections, is easily applicable with closed expressions to select specific data acquisition parameters.

Cryo-electron microscopy projection image analysis and tomography is used to describe the overall architecture of influenza B/Lee/40. Algebraic reconstruction techniques with utilization of volume elements (blobs) are employed to reconstruct tomograms of this pleomorphic virus and distinguish viral surface spikes. The purpose of this research is to examine the architecture of influenza type B virions by cryo-electron tomography and projection image analysis. The aims are to explore the degree of ribonucleoprotein disorder in irregular shaped virions; and to quantify the number and distribution of glycoprotein surface spikes (hemagglutinin and neuraminidase) on influenza B. Projection image analysis of virion morphology shows that the majority (∼83%) of virions are spherical with an average diameter of 134±19 nm. The aspherical virions are larger (average diameter = 155±47 nm), exhibit disruption of the ribonucleoproteins, and show a partial loss of surface protein spikes. A count of glycoprotein spikes indicates that a typical 130 nm diameter type B virion contains ∼460 surface spikes. Configuration of the ribonucleoproteins and surface glycoprotein spikes are visualized in tomogram reconstructions and EM densities visualize extensions of the spikes into the matrix. The importance of the viral matrix in organization of virus structure through interaction with the ribonucleoproteins and the anchoring of the glycoprotein spikes to the matrix is demonstrated.

Recognizing that the microscope depth of field is a significant resolution-limiting factor in threedimensional cryoelectron microscopy, Jensen and Kornberg proposed a concept they called defocus-gradient corrected backprojection (DGCBP) and illustrated by computer simulations that DGCBP can effectively eliminate the depth of field limitation. They did not provide a mathematical justification for their concept. Our paper provides this, by showing (in the idealized case of noiseless data being available for all projection directions) that the reconstructions obtained based on DGCBP from data produced with distance-dependent blurring are essentially the same as what is obtained by a classical method of reconstruction of a 3D object from its line integrals. The approach is general enough to be applicable for correcting for any distancedependent blurring during projection data collection. We present a new implementation of the DGCBP concept, one that closely follows the mathematics of its justifications, and illustrate it using mathematically-described phantoms and their reconstructions from finitely-many distancedependently blurred projections.