Low-Dosage Maximum-A-Posteriori Focusing and Stigmation

Abstract

Radiation damage is often an issue during high-resolution imaging, making low-dose focusing and stigmation essential, in particular when no part of the sample can be "sacrificed" for this. An example is serial block-face electron microscopy, where the imaging resolution must be kept optimal during automated acquisition that can last months. Here, we present an algorithm, which we call "Maximum-A-Posteriori Focusing and Stigmation (MAPFoSt)," that was designed to make optimal use of the available signal. We show that MAPFoSt outperforms the built-in focusing algorithm of a commercial scanning electron microscope even at a tenfold reduced total dose. MAPFoSt estimates multiple aberration modes (focus and the two astigmatism coefficients) using just two test images taken at different focus settings. Using an incident electron dose density of 2,500 electrons/pixel and a signal-to-noise ratio of about one, all three coefficients could be estimated to within <7% of the depth of focus, using 19 detected secondary electrons per pixel. A generalization to higher-order aberrations and to other forms of imaging in both two and three dimensions appears possible.

Enhanced FIB-SEM systems for large-volume 3D imaging

“…The in-line optimization procedure described below effectively overcomes the deficiencies in conventional methods. Recognizing these problems, a functionally similar approach was previously described (Binding et al, 2013). …”

“…When comparing neighboring frames in a 3D FIB-SEM image stack, there proved to be only minimal variations in either sample features or signal intensity, so focus index is conveniently sensitive to the beam spot size. Alternatively, the FI can be obtained by other methods (Binding et al, 2013) that quantify the ‘sharpness’ of an image.…”

Volume scanning electron microscopy for imaging biological ultrastructure

“…Focus and astigmatism drifts that compromise image quality may occur when a SBEM system is continuously operating for weeks or months (as in Briggman et al., ). While manual adjustments are possible by repeatedly pausing the acquisition and refocusing, more effective algorithmic solutions have been developed that allow automatic correction (Binding et al., ; various algorithms in commercial software).…”

Challenges of microtome‐based serial block‐face scanning electron microscopy in neuroscience

“…Currently, Winfried Denk is pushing the extremes of SBEM with the goal of cutting and imaging an entire mouse brain (Mikula et al ., ; Perkel, ). Besides the development of a completely new microtome capable of cutting centimetre‐sized blocks, they are also working on improving the stability of image acquisition by developing methods for automated correction of focus and stigmatism (Binding et al ., ).…”