Deep generative priors for biomolecular 3D heterogeneous reconstruction from cryo-EM projections.

Cryo-electron microscopy has become a powerful tool to determine three-dimensional (3D) structures of rigid biological macromolecules from noisy micrographs with single-particle reconstruction. Recently, deep neural networks, e.g., CryoDRGN, have demonstrated conformational and compositional heterogeneity of complexes. However, the lack of ground-truth conformations poses a challenge to assess the performance of heterogeneity analysis methods. In this work, variational autoencoders (VAE) with three types of deep generative priors were learned for latent variable inference and heterogeneous 3D reconstruction via Bayesian inference. More specifically, VAEs with "Variational Mixture of Posteriors" priors (VampPrior-SPR), non-parametric exemplar-based priors (ExemplarPrior-SPR) and priors from latent score-based generative models (LSGM-SPR) were quantitatively compared with CryoDRGN. We built four simulated datasets composed of hypothetical continuous conformation or discrete states of the hERG K + channel. Empirical and quantitative comparisons of inferred latent representations were performed with affine-transformation-based metrics. These models with more informative priors gave better regularized, interpretable factorized latent representations with better conserved pairwise distances, less deformed latent distributions and lower within-cluster variances. They were also tested on experimental datasets to resolve compositional and conformational heterogeneity (50S ribosome assembly, cowpea chlorotic mottle virus, and pre-catalytic spliceosome) with comparable high resolution. Codes and data are available: https://github.com/benjamin3344/DGP-SPR.

Compact Ho:YLF-pumped ZnGeP2-based optical parametric amplifiers tunable in the molecular fingerprint regime.

We report on a compact mid-infrared laser architecture, comprising a chain of $ {\rm ZnGeP}_2 $ZnGeP2-based optical parametric amplifiers (OPAs), which afford a higher energy yield ($ \mathbin{\lower.3ex\hbox{$\buildrel \lt \over{\smash{\scriptstyle\sim}\vphantom{_x}}$}} 60\;\unicode{x00B5} {\rm J} $∼x<60µJ at 1 kHz) compared to most conventional OPA gain media transparent in the 2-8-µm wavelength range. Specifically, our OPA scheme allows ready tunability in the molecular fingerprint regime and is tailored for strong-field excitation and coherent control of both stretch and bend (or torsional) vibrational modes in molecules. The OPAs are pumped and directly seeded (via supercontinuum generation) by a 2-µm, 3-ps Ho:YLF regenerative amplifier. The compressibility of the OPA output is demonstrated by a representative measurement of the near-Gaussian temporal profile of a dispersion-compensated 105-fs idler pulse at a central wavelength of 5.1 µm, corresponding to ${\sim}6 $∼6 optical cycles. Detailed numerical simulations closely corroborate the experimental measurements, providing a benchmark and a platform to further explore the parameter space for future design, optimization, and implementation of high-energy, ultrafast, mid-infrared laser schemes.

Serial protein crystallography in an electron microscope.

Serial X-ray crystallography at free-electron lasers allows to solve biomolecular structures from sub-micron-sized crystals. However, beam time at these facilities is scarce, and involved sample delivery techniques are required. On the other hand, rotation electron diffraction (MicroED) has shown great potential as an alternative means for protein nano-crystallography. Here, we present a method for serial electron diffraction of protein nanocrystals combining the benefits of both approaches. In a scanning transmission electron microscope, crystals randomly dispersed on a sample grid are automatically mapped, and a diffraction pattern at fixed orientation is recorded from each at a high acquisition rate. Dose fractionation ensures minimal radiation damage effects. We demonstrate the method by solving the structure of granulovirus occlusion bodies and lysozyme to resolutions of 1.55 Å and 1.80 Å, respectively. Our method promises to provide rapid structure determination for many classes of materials with minimal sample consumption, using readily available instrumentation.

Towards OCT-Navigated Tissue Ablation with a Picosecond Infrared Laser (PIRL) and Mass-Spectrometric Analysis.

Medical lasers are commonly used in interventions to ablate tumor tissue. Recently, the picosecond infrared laser has been introduced, which greatly decreases damaging of surrounding healthy tissue. Further, its ablation plume contains intact biomolecules which can be collected and analyzed by mass spectrometry. This allows for a specific chracterization of the tissue. For a precise treatment, however, a suitable guidance is needed. Further, spatial information is required if the tissue is to be characterized at different parts in the ablated area. Therefore, we propose a system which employs optical coherence tomography as the guiding imaging modality. We describe a prototypical system which provides automatic ablation of areas defined in the image data. For this purpose, we use a calibration with a robot which drives the laser fiber and collects the arising plume. We demonstrate our system on porcine tissue samples.

Robust reconstruction of time-resolved diffraction from ultrafast streak cameras.

In conjunction with ultrafast diffraction, streak cameras offer an unprecedented opportunity for recording an entire molecular movie with a single probe pulse. This is an attractive alternative to conventional pump-probe experiments and opens the door to studying irreversible dynamics. However, due to the "smearing" of the diffraction pattern across the detector, the streaking technique has thus far been limited to simple mono-crystalline samples and extreme care has been taken to avoid overlapping diffraction spots. In this article, this limitation is addressed by developing a general theory of streaking of time-dependent diffraction patterns. Understanding the underlying physics of this process leads to the development of an algorithm based on Bayesian analysis to reconstruct the time evolution of the two-dimensional diffraction pattern from a single streaked image. It is demonstrated that this approach works on diffraction peaks that overlap when streaked, which not only removes the necessity of carefully choosing the streaking direction but also extends the streaking technique to be able to study polycrystalline samples and materials with complex crystalline structures. Furthermore, it is shown that the conventional analysis of streaked diffraction can lead to erroneous interpretations of the data.

Compression of high-density 0.16 pC electron bunches through high field gradients for ultrafast single shot electron diffraction: The Compact RF Gun.

We present an RF gun design for single shot ultrafast electron diffraction experiments that can produce sub-100 fs high-charge electron bunches in the 130 keV energy range. Our simulations show that our proposed half-cell RF cavity is capable of producing 137 keV, 27 fs rms (60 fs FWHM), 106 electron bunches with an rms spot size of 276 µm and a transverse coherence length of 2.0 nm. The required operation power is 9.2 kW, significantly lower than conventional rf cavity designs and a key design feature. This electron source further relies on high electric field gradients at the cathode to simultaneously accelerate and compress the electron bunch to open up new space-time resolution domains for atomically resolved dynamics.

Fixed target matrix for femtosecond time-resolved and in situ serial micro-crystallography.

We present a crystallography chip enabling in situ room temperature crystallography at microfocus synchrotron beamlines and X-ray free-electron laser (X-FEL) sources. Compared to other in situ approaches, we observe extremely low background and high diffraction data quality. The chip design is robust and allows fast and efficient loading of thousands of small crystals. The ability to load a large number of protein crystals, at room temperature and with high efficiency, into prescribed positions enables high throughput automated serial crystallography with microfocus synchrotron beamlines. In addition, we demonstrate the application of this chip for femtosecond time-resolved serial crystallography at the Linac Coherent Light Source (LCLS, Menlo Park, California, USA). The chip concept enables multiple images to be acquired from each crystal, allowing differential detection of changes in diffraction intensities in order to obtain high signal-to-noise and fully exploit the time resolution capabilities of XFELs.

The photocycle and ultrafast vibrational dynamics of bacteriorhodopsin in lipid nanodiscs.

The photocycle and vibrational dynamics of bacteriorhodopsin in a lipid nanodisc microenvironment have been studied by steady-state and time-resolved spectroscopies. Linear absorption and circular dichroism indicate that the nanodiscs do not perturb the structure of the retinal binding pocket, while transient absorption and flash photolysis measurements show that the photocycle which underlies proton pumping is unchanged from that in the native purple membranes. Vibrational dynamics during the initial photointermediate formation are subsequently studied by ultrafast broadband transient absorption spectroscopy, where the low scattering afforded by the lipid nanodisc microenvironment allows for unambiguous assignment of ground and excited state nuclear dynamics through Fourier filtering of frequency regions of interest and subsequent time domain analysis of the retrieved vibrational dynamics. Canonical ground state oscillations corresponding to high frequency ethylenic and C-C stretches, methyl rocks, and hydrogen out-of-plane wags are retrieved, while large amplitude, short dephasing time vibrations are recovered predominantly in the frequency region associated with out-of-plane dynamics and low frequency torsional modes implicated in isomerization.

[Perspectives of laser-assisted keratoplasty: current overview and first preliminary results with the picosecond infrared laser (λ = 3 µm)]. / Perspektiven der laserassistierten Keratoplastik: Eine aktuelle Übersicht und erste experimentelle Erfahrungen mit dem Pikosekundeninfrarotlaser (λ = 3 µm).

BACKGROUND: This article provides a review of the current state of laser-assisted keratoplasty and describes a first proof of concept study to test the feasibility of a new mid-infrared (MIR) picosecond laser to perform applanation-free corneal trephination. METHODS: The procedure is based on a specially adapted laser system (PIRL-HP2-1064 OPA-3000, Attodyne, Canada) which works with a wavelength of 3,000 ± 90 nm, a pulse duration of 300 ps and a repetition rate of 1 kHz. The picosecond infrared laser (PIRL) beam is delivered to the sample by a custom-made optics system with an implemented scanning mechanism. Corneal specimens were mounted on an artificial anterior chamber and subsequent trephination was performed with the PIRL under stable intraocular pressure conditions. RESULTS: A defined corneal ablation pattern, e.g. circular, linear, rectangular or disc-shaped, can be selected and its specific dimensions are defined by the user. Circular and linear ablation patterns were employed for the incisions in this study. Linear and circular penetrating PIRL incisions were examined by macroscopic inspection, histology, confocal microscopy and environmental scanning electron microscopy (ESEM) for characterization of the incisional quality. Using PIRL reproducible and stable incisions could be made in human and porcine corneal samples with minimal damage to the surrounding tissue. CONCLUSION: The PIRL laser radiation in the mid-infrared spectrum with a wavelength of 3 µm is exactly tuned to one of the dominant vibrational excitation bands of the water molecule, serves as an effective tool for applanation-free corneal incision and might broaden the armamentarium of corneal transplant surgery.

Full characterization of RF compressed femtosecond electron pulses using ponderomotive scattering.

High bunch charge, femtosecond, electron pulses were generated using a 95 kV electron gun with an S-band RF rebunching cavity. Laser ponderomotive scattering in a counter-propagating beam geometry is shown to provide high sensitivity with the prerequisite spatial and temporal resolution to fully characterize, in situ, both the temporal profile of the electron pulses and RF time timing jitter. With the current beam parameters, we determined a temporal Instrument Response Function (IRF) of 430 fs FWHM. The overall performance of our system is illustrated through the high-quality diffraction data obtained for the measurement of the electron-phonon relaxation dynamics for Si (001).

Two-dimensional fifth-order Raman spectroscopy of liquid formamide: Experiment and Theory.

The fifth-order two-dimensional (2D) Raman spectrum of liquid formamide has been obtained. The absolute signal levels, qualitative features, as well as quantitative aspects of the 2D spectrum are found to be in good agreement with recent molecular dynamics calculations. The most important singular feature is the relatively strong rephasing signal observed along the diagonal. This finding illustrates the more structured nature of the hydrogen bond network of liquid formamide in comparison with simple liquids as exemplified by CS(2). The theoretical calculations have been extended to include comparisons of different potentials that illustrate the sensitivity of the experiment to the anharmonic motions in the liquid state. The theoretical results point out the key features in the 2D spectrum that probe the essential details in the intermolecular potential. The experiment has been demonstrated to provide new insight into collective effects operating in hydrogen bonded liquids and opens up the exploration of other liquids with this approach.

Femtosecond electron pulse characterization using laser ponderomotive scattering.

We demonstrate a method for the measurement of the instantaneous duration of femtosecond electron pulses using the ponderomotive force of an intense ultrashort laser pulse. An analysis procedure for the extraction of the electron pulse duration from the transient change of the transverse electron beam profile is proposed. The durations of the electron pulses generated in our setup were determined to be 410+/-30 fs.

Diffractive optics implementation of six-wave mixing.

Diffractive optics are applied to six-wave mixing processes to provide a single optic approach to attaining the required, relatively complex, phase-matching geometry to discriminate against lower-order nonlinear responses. The diffractive optics were designed specifically for broad-bandwidth operation and passive phase locking of the appropriate pulse pairs for use in femtosecond two-dimensional Raman studies of the dynamic structure of liquids. The fifth-order signal was studied in liquid CS>(2); two different colors were used for the excitation and the probe to reduce background scatter, as were two different phase-matching geometries with different degrees of suppression of cascaded third-order processes.