The Center for Biomolecular Structure (CBMS) Bioimaging Workshop was held from September 25–29, 2023, at the National Synchrotron Light Source II (NSLS-II) at Brookhaven National Laboratory in Upton, NY. The aim of the annual workshop is to provide hands-on training in X-ray Fluorescence (XRF) microscopy and X-ray Absorption Micro-Spectroscopy (micro-XAS) to bioenvironmental scientists. In 2023, ten new researchers were trained on the techniques that they are interested in applying to their research.
The first day of the workshop consisted of lectures on fundamental concepts of X-ray methods and hands-on training on sample preparation. During the next three days, the participants performed measurements at three X-ray microscopy beamlines within the Imaging and Microscopy Program (IMP) at NSLS-II. These beamlines included the X-ray Fluorescence Microprobe (XFM) beamline, the Sub-Micron X-ray Fluorescence Microprobe (SRX) beamline, and the Tender Energy X-ray Absorption Spectroscopy (TES) beamline. The IMP, which also includes the Hard X-ray Nanoprobe (HXN) beamline, provides complementary X-ray imaging and spectroscopy techniques with length scales ranging from 12 nm to a few microns, allowing the imaging of a wide range of biological samples and substructures.1–4 While the data were being collected and analyzed, the participants spent additional time in the chemistry laboratories preparing additional samples. Each user group rotated through all three beamlines. On the final day of the workshop, participants shared their results and experience of using state-of-the-art tools at NSLS-II.
This year’s researchers included graduate students, postdocs, professors, and scientists from both academic institutions and national laboratories. The research themes of the participants included plant-microbe interactions, bacterial-fungal interactions, the impact of different fertilizer regimes on plant nutrition, oxidative stress in sickle cell disease, and bio-mining of lanthanides from red mud.
Sample preparation is key to a successful experiment
Throughout the workshop, the importance of careful sample preparation was emphasized. Because XRF microscopy is sensitive to elements down to attogram concentrations and for special cases well below an attogram,5 it is easy to introduce artifacts and impurities into the sample during handling. The sample preparation sessions emphasized “tips and tricks” to preserve the morphology and elemental distribution at the experimental endpoint. Participants were taught to question and understand every step of the sample preparation process including potential pitfalls when introducing chemical fixatives, washing samples with buffers, and mounting samples onto substrates for measurements. As part of the sample preparation training, participants consulted with beamline staff ahead of the workshop.
Introduction to multi-modal and multi-length scale experiments
Depending on the research question under investigation, it is necessary to acquire XRF microscopy data at several spatial scales; for example, looking at a plant leaf on the millimeter scale using micron resolution and then zooming in to ~10–20 μm cells using sub-micron resolution. Science cases for these approaches were presented during the workshop. To measure the same sample at TES, XFM, and SRX, a plant root cross-section was mounted onto a 5 mm2 silicon nitride window and inserted into in-house fabricated sample holders and adapters. This multi-beamline adaptation allowed for low-energy phosphorus XRF mapping at TES, and zinc XRF mapping at SRX (for submicron resolution) and XFM (for micron resolution). NSLS-II offers its users opportunities to utilize other multi-modal techniques e.g., XRF microscopy combined with X-ray diffraction or X-ray phase contrast imaging.
Expanding access to the biological and environmental research community and minority-serving institutions (DEIA)
The CBMS Bioimaging Workshop was launched to recruit and train biological and environmental researchers thereby increasing this fraction of the user population at the imaging and microscopy beamlines. This mission also includes recruiting individuals from more diverse groups (~50% of the accepted participants in the 2023 training group). Participating researchers gained access to obtain preliminary data for submission of a NSLS-II General User proposal for future beam time at the facility. Approximately 60% of the participants submitted NSLS-II general user proposals during the first proposal call after the workshop while the remainder anticipate submitting a NSLS-II General User proposal in the future.
X-ray microscopy is a fast-developing field. The improvements in X-ray optics and detectors have increased the speed, quantity, and resolution of the data. This annual workshop is an excellent forum for researchers to learn how to correctly prepare samples, collect data, and interpret the data. Future workshops will include phase contrast imaging, oxidation-state mapping, and cryo-scanning transmission X-ray microscopy. We anticipate the growth of an experienced bioimaging research community to maximize the techniques and expertise offered at NSLS-II.
Center for BioMolecular Structure Bioimaging Workshop participants
Acknowledgements
The Center for BioMolecular Structure (CBMS) is primarily supported by the National Institutes of Health, National Institute of General Medical Sciences (NIGMS) through a Center Core P30 Grant (P30GM133893), and by the DOE Office of Biological and Environmental Research (KP1607011). As part of NSLS-II, a national user facility at Brookhaven National Laboratory, work performed at the CBMS is supported in part by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences Program under contract number and DE-SC0012704.
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