Colorimetric histology using plasmonically active microscope slides.

The human eye can distinguish as many as 10,000 different colours but is far less sensitive to variations in intensity1, meaning that colour is highly desirable when interpreting images. However, most biological samples are essentially transparent, and nearly invisible when viewed using a standard optical microscope2. It is therefore highly desirable to be able to produce coloured images without needing to add any stains or dyes, which can alter the sample properties. Here we demonstrate that colorimetric histology images can be generated using full-sized plasmonically active microscope slides. These slides translate subtle changes in the dielectric constant into striking colour contrast when samples are placed upon them. We demonstrate the biomedical potential of this technique, which we term histoplasmonics, by distinguishing neoplastic cells from normal breast epithelium during the earliest stages of tumorigenesis in the mouse MMTV-PyMT mammary tumour model. We then apply this method to human diagnostic tissue and validate its utility in distinguishing normal epithelium, usual ductal hyperplasia, and early-stage breast cancer (ductal carcinoma in situ). The colorimetric output of the image pixels is compared to conventional histopathology. The results we report here support the hypothesis that histoplasmonics can be used as a novel alternative or adjunct to general staining. The widespread availability of this technique and its incorporation into standard laboratory workflows may prove transformative for applications extending well beyond tissue diagnostics. This work also highlights opportunities for improvements to digital pathology that have yet to be explored.

Observations of phase changes in monoolein during high viscous injection.

Serial crystallography of membrane proteins often employs high-viscosity injectors (HVIs) to deliver micrometre-sized crystals to the X-ray beam. Typically, the carrier medium is a lipidic cubic phase (LCP) media, which can also be used to nucleate and grow the crystals. However, despite the fact that the LCP is widely used with HVIs, the potential impact of the injection process on the LCP structure has not been reported and hence is not yet well understood. The self-assembled structure of the LCP can be affected by pressure, dehydration and temperature changes, all of which occur during continuous flow injection. These changes to the LCP structure may in turn impact the results of X-ray diffraction measurements from membrane protein crystals. To investigate the influence of HVIs on the structure of the LCP we conducted a study of the phase changes in monoolein/water and monoolein/buffer mixtures during continuous flow injection, at both atmospheric pressure and under vacuum. The reservoir pressure in the HVI was tracked to determine if there is any correlation with the phase behaviour of the LCP. The results indicated that, even though the reservoir pressure underwent (at times) significant variation, this did not appear to correlate with observed phase changes in the sample stream or correspond to shifts in the LCP lattice parameter. During vacuum injection, there was a three-way coexistence of the gyroid cubic phase, diamond cubic phase and lamellar phase. During injection at atmospheric pressure, the coexistence of a cubic phase and lamellar phase in the monoolein/water mixtures was also observed. The degree to which the lamellar phase is formed was found to be strongly dependent on the co-flowing gas conditions used to stabilize the LCP stream. A combination of laboratory-based optical polarization microscopy and simulation studies was used to investigate these observations.

Spatial coherence of electron bunches extracted from an arbitrarily shaped cold atom electron source.

We describe the spatial coherence properties of a cold atom electron source in the framework of a quasihomogeneous wavefield. The model is used as the basis for direct measurements of the transverse spatial coherence length of electron bunches extracted from a cold atom electron source. The coherence length is determined from the measured visibility of a propagated electron distribution with a sinusoidal profile of variable spatial frequency. The electron distribution was controlled via the intensity profile of an atomic excitation laser beam patterned with a spatial light modulator. We measure a lower limit to the coherence length at the source of lc = 7.8 ± 0.9 nm.

Internal structure of an intact Convallaria majalis pollen grain observed with X-ray Fresnel coherent diffractive imaging.

We have applied Fresnel Coherent Diffractive Imaging (FCDI) to image an intact pollen grain from Convallaria majalis. This approach allows us to resolve internal structures without the requirement to chemically treat or slice the sample into thin sections. Coherent X-ray diffraction data from this pollen grain-composed of a hologram and higher resolution scattering information-was collected at a photon energy of 1820 eV and reconstructed using an iterative algorithm. A comparison with images recorded using transmission electron microscopy demonstrates that, while the resolution of these images is limited by the available flux and mechanical stability, we observed structures internal to the pollen grain-the intine/exine separations and pore dimensions-finer than 60 nm. The potential of this technique for further biological imaging applications is discussed.

Atom-scale ptychographic electron diffractive imaging of boron nitride cones.

Ptychographic coherent diffractive imaging (CDI) has been extensively applied using both x rays and electrons. The extension to atomic resolution has been elusive. This Letter demonstrates ptychographic electron diffractive imaging at atomic resolution, permitting identification of structure in a boron nitride helical cone at a resolution of order 1 Å, beyond that of comparative Z-contrast images. A scanning transmission electron microscope is used to create a diverging illumination in a defocused Fresnel CDI geometry, providing a robust strategy leading to a unique solution.

Experimental characterization of the coherence properties of hard x-ray sources.

The experimental characterization of the coherence properties of hard X-ray sources is reported and discussed. The source is described by its Mutual Optical Intensity (MOI). The coherent-mode decomposition is applied to the MOI described by a Gaussian-Schell model. The method allows for a direct, quantitative characterization of the degree of coherence of both synchrotron and laboratory sources. The latter represents the first example of characterizing a low coherence hard x-ray source.

Dynamic sample imaging in coherent diffractive imaging.

As the resolution in coherent diffractive imaging improves, interexposure and intraexposure sample dynamics, such as motion, degrade the quality of the reconstructed image. Selecting data sets that include only exposures where tolerably little motion has occurred is an inefficient use of time and flux, especially when detector readout time is significant. We provide an experimental demonstration of an approach in which all images of a data set exhibiting sample motion are combined to improve the quality of a reconstruction. This approach is applicable to more general sample dynamics (including sample damage) that occur during measurement.

Phase-diverse coherent diffractive imaging: high sensitivity with low dose.

This Letter demonstrates that coherent diffractive imaging (CDI), in combination with phase-diversity methods, provides reliable and artefact free high-resolution images. Here, using x rays, experimental results show a threefold improvement in the available image contrast. Furthermore, in conditions requiring low imaging dose, it is demonstrated that phase-diverse CDI provides a factor of 2 improvement in comparison to previous CDI techniques.

Astigmatic phase retrieval: an experimental demonstration.

We present the first experimental demonstration of the astigmatic phase retrieval technique, in which the diffracted wavefield is distorted by cylindrical curvature in two orthogonal directions. A charge-one vortex, a charge-two vortex, and a simple test image are all correctly reconstructed.

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 coherence approach to phase-contrast microscopy: theory.

Optical coherence theory is used to describe image formation in a telecentric optical system. By assuming a weakly interacting object and by considering points that are not too far from the optical axis, an optical transfer function description is obtained for imaging both the phase and the amplitude components of the object. A dimensionless coordinate system is identified to allow the transfer functions to be expressed independently of the details of the imaging system. Phase-contrast imaging is found to have an essentially coherent behaviour when the coherence length is a factor of 15 larger than the system resolution, and that the coherent region of the illumination therefore does not need to encompass the object.

Nanocrystallography measurements of early stage synthetic malaria pigment.

The recent availability of extremely intense, femtosecond X-ray free-electron laser (XFEL) sources has spurred the development of serial femtosecond nanocrystallography (SFX). Here, SFX is used to analyze nanoscale crystals of ß-hematin, the synthetic form of hemozoin which is a waste by-product of the malaria parasite. This analysis reveals significant differences in ß-hematin data collected during SFX and synchrotron crystallography experiments. To interpret these differences two possibilities are considered: structural differences between the nanocrystal and larger crystalline forms of ß-hematin, and radiation damage. Simulation studies show that structural inhomogeneity appears at present to provide a better fit to the experimental data. If confirmed, these observations will have implications for designing compounds that inhibit hemozoin formation and suggest that, for some systems at least, additional information may be gained by comparing structures obtained from nanocrystals and macroscopic crystals of the same molecule.

Measurements of Long-range Electronic Correlations During Femtosecond Diffraction Experiments Performed on Nanocrystals of Buckminsterfullerene.

The precise details of the interaction of intense X-ray pulses with matter are a topic of intense interest to researchers attempting to interpret the results of femtosecond X-ray free electron laser (XFEL) experiments. An increasing number of experimental observations have shown that although nuclear motion can be negligible, given a short enough incident pulse duration, electronic motion cannot be ignored. The current and widely accepted models assume that although electrons undergo dynamics driven by interaction with the pulse, their motion could largely be considered 'random'. This would then allow the supposedly incoherent contribution from the electronic motion to be treated as a continuous background signal and thus ignored. The original aim of our experiment was to precisely measure the change in intensity of individual Bragg peaks, due to X-ray induced electronic damage in a model system, crystalline C60. Contrary to this expectation, we observed that at the highest X-ray intensities, the electron dynamics in C60 were in fact highly correlated, and over sufficiently long distances that the positions of the Bragg reflections are significantly altered. This paper describes in detail the methods and protocols used for these experiments, which were conducted both at the Linac Coherent Light Source (LCLS) and the Australian Synchrotron (AS) as well as the crystallographic approaches used to analyse the data.

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.

X-ray laser-induced electron dynamics observed by femtosecond diffraction from nanocrystals of Buckminsterfullerene.

X-ray free-electron lasers (XFELs) deliver x-ray pulses with a coherent flux that is approximately eight orders of magnitude greater than that available from a modern third-generation synchrotron source. The power density of an XFEL pulse may be so high that it can modify the electronic properties of a sample on a femtosecond time scale. Exploration of the interaction of intense coherent x-ray pulses and matter is both of intrinsic scientific interest and of critical importance to the interpretation of experiments that probe the structures of materials using high-brightness femtosecond XFEL pulses. We report observations of the diffraction of extremely intense 32-fs nanofocused x-ray pulses by a powder sample of crystalline C60. We find that the diffraction pattern at the highest available incident power significantly differs from the one obtained using either third-generation synchrotron sources or XFEL sources operating at low output power and does not correspond to the diffraction pattern expected from any known phase of crystalline C60. We interpret these data as evidence of a long-range, coherent dynamic electronic distortion that is driven by the interaction of the periodic array of C60 molecular targets with intense x-ray pulses of femtosecond duration.

Emulated super-resolution using quantitative phase microscopy.

In the case of coherent illumination, knowledge of the phase and the amplitude of a light wave constitutes complete information. Phase and amplitude information can now be simply acquired using the technique of quantitative phase microscopy. It has been shown that this information allows other imaging modalities to be emulated. In this paper we consider how this information may be used to perform a form of super-resolution by emulating the effect of an annular pupil.

Rapid, low dose X-ray diffractive imaging of the malaria parasite Plasmodium falciparum.

Phase-diverse X-ray coherent diffractive imaging (CDI) provides a route to high sensitivity and spatial resolution with moderate radiation dose. It also provides a robust solution to the well-known phase-problem, making on-line image reconstruction feasible. Here we apply phase-diverse CDI to a cellular sample, obtaining images of an erythrocyte infected by the sexual stage of the malaria parasite, Plasmodium falciparum, with a radiation dose significantly lower than the lowest dose previously reported for cellular imaging using CDI. The high sensitivity and resolution allow key biological features to be identified within intact cells, providing complementary information to optical and electron microscopy. This high throughput method could be used for fast tomographic imaging, or to generate multiple replicates in two-dimensions of hydrated biological systems without freezing or fixing. This work demonstrates that phase-diverse CDI is a valuable complementary imaging method for the biological sciences and ready for immediate application.

Continuous X-ray diffractive field in protein nanocrystallography.

The recent development of X-ray free-electron laser sources has created new opportunities for the structural analysis of protein nanocrystals. The extremely small sizes of the crystals, as well as imperfections of the crystal structure, result in an interference phenomenon in the diffraction pattern. With decreasing crystallite size the structural imperfections play a role in the formation of the diffraction pattern that is comparable in importance to the size effects and should be taken into account during the data analysis and structure reconstruction processes. There now exists a need to develop new methods of protein structure determination that do not depend on the availability of good-quality crystals and that can treat proteins under conditions close to the active form. This paper demonstrates an approach that is specifically tailored to nanocrystalline samples and offers a unique crystallographic solution.

Whole-cell phase contrast imaging at the nanoscale using Fresnel coherent diffractive imaging tomography.

X-ray tomography can provide structural information of whole cells in close to their native state. Radiation-induced damage, however, imposes a practical limit to image resolution, and as such, a choice between damage, image contrast, and image resolution must be made. New coherent diffractive imaging techniques, such Fresnel Coherent Diffractive Imaging (FCDI), allows quantitative phase information with exceptional dose efficiency, high contrast, and nano-scale resolution. Here we present three-dimensional quantitative images of a whole eukaryotic cell by FCDI at a spatial resolution below 70 nm with sufficient phase contrast to distinguish major cellular components. From our data, we estimate that the minimum dose required for a similar resolution is close to that predicted by the Rose criterion, considerably below accepted estimates of the maximum dose a frozen-hydrated cell can tolerate. Based on the dose efficiency, contrast, and resolution achieved, we expect this technique will find immediate applications in tomographic cellular characterisation.