Point‐projection imaging of macromolecular contours

Abstract

A point-projection microscope is described which uses an electric field of several volts per nanometre to generate ions from a layer of benzene condensed in high vacuum on to the apex of a cryogenically cooled field-emitter tip. Clusters of ferritin (a spherical protein molecule) embedded in the layer, have been imaged by exposing their contours during controlled fielddesorption. Image contrast is high. A magnification of 2 x 105 has been achieved at a spatial resolution estimated to be better than 3 nm. Three-dimensional reconstructions of cluster morphology from a series of molecular contours are in qualitative agreement with transmissionelectron micrographs of the ferritin covered emitter apex. Reproducibility of the cluster contours, and TEM images of the apex before and after imaging suggest that the imaging procedure is nondestructive. The Royal Microscopical Society 3 4 J. A . Panitz

Chemical mapping of mammalian cells by atom probe tomography

“…Despite limited sample preparation methods, Panitz and co-workers (Panitz, 1982; Panitz, 1983; Panitz and Giaver, 1981) attempted to visualize strands of DNA attached via a charged biopolymer to a metal substrate, with marginal success. The group also pioneered specimen preparation methods that allowed for imaging of monolayers of ferritin that were buried in a volume of benzene ice.…”

An atomic-scale view at the composition of amyloid-beta fibrils by atom probe tomography

“…Different from metals and semiconductors, biological materials are characterized by a complex bonding energy landscape, ranging from very weak Van Der Waals bondings (0.1 eV) over ionic to several eV strong covalent bonds. Their integrity and decomposition when exposed to high voltage fields and laser induced temperature increase can be very sensitive to the APT experiment conditions and so we were cautious when setting the laser pulse energy to minimize thermal damage, this being important to achieve well-behaved field evaporation 45,52,54,55,57 . We varied the laser pulse energy from 10 pJ up to 40 pJ (Fig 3) and the repetition rate between 125 kHz to 500 kHz (Fig 4) to explore their respective impact upon the quality of the mass-to-charge spectrum, particularly with respect to thermal tails coming from evaporation after the pulse has ended because of remaining thermal energy in the specimen and DC evaporation (described below).…”

“…Previous studies have demonstrated near-atomic resolution of chemically-resolved data of bulk bio-mineralized hard biological matter such as teeth and bone [45][46][47][48][49][50][51] . Progress was also made on more soft materials such as cells 52,53 and also with ferritin as example for proteins [54][55][56] . In early designs of the atom probe microscope, single amino acids, nucleic acids and other polymers were successfully analyzed [57][58][59][60] .…”

In Search of the Chimera: Molecular Imaging in the Atom-Probe

“…Stable and reproducible images of a ferritin monolayer on a tungsten surface were obtained in the Imaging Atom-Probe by embedding ferritin within a layer of vitreous benzene ice condensed onto an 80 K surface using gas phase benzene as a blanket gas [4]. As the field is increased, benzene is desorbed from the surface as cluster ions (C 6 H 6 ) n + , n=1,2) which expose the contour of ferritin molecules as a function of depth within the benzene layer (Fig.…”

“…Although Atom-Probe analysis has been confined to surface studies and problems in the materials sciences the lure of imaging and analyzing individual molecules has been strong. See a review of attempts to image individual molecules in the Field-Emission Electron Microscope and the Field-Ion Microscope for details [4].…”