X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded1-3. It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction ‘snapshots’ are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source4. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes5. More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (~200 nm to 2 μm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes6. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage.

X-ray lasers offer new capabilities in understanding the structure of biological systems, complex materials and matter under extreme conditions1–4. Very short and extremely bright, coherent X-ray pulses can be used to outrun key damage processes and obtain a single diffraction pattern from a large macromolecule, a virus or a cell before the sample explodes and turns into plasma1. The continuous diffraction pattern of non-crystalline objects permits oversampling and direct phase retrieval2. Here we show that high-quality diffraction data can be obtained with a single X-ray pulse from a non-crystalline biological sample, a single mimivirus particle, which was injected into the pulsed beam of a hard-X-ray free-electron laser, the Linac Coherent Light Source5. Calculations indicate that the energy deposited into the virus by the pulse heated the particle to over 100,000 K after the pulse had left the sample. The reconstructed exit wavefront (image) yielded 32-nm full-period resolution in a single exposure and showed no measurable damage. The reconstruction indicates inhomogeneous arrangement of dense material inside the virion. We expect that significantly higher resolutions will be achieved in such experiments with shorter and brighter photon pulses focused to a smaller area. The resolution in such experiments can be further extended for samples available in multiple identical copies.

Strong-field physics is currently experiencing a shift towards the use of mid-IR driving wavelengths. This is because they permit conducting experiments unambiguously in the quasistatic regime and enable exploiting the effects related to ponderomotive scaling of electron recollisions. Initial measurements taken in the mid-IR immediately led to a deeper understanding of photoionization and allowed a discrimination among different theoretical models. Ponderomotive scaling of rescattering has enabled new avenues towards time-resolved probing of molecular structure. Essential for this paradigm shift was the convergence of two experimental tools: (1) intense mid-IR sources that can create high-energy photons and electrons while operating within the quasistatic regime and (2) detection systems that can detect the generated highenergy particles and image the entire momentum space of the interaction in full coincidence. Here, we present a unique combination of these two essential ingredients, namely, a 160-kHz mid-IR source and a reaction microscope detection system, to present an experimental methodology that provides an unprecedented three-dimensional view of strong-field interactions. The system is capable of generating and detecting electron energies that span a 6 order of magnitude dynamic range. We demonstrate the versatility of the system by investigating electron recollisions, the core process that drives strong-field phenomena, at both low (meV) and high (hundreds of eV) energies. The low-energy region is used to investigate recently discovered low-energy structures, while the high-energy electrons are used to probe atomic structure via laser-induced electron diffraction. Moreover, we present, for the first time, the correlated momentum distribution of electrons from nonsequential double ionization driven by mid-IR pulses.

Fully differential data for H2 dissociation in ultrashort (6 fs, 760 nm), linearly polarized, intense (0.44 PW/cm{2}) laser pulses with a stabilized carrier-envelope phase (CEP) were recorded with a reaction microscope. Depending on the CEP, the molecular orientation, and the kinetic energy release (KER), we find asymmetric proton emission at low KERs (0-3 eV), basically predicted by Roudnev and Esry, and much stronger than reported by Kling et al. Wave packet propagation calculations reproduce the salient features and discard, together with the observed KER-independent electron asymmetry, the first ionization step to be the reason for the asymmetric proton emission.

Vector momentum distributions of two electrons created in double ionization of Ar by 25 fs, 0.25 PW/cm(2) laser pulses at 795 nm have been measured using a "reaction microscope." At this intensity, where nonsequential ionization dominates, distinct correlation patterns are observed in the two-electron momentum distributions. A kinematical analysis of these spectra within the classical "recollision model" revealed an (e,2e)-like process and excitation with subsequent tunneling of the second electron as two different ionization mechanisms. This allows a qualitative separation of the two mechanisms demonstrating that excitation-tunneling is the dominant contribution to the total double ionization yield.