Cryo-EM phase-plate images reveal unexpected levels of apparent specimen damage.

Apoferritin (apoF) is commonly used as a test specimen in single-particle electron cryo-microscopy (cryo-EM), since it consistently produces density maps that go to 3 Å resolution or higher. When we imaged apoF with a laser phase plate (LPP), however, we observed more severe particle-to-particle variation in the images than we had previously thought to exist. Similarly, we found that images of ribulose bisphosphate carboxylase/oxygenase (rubisco) also exhibited a much greater amount of heterogeneity than expected. By comparison to simulations of images, we verified that the heterogeneity is not explained by the known features of the LPP, shot noise, or differences in particle orientation. We also demonstrate that our specimens are comparable to those previously used in the literature, based on using the final-reconstruction resolution as the metric for evaluation. All of this leads us to the hypothesis that the heterogeneity is due to damage that has occurred either during purification of the specimen or during preparation of the grids. It is not, however, our goal to explain the causes of heterogeneity; rather, we report that using the LPP has made the apparent damage too obvious to be ignored. In hindsight, similar heterogeneity can be seen in images of apoF and the 20S proteasome which others had recorded with a Volta phase plate. We therefore conclude that the increased contrast of phase-plate images (at low spatial frequencies) should also make it possible to visualize, on a single-particle basis, various forms of biologically functional heterogeneity in structure that had previously gone unnoticed.

Modern approaches to improving phase contrast electron microscopy.

Although defocus can be used to generate partial phase contrast in transmission electron microscope images, cryo-electron microscopy (cryo-EM) can be further improved by the development of phase plates which increase contrast by applying a phase shift to the unscattered part of the electron beam. Many approaches have been investigated, including the ponderomotive interaction between light and electrons. We review the recent successes achieved with this method in high-resolution, single-particle cryo-EM. We also review the status of using pulsed or near-field enhanced laser light as alternatives, along with approaches that use scanning transmission electron microscopy (STEM) with a segmented detector rather than a phase plate.

Modern approaches to improving phase contrast electron microscopy.

Although defocus can be used to generate partial phase contrast in transmission electron microscope images, cryo-electron microscopy (cryo-EM) can be further improved by the development of phase plates which increase contrast by applying a phase shift to the unscattered part of the electron beam. Many approaches have been investigated, including the ponderomotive interaction between light and electrons. We review the recent successes achieved with this method in high-resolution, single-particle cryo-EM. We also review the status of using pulsed or near-field enhanced laser light as alternatives, along with approaches that use scanning transmission electron microscopy (STEM) with a segmented detector rather than a phase plate.

Extended Rotational Coherence of Polar Molecules in an Elliptically Polarized Trap.

We demonstrate long rotational coherence of individual polar molecules in the motional ground state of an optical trap. In the present, previously unexplored regime, the rotational eigenstates of molecules are dominantly quantized by trapping light rather than static fields, and the main source of decoherence is differential light shift. In an optical tweezer array of NaCs molecules, we achieve a three-orders-of-magnitude reduction in differential light shift by changing the trap's polarization from linear to a specific "magic" ellipticity. With spin-echo pulses, we measure a rotational coherence time of 62(3) ms (one pulse) and 250(40) ms (up to 72 pulses), surpassing the projected duration of resonant dipole-dipole entangling gates by orders of magnitude.

Overcoming resolution loss due to thermal magnetic field fluctuations from phase plates in transmission electron microscopy.

We identify thermal magnetic field fluctuations, caused by thermal electron motion ("Johnson noise") in electrically conductive materials, as a potential resolution limit in transmission electron microscopy with a phase plate. Specifically, resolution loss can occur if the electron diffraction pattern is magnified to extend phase contrast to lower spatial frequencies, and if conductive materials are placed too close to the electron beam. While our initial implementation of a laser phase plate (LPP) was significantly affected by these factors, a redesign eliminated the problem and brought the performance close to the expected level. The resolution now appears to be limited by residual Johnson noise arising from the electron beam liner tube in the region of the LPP, together with the chromatic aberration of the relay optics. These two factors can be addressed during future development of the LPP.

Overcoming resolution loss due to thermal magnetic field fluctuations from phase plates in transmission electron microscopy.

We identify thermal magnetic field fluctuations, caused by thermal electron motion ("Johnson noise") in electrically conductive materials, as a potential resolution limit in transmission electron microscopy with a phase plate. Specifically, resolution loss can occur if the electron diffraction pattern is magnified to extend phase contrast to lower spatial frequencies, and if conductive materials are placed too close to the electron beam. While our initial implementation of a laser phase plate (LPP) was significantly affected by these factors, a redesign eliminated the problem and brought the performance close to the expected level. The resolution now appears to be limited by residual Johnson noise arising from the electron beam liner tube in the region of the LPP, together with the chromatic aberration of the relay optics. These two factors can be addressed during future development of the LPP.

Assembly of a Rovibrational Ground State Molecule in an Optical Tweezer.

We demonstrate the coherent creation of a single NaCs molecule in its rotational, vibrational, and electronic (rovibronic) ground state in an optical tweezer. Starting with a weakly bound Feshbach molecule, we locate a two-photon transition via the |c^{3}Σ_{1},v^{'}=26⟩ excited state and drive coherent Rabi oscillations between the Feshbach state and a single hyperfine level of the NaCs rovibronic ground state |X^{1}Σ,v^{''}=0,N^{''}=0⟩ with a binding energy of D_{0}=h×147044.63(11) GHz. We measure a lifetime of 3.4±1.6 s for the rovibronic ground state molecule, which possesses a large molecule-frame dipole moment of 4.6D and occupies predominantly the motional ground state. These long-lived, fully quantum-state-controlled individual dipolar molecules provide a key resource for molecule-based quantum simulation and information processing.

Forming a Single Molecule by Magnetoassociation in an Optical Tweezer.

We demonstrate the formation of a single NaCs molecule in an optical tweezer by magnetoassociation through an s-wave Feshbach resonance at 864.11(5) G. Starting from single atoms cooled to their motional ground states, we achieve conversion efficiencies of 47(1)%, and measure a molecular lifetime of 4.7(7) ms. By construction, the single molecules are predominantly [77(5)%] in the center-of-mass motional ground state of the tweezer. Furthermore, we produce a single p-wave molecule near 807 G by first preparing one of the atoms with one quantum of motional excitation. Our creation of a single weakly bound molecule in a designated internal state in the motional ground state of an optical tweezer is a crucial step towards coherent control of single molecules in optical tweezer arrays.