D-CAT: Density and Clustering Annotation Tool for three dimensional electron microscopic volumes.

Three dimensional (3D) electron microscopy techniques have become valuable tools for investigating cellular architecture and the processes that govern it. A vast amount of information is available in every 3D tomogram but the options for presenting this information in a clear and visually appealing way are limited. To address this, we developed D-CAT; a MatLab-application to accurately visualize the distribution of membrane proteins and/or membrane-bound structures. Presence (density) and distribution (clustering, depletion) are presented as color-coded areas on membranes. By using IMOD models both as input and output format, we ensure that the application fits within workflows common in the field of 3D electron microscopy.

STEM tomography in cell biology.

Transmission electron tomography has been used in biological sciences for quite some time and proven to be a valuable tool. However, to date, the different Scanning Transmission modes are almost not used for electron tomography on resin-embedded biological material. We explored different STEM modes on epon-embedded, osmium-uranyl-lead-stained biological material. Bright Field-TEM and High Angle Annular Dark Field-STEM tomograms from the same areas were recorded and compared. Contrast and signal-to-noise ratios were calculated. Template matching was used to validate results obtained in Bright Field-TEM and High Angle Annular Dark Field-STEM tomograms. It is concluded that High Angle Annular Dark Field-STEM gives a five times better contrast and signal-to-noise ratio than Bright Field-TEM. Template matching showed that 1.3 times more information could be extracted from High Angle Annular Dark Field-STEM tomograms than from Bright Field-TEM tomograms.

Correction of autofocusing errors due to specimen tilt for automated electron tomography.

Transmission electron microscopy images acquired under tilted-beam conditions experience an image shift as a function of defocus settings - a fact that is exploited as a method for defocus determination in most of the automated tomography data collection systems. Although the method was shown to be highly accurate for a large variety of specimens, we point out that in its original design it can strictly only be applied to images of untilted samples. The application to tilted samples and thus in automated electron tomography is impaired mainly due to a defocus change across the images, resulting in reduced accuracy. In this communication we present a method that can be used to improve the accuracy of the basic autofocusing procedures currently used in systems for automated electron tomography.

Automated high-throughput electron tomography by pre-calibration of image shifts.

Electron tomography is a versatile method for obtaining three-dimensional (3D) images with transmission electron microscopy. The technique is suitable to investigate cell organelles and tissue sections (100-500 nm thick) with 4-20 nm resolution. 3D reconstructions are obtained by processing a series of images acquired with the samples tilted over different angles. While tilting the sample, image shifts and defocus changes of several microm can occur. The current generation of automated acquisition software detects and corrects for these changes with a procedure that incorporates switching the electron optical magnification. We developed a novel method for data collection based on the measurement of shifts prior to data acquisition, which results in a five-fold increase in speed, enabling the acquisition of 151 images in less than 20 min. The method will enhance the quality of a tilt series by minimizing the amount of required focus-change compensation by aligning the optical axis to the tilt axis of the specimen stage. The alignment is achieved by invoking an amount of image shift as deduced from the mathematical model describing the effect of specimen tilt. As examples for application in biological and materials sciences 3D reconstructions of a mitochondrion and a zeolite crystal are presented.