Imaging biological samples by integrated differential phase contrast (iDPC) STEM technique

Abstract

Scanning transmission electron microscopy (STEM) is a powerful imaging technique and has been widely used in current material science research. The attempts of applying STEM into biological research have been going on for decades while applications have still been limited because of the existing bottlenecks in dose efficiency and non-linearity in contrast. Recently, integrated differential phase contrast (iDPC) STEM technique emerged and achieved a linear phase contrast imaging condition, while resolving signals of light elements next to heavy ones even at low electron dose. This enables successful investigation of beam sensitive materials. Here, we investigate iDPC-STEM advantages in biology, in particular, chemically fixed and resin embedded biological tissues. By comparing results to the conventional TEM, we have found that iDPC-STEM not only shows better contrast but also resolves more structural details at molecular level, including conditions of extremely low dose and minimal heavy-atom staining. For thick sample sections, iDPC-STEM is particularly advantageous. Unlike TEM, it avoids contrast inversion canceling effects, and by adjusting the depth of focus, fully preserves the contrast of relevant features along with the sample. In addition, using depth-sectioning, iDPC-STEM enables resolving in-depth structural variation. Our work suggests that promising, wide and attractive applications of iDPC-STEM in biological research are opening.

Single-particle cryo-EM structures from iDPC–STEM at near-atomic resolution

“…Largest benefits can be expected when no or little averaging of cryo-images is possible. In the case of biological vitrified samples, this feature may be particularly useful for the visualization of complex environments such as cellular lysates and biological cells 20 . In CTEM, the thickness of the specimen imposes a substantial limitation on such samples as contrast blurs due to incoherency and inelastic scattering.…”

“…By using iDPC-STEM with a low-dose exposure of as little as 40 e − /Å 2 , a resolution of 1.8 Å was obtained successfully from a single micrograph for such materials 19 . Recently, large biological sections, including thick ones (~500 nm), have also been imaged 20 . For these dose-sensitive low-contrast specimens, iDPC-STEM enables direct interpretation of the image without the need of defocusing and a subsequent contrast-transfer function (CTF) correction of the images 21 .…”