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Cryogenic electron tomography (cryo-ET) is the highest resolution imaging technique applicable to the life sciences, enabling subnanometer visualization of specimens preserved in their near native states. The rapid plunge freezing process used to prepare samples lends itself to time-resolved studies, which researchers have pursued for in vitro samples for decades. Here, we focus on developing a freezing apparatus for time-resolved studies in situ. The device mixes cellular samples with solution-phase stimulants before spraying them directly onto an electron microscopy grid that is transiting into cryogenic liquid ethane. By varying the flow rates of cell and stimulant solutions within the device, we can control the reaction time from tens of milliseconds to over a second before freezing. In a proof-of-principle demonstration, the freezing method is applied to a model bacterium, Caulobacter crescentus, mixed with an acidic buffer. Through cryo-ET we resolved structural changes throughout the cell, including surface-layer protein dissolution, outer membrane deformation, and cytosolic rearrangement, all within 1.5 s of reaction time. This new approach, Time-Resolved cryo-ET (TR-cryo-ET), enhances the capabilities of cryo-ET by incorporating a subsecond temporal axis and enables the visualization of induced structural changes at the molecular, organelle, or cellular level. 

Hypervalent iron intermediates have been invoked in the catalytic cycles of many metalloproteins, and thus, it is crucial to understand how the coupling between such species and their environment can impact their chemical and physical properties in such contexts. In this work, we take advantage of the solvent kinetic isotope effect (SKIE) to gain insight into the nonradiative deactivation of electronic excited states of the aqueous ferrate(VI) ion. We observe an exceptionally large SKIE of 9.7 for the nanosecond-scale relaxation of the lowest energy triplet ligand field state to the ground state. Proton inventory studies demonstrate that a single solvent O–H bond is coupled to the ion during deactivation, likely due to the sparse vibrational structure of ferrate(VI). Such a mechanism is consistent with that reported for the deactivation of f–f excited states of aqueous trivalent lanthanides, which exhibit comparably large SKIE values. This phenomenon is ascribed entirely to dissipation of energy into a higher overtone of a solvent acceptor mode, as any impact on the apparent relaxation rate due to a change in solvent viscosity is negligible. 

Ferrate(VI) has the potential to play a key role in future water supplies. Its salts have been suggested as “green” alternatives to current advanced oxidation and disinfection methods in water treatment, especially when combined with ultraviolet light to stimulate generation of highly oxidizing Fe(V) and Fe(IV) species. However, the nature of these intermediates, the mechanisms by which they form, and their roles in downstream oxidation reactions remain unclear. Here, we use a combination of optical and X-ray transient absorption spectroscopies to study the formation, interconversion, and relaxation of several excited-state and metastable high-valent iron species following excitation of aqueous potassium ferrate(VI) by ultraviolet and visible light. Branching from the initially populated ligand-to-metal charge transfer state into independent photophysical and photochemical pathways occurs within tens of picoseconds, with the quantum yield for the generation of reactive Fe(V) species determined by relative rates of the competing intersystem crossing and reverse electron transfer processes. Relaxation of the metal-centered states then occurs within 4 ns, while the formation of metastable Fe(V) species occurs in several steps with time constants of 250 ps and 300 ns. Results here improve the mechanistic understanding of the formation and fate of Fe(V) and Fe(IV), which will accelerate the development of novel advanced oxidation processes for water treatment applications.