Tim Salditt

Quantitative biological imaging by ptychographic x-ray diffraction microscopy

Recent advances in coherent x-ray diffractive imaging have paved the way to reliable and quantitative imaging of noncompact specimens at the nanometer scale. Introduced a year ago, an advanced implementation of ptychographic coherent diffractive imaging has removed much of the previous limitations regarding sample preparation and illumination conditions. Here, we apply this recent approach toward structure determination at the nanoscale to biological microscopy. We show that the projected electron density of unstained and unsliced freeze-dried cells of the bacterium Deinococcus radiodurans can be derived from the reconstructed phase in a straightforward and reproducible way, with quantified and small errors. Thus, the approach may contribute in the future to the understanding of the highly disputed nucleoid structure of bacterial cells. In the present study, the estimated resolution for the cells was 85 nm (half-period length), whereas 50-nm resolution was demonstrated for lithographic test structures. With respect to the diameter of the pinhole used to illuminate the samples, a superresolution of about 15 was achieved for the cells and 30 for the test structures, respectively. These values should be assessed in view of the low dose applied on the order of ≃1.3·105 Gy, and were shown to scale with photon fluence.

Hard x-ray nanobeam characterization by coherent diffraction microscopy

We have carried out a ptychographic scanning coherent diffraction imaging experiment on a test object in order to characterize the hard x-ray nanobeam in a scanning x-ray microscope. In addition to a high resolution image of the test object, a detailed quantitative picture of the complex wave field in the nanofocus is obtained with high spatial resolution and dynamic range. Both are the result of high statistics due to the large number of diffraction patterns. The method yields a complete description of the focus, is robust against inaccuracies in sample positioning, and requires no particular shape or prior knowledge of the test object.

Hard X-ray imaging of bacterial cells: nano-diffraction and ptychographic reconstruction.

Ptychographic coherent X-ray diffractive imaging (PCDI) has been combined with nano-focus X-ray diffraction to study the structure and density distribution of unstained and unsliced bacterial cells, using a hard X-ray beam of 6.2keV photon energy, focused to about 90nm by a Fresnel zone plate lens. While PCDI provides images of the bacteria with quantitative contrast in real space with a resolution well below the beam size at the sample, spatially resolved small angle X-ray scattering using the same Fresnel zone plate (cellular nano-diffraction) provides structural information at highest resolution in reciprocal space up to 2nm(-1). We show how the real and reciprocal space approach can be used synergistically on the same sample and with the same setup. In addition, we present 3D hard X-ray imaging of unstained bacterial cells by a combination of ptychography and tomography.

Collective Dynamics of Lipid Membranes Studied by Inelastic Neutron Scattering

We have studied the collective short wavelength dynamics in deuterated 1,2-dimyristoyl-sn-glycero-3-phoshatidylcholine (DMPC) bilayers by inelastic neutron scattering. The corresponding dispersion relation variant Plancks over 2pi omega(Q) is presented for the gel and the fluid phase of this model system. The temperature dependence of the inelastic excitations indicates a phase coexistence between the two phases over a broad range and leads to a different assignment of excitations from that reported in a preceding inelastic x-ray scattering study [Phys. Rev. Lett. 86, 740 (2001)]]. As a consequence, we find that the minimum in the dispersion relation is actually deeper in the gel than in the fluid phase. Finally, we can clearly identify an additional nondispersive (optical) mode predicted by molecular dynamics simulations [Phys. Rev. Lett. 87, 238101 (2001)]].

Sub-5 nm hard x-ray point focusing by a combined Kirkpatrick-Baez mirror and multilayer zone plate

Compound optics such as lens systems can overcome the limitations concerning resolution, efficiency, or aberrations which fabrication constraints would impose on any single optical element. In this work we demonstrate unprecedented sub-5 nm point focusing of hard x-rays, based on the combination of a high gain Kirkpatrick-Baez (KB) mirror system and a high resolution W/Si multilayer zone plate (MZP) for ultra-short focal length f. The pre-focusing allows limiting the MZP radius to below 2 μm, compatible with the required 5 nm structure width and essentially unlimited aspect ratios, provided by enabling fabrication technology based on pulsed laser deposition (PLD) and focused ion beam (FIB).

Energetics of stalk intermediates in membrane fusion are controlled by lipid composition

We have used X-ray diffraction on the rhombohedral phospholipid phase to reconstruct stalk structures in different pure lipids and lipid mixtures with unprecedented resolution, enabling a quantitative analysis of geometry, as well as curvature and hydration energies. Electron density isosurfaces are used to study shape and curvature properties of the bent lipid monolayers. We observe that the stalk structure is highly universal in different lipid systems. The associated curvatures change in a subtle, but systematic fashion upon changes in lipid composition. In addition, we have studied the hydration interaction prior to the transition from the lamellar to the stalk phase. The results indicate that facilitating dehydration is the key to promote stalk formation, which becomes favorable at an approximately constant interbilayer separation of 9.0 ± 0.5 Å for the investigated lipid compositions.

Molecular motions in lipid bilayers studied by the neutron backscattering technique.

We report a high energy-resolution neutron backscattering study to investigate slow motions on nanosecond time scales in highly oriented solid supported phospholipid bilayers of the model system DMPC-d54 (deuterated 1,2-dimyristoyl-sn-glycero-3-phoshatidylcholine), hydrated with heavy water. This technique allows to discriminate the onset of mobility at different length scales for the different molecular components, as, e.g., the lipid acyl-chains and the hydration water in between the membrane stacks, respectively, and provides a benchmark test regarding the feasibility of neutron backscattering investigations on these sample systems. We discuss freezing of the lipid acyl-chains, as observed by this technique, and observe a second freezing transition which we attribute to the hydration water.

Optogenetic stimulation of the auditory pathway

Auditory prostheses can partially restore speech comprehension when hearing fails. Sound coding with current prostheses is based on electrical stimulation of auditory neurons and has limited frequency resolution due to broad current spread within the cochlea. In contrast, optical stimulation can be spatially confined, which may improve frequency resolution. Here, we used animal models to characterize optogenetic stimulation, which is the optical stimulation of neurons genetically engineered to express the light-gated ion channel channelrhodopsin-2 (ChR2). Optogenetic stimulation of spiral ganglion neurons (SGNs) activated the auditory pathway, as demonstrated by recordings of single neuron and neuronal population responses. Furthermore, optogenetic stimulation of SGNs restored auditory activity in deaf mice. Approximation of the spatial spread of cochlear excitation by recording local field potentials (LFPs) in the inferior colliculus in response to suprathreshold optical, acoustic, and electrical stimuli indicated that optogenetic stimulation achieves better frequency resolution than monopolar electrical stimulation. Virus-mediated expression of a ChR2 variant with greater light sensitivity in SGNs reduced the amount of light required for responses and allowed neuronal spiking following stimulation up to 60 Hz. Our study demonstrates a strategy for optogenetic stimulation of the auditory pathway in rodents and lays the groundwork for future applications of cochlear optogenetics in auditory research and prosthetics.

A highly unusual palindromic transmembrane helical hairpin formed by SARS coronavirus E protein.

Abstract The agent responsible for the recent severe acute respiratory syndrome (SARS) outbreak is a previously unidentified coronavirus. While there is a wealth of epidemiological studies, little if any molecular characterization of SARS coronavirus (SCoV) proteins has been carried out. Here we describe the molecular characterization of SCoV E protein, a critical component of the virus responsible for virion envelope morphogenesis. We conclusively show that SCoV E protein contains an unusually short, palindromic transmembrane helical hairpin around a previously unidentified pseudo-center of symmetry, a structural feature which seems to be unique to SCoV. The hairpin deforms lipid bilayers by way of increasing their curvature, providing for the first time a molecular explanation of E proteins pivotal role in viral budding. The molecular understanding of this critical component of SCoV may represent the beginning of a concerted effort aimed at inhibiting its function, and consequently, viral infectivity.

Thermal fluctuations and stability of solid-supported lipid membranes

In this review article we discuss recent work on the thermal stability, thermal fluctuations and interaction forces in lipid membrane stacks. The lipid bilayers are deposited on solid surfaces for investigation by interface sensitive x-ray and neutron scattering on length scales ranging from the molecular to the mesoscopic scale. We discuss how the structure, composition, fluctuations, and interactions in lipid and biomimetic membranes can be studied in the fluid Lα phase. Results on the interaction potentials, height–height correlation functions, elasticity parameters, fluid lipid chain ordering, and collective lipid chain motions are discussed.