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Employing a fiducial alignment mark on the transducer to orient the robot's frame of reference and using simple thresholding algorithms to segment the 3D images, we tested the accuracy of using the scanner to automatically direct a robot arm that touched two needle tips together within a water tank.
Electron tomograms were generated by fiducial alignment in the tilt series and videos reconstituted from consecutive snapshots with ImageJ (Rasband, 1997 2013).
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Dicing of images was done semi automatically as scientists told the algorithm where fiducials for alignment were subsequent to scanning.
While subnanometer resolution can be achieved in cryoSPT with standard fiducial-based alignment (Schur et al. 2013), circumventing the known errors inherent in this methodology can improve resolution (Bartesaghi et al. 2012).
Alternatively, many fiducial-less alignment methods have been developed (Liu et al. 1995; Brandt et al. 2001; Renken and McEwen 2003; Castaño-Díez et al. 2007) but usually require that the specimen itself exhibits high-contrast features.
Indeed, if made easily applicable, overcoming the limitations of fiducial-based alignment (Iwasaki et al. 2005; Bartesaghi et al. 2012; Zhang and Ren 2012) might facilitate achieving subtomogram averages beyond the 8 12 Å resolution range routinely for various types of specimens.
Re-projections from the tilt series were generated using IMOD software from the Boulder Laboratory for 3D Electron Microscopy of Cells, using the gold particles as fiducials for alignment [68].
Fiducial-free alignment of tilt images was carried out in Protomo (Winkler and Taylor, 2006), and the two reconstructions from the dual-axis tilt series were merged in IMOD (Kremer et al., 1996; Mastronarde, 1997).
Fiducial-less alignment of the tilt-series was performed by feature tracking with TOM software (Nickell et al., 2005; Korinek et al., 2011) or patch tracking with IMOD software (Mastronarde, 1997), and tomograms were reconstructed using the weighted back projection algorithm.
The CCD temperature changes are coincident with shifts in the fiducial transfer system alignment that are visible in our X-ray/Optical/Radio cross-correlation monitoring.
We report state-of-the-art results for pose-invariant face recognition on CMU PIE and FERET and comparable results on MultiPIE when using only four fiducial points for alignment and intensity features.
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