Selective Oligonucleotide and MRNA Pull-Down with Shielded Covalent Probes

2556-Pos Board B575 Bringing Deconvolution Algorithmic Techniques to the Electron Microscope Ben Lich, Xiaodong Zhuge, Pavel Potocek, Faysal Boughorbel, Cliff Mathisen. FEI Company, Eindhoven, Netherlands. The Scanning Electron Microscope (SEM) as a three dimensional imaging tool is increasingly popular for studies in neurobiology. Serial slicing methods based on diamond-knife cutting are, however, reaching practical limitations in terms of achievable z-resolution and voxel isotropy. While Focused-Ion Beam SEM serial block face imaging can improve the z-resolution to 5nm, this technology is restricted in terms of the total volume of material that can be processed. In this work we describe the Multi Energy Confocal Scanning Electron Microscope or ‘‘MECSEM’’ method that achieves high isotropic resolution by a combination of backscatter image sequence acquisition and deconvolution (DC). The method is built on improved understanding of beam-sample interaction for classically prepared samples. Furthermore the point spread function (PSF) of backscatter electrons (BSE) in these materials appears to be well confined laterally. As the range of penetration in the sample is dependent on the energy of the primary beam, acquiring an image sequence with increasing landing energies leads to the acquisition of images from increasing depth. The lateral confinement of BSE PSFs allows for restricting the DC to a layer separation task (z direction). As the structure of the PSF is difficult to obtain experimentally it will be considered unknown leading to a blind DC problem requiring the recovery of both depth layers and PSFs. To verify the reconstruction results we combine this technique with classical FIB-SEM serial block imaging using the through the lens detector in BSE mode on an FEI Helios 650 DualBeam. The z-resolution was controlled by varying the primary beam energy. The comparison with finer FIB reconstruction shows identical structures on the studied samples proving the reliability of the 3D technique, while the achieved depth using DC can reach up to 180nm.