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Abstract Serial crystallography (SX) has revolutionized structural biology by enabling high-resolution structure determination for important classes of proteins, including the study of relevant biomolecular reaction mechanisms. However, one of the ongoing challenges in this field remains the efficient use of precious macromolecule samples whose availability is often limited. Reducing sample consumption is thus critical in maximizing the potential of SX conducted at powerful X-ray sources such as synchrotrons and X-ray free-electron lasers (XFEL) to expand to a broader range of significant biological samples, gaining insights into unraveled biological reaction mechanisms. This review focuses on three primary sample delivery systems: fixed-targets, liquid injection, and hybrid methods, each with distinct advantages and limitations concerning sample consumption. The progress and challenges associated with these methods, highlighting advancements in reducing sample consumption and thus enabling the study of more diverse biological samples, are summarized. We compare the currently reported sample delivery methods in view of the minimum amount of sample required to obtain a full data set and discuss how the current approaches compare to this theoretical minimum. With this overview, we aim to provide a critical and comprehensive assessment of the current methods and experimental realizations for sample delivery in SX with proteins. 

Two-photon excited fluorescence (TPEF) is a powerful technique that enables the examination of intrinsic retinal fluorophores involved in cellular metabolism and the visual cycle. Although previous intensity-based TPEF studies in non-human primates have successfully imaged several classes of retinal cells and elucidated aspects of both rod and cone photoreceptor function, fluorescence lifetime imaging (FLIM) of the retinal cells under light-dark visual cycle has yet to be fully exploited. Here we demonstrate a FLIM assay of photoreceptors and retinal pigment epithelium (RPE) that reveals key insights into retinal physiology and adaptation. We found that photoreceptor fluorescence lifetimes increase and decrease in sync with light and dark exposure, respectively. This is likely due to changes in all-trans-retinol and all-trans-retinal levels in the outer segments, mediated by phototransduction and visual cycle activity. During light exposure, RPE fluorescence lifetime was observed to increase steadily over time, as a result of all-trans-retinol accumulation during the visual cycle and decreasing metabolism caused by the lack of normal perfusion of the sample. Our system can measure the fluorescence lifetime of intrinsic retinal fluorophores on a cellular scale, revealing differences in lifetime between retinal cell classes under different conditions of light and dark exposure.