Transmission imaging of biological tissue with the Delft multi‐beam SEM

Abstract

A major bottleneck for large‐scale and volume EM is the imaging speed. The total acquisition time needed for a single sample can easily take days, or even weeks using standard single‐beam SEM's. Multi‐beam microscopes have been developed to increase imaging speed[1,2], but it remains a challenge to achieve electron detection similar to a regular SEM in terms of signal type, contrast and resolution. We have developed a SEM employing 196 electron beams using a standard column of a FEI Nova NanoSEM 200. The 196 electron beams are generated from a single high‐brightness Schottky electron source, making use of a square aperture lens array grid of 14 by 14 holes. Modified source optics allows focusing of all beams in the sample plane, with the same probe current and probe size as in a single‐beam SEM. Both secondary and transmission electron signals can be detected in the system, of which an overview is shown in figure 1. For the detection of the transmitted electrons, the sample of interest is placed on a scintillating screen and the light generated by each beam is collected through an optical objective lens. This light is focused on a CMOS camera placed outside the SEM chamber and the image is produced through online processing of the intensity of each beam. An example of rat pancreas tissue imaged by this method is shown in figure 2. The secondary electrons are focused on a scintillating screen in the variable aperture plane, making use of a retarding lens and the electron optics used for the focusing of the primary beams. This signal is again focused on a CMOS camera and the same process for imaging is performed as for the transmitted electrons. We present proof‐of‐principle results showing that sub‐10nm resolution can be obtained for transmission imaging of stained rat pancreas tissue. We will discuss our efforts towards improving the detection methods and the data processing speed. Furthermore, work will be shown on quantifying and comparing the signals obtained from secondary, transmitted and backscattered electrons on stained tissue sections, as imaged by a conventional SEM.