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.
Ziese
U
U
Molecular Cell Biology, Utrecht University, 3584 CH Utrecht, The Netherlands.
Janssen
A H
AH
Murk
J-L
JL
Geerts
W J C
WJ
Van der Krift
T
T
Verkleij
A J
AJ
Koster
A J
AJ
D000818:Animals; D003713:Dendritic Cells / Q000648:ultrastructure; D021621:Imaging, Three-Dimensional / Q000379:methods*; D051379:Mice; D008854:Microscopy, Electron / Q000379:methods; D008928:Mitochondria / Q000648:ultrastructure; D014054:Tomography / Q000379:methods*; D017641:Zeolites / Q000737:chemistry
D017641:Zeolites