Ptychographic imaging of NaD1 induced yeast cell death.

Characterising and understanding the mechanisms involved in cell death are especially important to combating threats to human health, particularly for the study of antimicrobial peptides and their effectiveness against pathogenic fungi. However, imaging these processes often relies on the use of synthetic molecules which bind to specific cellular targets to produce contrast. Here we study yeast cell death, induced by the anti-fungal peptide, NaD1. By treating yeast as a model organism we aim to understand anti-fungal cell death processes without relying on sample modification. Using a quantitative phase imaging technique, ptychography, we were able to produce label free images of yeast cells during death and use them to investigate the mode of action of NaD1. Using this technique we were able to identify a significant phase shift which provided a clear signature of yeast cell death. Additionally, ptychography identifies cell death much earlier than a comparative fluorescence study, providing new insights into the cellular changes that occur during cell death. The results indicate ptychography has great potential as a means of providing additional information about cellular processes which otherwise may be masked by indirect labelling approaches.

High-resolution X-ray imaging of Plasmodium falciparum-infected red blood cells.

Methods for imaging cellular architecture and ultimately macromolecular complexes and individual proteins, within a cellular environment, are an important goal for cell and molecular biology. Coherent diffractive imaging (CDI) is a method of lensless imaging that can be applied to any individual finite object. A diffraction pattern from a single biological structure is recorded and an iterative Fourier transform between real space and reciprocal space is used to reconstruct information about the architecture of the sample to high resolution. As a test system for cellular imaging, we have applied CDI to an important human pathogen, the malaria parasite, Plasmodium falciparum. We have employed a novel CDI approach, known as Fresnel CDI, which uses illumination with a curved incident wavefront, to image red blood cells infected with malaria parasites. We have examined the intrinsic X-ray absorption contrast of these cells and compared them with cells contrasted with heavy metal stains or immunogold labeling. We compare CDI images with data obtained from the same cells using scanning electron microscopy, light microscopy, and scanning X-ray fluorescence microscopy. We show that CDI can offer new information both within and at the surface of complex biological specimens at a spatial resolution of better than 40 nm. and we demonstrate an imaging modality that conveniently combines scanning X-ray fluorescence microscopy with CDI. The data provide independent confirmation of the validity of the coherent diffractive image and demonstrate that CDI offers the potential to become an important and reliable new high-resolution imaging modality for cell biology. CDI can detect features at high resolution within unsectioned cells.

A Direct Approach to In-Plane Stress Separation using Photoelastic Ptychography.

The elastic properties of materials, either under external load or in a relaxed state, influence their mechanical behaviour. Conventional optical approaches based on techniques such as photoelasticity or thermoelasticity can be used for full-field analysis of the stress distribution within a specimen. The circular polariscope in combination with holographic photoelasticity allows the sum and difference of principal stress components to be determined by exploiting the temporary birefringent properties of materials under load. Phase stepping and interferometric techniques have been proposed as a method for separating the in-plane stress components in two-dimensional photoelasticity experiments. In this paper we describe and demonstrate an alternative approach based on photoelastic ptychography which is able to obtain quantitative stress information from far fewer measurements than is required for interferometric based approaches. The complex light intensity equations based on Jones calculus for this setup are derived. We then apply this approach to the problem of a disc under diametrical compression. The experimental results are validated against the analytical solution derived by Hertz for the theoretical displacement fields for an elastic disc subject to point loading.

Mapping granular structure in the biological adhesive of Phragmatopoma californica using phase diverse coherent diffractive imaging.

This paper demonstrates the application of the high sensitivity, low radiation dose imaging method recently presented as phase diverse coherent diffraction imaging, to the study of biological and other weakly scattering samples. The method is applied, using X-ray illumination, to quantitative imaging of the granular precursors of underwater adhesive produced by the marine sandcastle worm, Phragmatopoma californica. We are able to observe the internal structure of the adhesive precursors in a number of states.