Three-dimensional deconvolution processing for STEM cryotomography

The complex environment of biological cells and tissues has motivated development of three-dimensional (3D) imaging in both light and electron microscopies. To this end, one of the primary tools in fluorescence microscopy is that of computational deconvolution. Wide-field fluorescence images are often corrupted by haze due to out-of-focus light, i.e., to cross-talk between different object planes as represented in the 3D image. Using prior understanding of the image formation mechanism, it is possible to suppress the cross-talk and reassign the unfocused light to its proper source post facto. Electron tomography based on tilted projections also exhibits a cross-talk between distant planes due to the discrete angular sampling and limited tilt range. By use of a suitably synthesized 3D point spread function, we show here that deconvolution leads to similar improvements in volume data reconstructed from cryoscanning transmission electron tomography (CSTET), namely a dramatic in-plane noise reduction and improved representation of features in the axial dimension. Contrast enhancement is demonstrated first with colloidal gold particles and then in representative cryotomograms of intact cells. Deconvolution of CSTET data collected from the periphery of an intact nucleus revealed partially condensed, extended structures in interphase chromatin.

Microscope 3D Point Spread Function Evaluation Method on a Confirmed Object Plane Perpendicular to the Optical Axis

A point spread function evaluation method for a microscope on the object plane that is perpendicular to the optical axis is proposed. The measurement of the incident beam direction from the dual position-sensitive-detector (PSD)-based units, the determination of the object plane perpendicularity and the paraxial region, and evaluation methods for the point spread function (PSF) are presented and integrated into the proposed method. The experimental verification demonstrates that the proposed method can achieve a 3D PSF on the perpendicular object plane, as well as magnification, paraxial region evaluation, and confirmation for any microscopic system.

A New Method to Retrieve the Three-Dimensional Refractive Index and Specimen Size Using the Transport Intensity Equation, Taking Diffraction into Account

Refractive index retrieval is possible using the transport intensity equation (TIE), which presents advantages over interferometric techniques. The TIE method is valid only for paraxial ray assumptions. However, diffraction can nullify these TIE model assumptions. Therefore, the refractive index is problematic for reconstruction in three-dimensions (3D) using a set of defocused images, as diffraction effects become prominent. We propose a method to recover the 3D refractive index by combining TIE and deconvolution. A brightfield (BF) microscope was then constructed to apply the proposed technique. A microsphere was used as a sample with well-known properties. The deconvolution of the BF-images of the sample using the microscope’s 3D point spread function led to significantly reduced diffraction effects. TIE was then applied for each set of three images. Applying TIE without taking into account diffraction failed to reconstruct the 3D refractive index. Taking diffraction into account, the refractive index of the sample was clearly recovered, and the sectioning effect of the microsphere was highlighted, leading to a determination of its size. This work is of great significance in improving the 3D reconstruction of the refractive index using the TIE method.

Design of Two-Dimensional Ultrasonic Phased Array Transducers

Two-dimensional (2D) phased arrays have the potential to significantly change the way in which engineering components in safety critical industries are inspected. In addition to enabling a three-dimensional (3D) volume of a component to be inspected from a single location, they could also be used in a C-scan configuration. The latter would enable any point in a component to be interrogated over a range of solid angles, allowing more accurate defect characterization and sizing. This paper describes the simulation and evaluation of grid, cross and circular 2D phased array element configurations. The aim of the cross and circle configurations is to increase the effective aperture for a given number of elements. Due to the multitude of possible array element configurations a model, based on Huygens’ principle, has been developed to allow analysis and comparison of candidate array designs. In addition to the element configuration, key issues such as element size, spacing, and frequency are discussed and quantitatively compared using the volume of the 3D point spread function (PSF) as a measurand. The results of this modeling indicate that, for a given number of elements, a circular array performs best and that the element spacing should be less than half a wavelength to avoid grating lobes. A prototype circular array has been built and initial results are presented. These show that a flat bottomed hole, half a wavelength in diameter, can be imaged. Furthermore, it is shown that the volume of the 3D reflection obtained experimentally from the end of the hole compares well with the volume of the 3D PSF predicted for the array at that point.