NIGMS - National Institute of General Medical Sciences Grants

NIGMS - National Institute of General Medical Sciences

Project Summary Advancing Probe Technology for Ultrasensitive RNA Imaging via Hybridization Chain Reaction Encoded in the genome of each organism, biological circuits direct development, maintain integrity in the face of attacks, control responses to environmental stimuli, and sometimes malfunction to cause disease. RNA in situ hybridization (RNA-ISH) methods provide drug developers, pathologists, and biologists with a critical window into the spatial organization of this circuitry, enabling imaging of RNA expression in an anatomical context. While it is desirable to perform multiplex experiments in which multiple targets are imaged quantitatively at high resolution in a single specimen, using traditional RNA-ISH methods in whole-mount vertebrate embryos and thick tissue sections, multiplexing is cumbersome, staining is non-quantitative, and spatial resolution is routinely compromised by diffusion of reporter molecules. These multi-decade technological shortcomings have significantly impeded the study of gene regulatory networks in systems most relevant to human development and disease. To overcome these challenges, in situ amplification based on the mechanism of hybridization chain reaction (HCR) draws on concepts from the new field of dynamic nucleic acid nanotechnology to redefine the state-of-the- art for RNA fluorescence in situ hybridization (RNA-FISH), achieving four breakthroughs in highly autofluorescent samples including whole-mount vertebrate embryos, thick brain slices, and FFPE tissue sections: 1) straight- forward multiplexing with 1-step quantitative signal amplification for up to 10 target mRNAs simultaneously; 2) analog mRNA relative quantitation with subcellular resolution in an anatomical context; 3) digital mRNA abso- lute quantitation with single-molecule resolution in an anatomical context; 4) automatic background suppression throughout the protocol, dramatically enhancing performance and ease-of-use. However, automatic background suppression is achieved using a dual-probe technology that does not apply to short RNA targets (e.g., miRNAs) that are only long enough to stably bind a single probe. Moreover, for single- molecule imaging of low-abundance mRNA targets, where each target molecule is resolved as a distinct dot, variable probe hybridization yield due to competing secondary structure within the target leads to a distribution of dot intensities that can overlap with autofluorescence, leading to false-negatives or false-positives. The proposed R&D will address these two critical technology gaps. To suppress background for imaging short RNA targets, we will develop a new probe architecture that minimizes non-specific binding while preserving robust HCR am- plification. To achieve high-fidelity single-molecule imaging across all classes of RNA targets, we will develop an automated probe design pipeline that combines physics-based simulations with bioinformatics to generate probe sequences with minimal off-target binding and high-yield hybridization to cognate RNA targets. These advances will enable biologists, drug developers, and pathologists to perform ultrasensitive imaging of all classes of target RNAs with anatomical context in the samples most relevant to human development and disease.

NIGMS - National Institute of General Medical Sciences

Project Summary/Abstract Protein therapeutics have revolutionized how we treat disease and now represent seven of the top ten drugs. However, their success has been limited with many of the more challenging diseases, which is perhaps not surprising as while proteins were evolved for many functions, they were not evolved to be therapeutics. What is needed to realize the full potential of these natural product macromolecules is a cost-effective method to chemi- cally manipulate and optimize their properties, just as has been and remains common with small molecule natural product-based therapeutics. We are developing such a method, based on the first semi-synthetic organism (SSO) that by virtue of harboring two synthetic nucleotides that form an unnatural base pair (UBP), stores and retrieves more information than any natural cell. The SSO uses the increased information to create new codons that are used to site-specifically encode non-canonical amino acids (ncAAs) during recombinant expression, and after purification, the ncAAs provide reactive handles with which to attach different functionalities of interest. We are using the SSO-platform to build next generation protein therapeutics, such as dual-drug protein-conjugates and bispecific engagers with unprecedented architectures. While the SSO-platform has been validated for indus- trial scale use, its broader application requires reducing production costs during high density growth by lowering the amount of the expensive unnatural triphosphates required and by increasing the yield of protein expressed. Our preliminary results have now identified the two aspects of the SSO’s physiology whose optimization promises to most significantly reduce costs and increase production yields. While the SSO was originally plagued by unstable expression of the transporter that imports the unnatural triphosphates, this has recently been remedied, leaving only its suboptimal recognition of the unnatural substrates as a barrier to reducing the amount of the most costly component of the platform that must be used, the unnatural triphosphates. Thus, the first aim of this application is to decrease the amount of unnatural triphosphate that must be added 10-fold by using directed evolution to optimize their recognition by the transporter. In addition, our preliminary data indicate that the presence of an unnatural nucleotide in the anticodon of the tRNAs used, disrupts its structure and/or ability to pair with a cognate unnatural codon. This is not surprising as the region flanking the anticodon of each tRNA evolved specifically to complement the structure and stability of the anticodon-codon minihelix, and obviously never evolved for anticodons with unnatural nucleotides. Thus, the second aim of this application is to evolve the corresponding regions of the tRNAs used by the SSO to facilitate decoding the unnatural codons. Successful completion of the two aims will result in an optimized SSO-platform that is efficient, cost-effective, and able to tailor proteins for therapeutic function as has only been previously possible with small molecules. This Phase I study is thus expected to lead to a Phase II study focused on developing next generation therapeutics for a wide variety of difficult to treat diseases.

NIGMS - National Institute of General Medical Sciences

Project Summary This proposal outlines a training program for an aspiring principal investigator with a focus on developing a comprehensive skillset to make impactful discoveries in our understanding of the mechanisms that overcome replication stress. Replication stress, the slowing and stalling of DNA synthesis, endangers accurate DNA replication and threatens genome stability. Replication stress is induced in healthy cells through various endogenous and exogenous factors, such as a shortage in deoxyribonucleotides or exposure to reactive oxygen species. In genotoxic cancer therapy, however, replication stress in tumor cells is anticipated, and pathways that mitigate replication stress lead to resistance to treatment and to recurrence of disease. Consequently, there is a critical need to understand the molecular mechanisms by which both healthy and cancerous cells overcome replication stress. The proposed project will impact human health by uncovering the molecular mechanisms of proteins that mitigate replication stress through their engagement in replication fork (RF) reversal. In RF reversal, the RF undergoes a structural change that involves the annealing of the nascent and parental DNA strands to drive the movement of the RF in the reverse direction. In the reversed configuration, the RF is stabilized and able to withstand loss of its integrity from replication stress through repair and checkpoint mechanisms. In human cells, two distinct RF reversal pathways have been described- one relying on SMARCAL1 with HLTF and ZRANB3 and the other on FBH1. However, the molecular mechanisms of the FBH1-pathway of RF reversal have remained unclear, leaving a significant knowledge gap in the field. As such, there is a critical need to delineate the molecular details of FBH1-mediated RF reversal. The central hypothesis of this project is that RAD54L, a critical protein in DNA repair by homologous recombination, is essential in the FBH1-mediated pathway of RF reversal. Our overall objectives are to explicate the molecular mechanisms of FBH1 and RAD54L in RF reversal in human cells (Aim 1), determine the physical and functional interaction between RAD54L and FBH1 in vitro (Aim 1), and investigate the consequences of impaired RF reversal in pre-neoplastic cells with an activated oncogene (Aim 2). The proposed research is significant because it is expected to provide scientific justification for the continued development of inhibitors targeting pathways that mitigate replication stress. The knowledge gained herein also will improve risk predictions for health of individuals with altered genes and proteins critical in RF reversal. The proposed research will incorporate expert training in cell biology, biochemistry, and model organisms in the F99 and K00 phases. The F99 phase will be completed at Colorado State University under the mentorship of Dr. Claudia Wiese with additional biochemical training from Dr. Patrick Sung at the University of Texas Health Science Center San Antonio. The K00 phase will be completed at a separate institution under the K00 mentor.

NIGMS - National Institute of General Medical Sciences

Project Summary The timely delivery of membrane-bound vesicles and tubules bearing transmembrane protein and lipid cargoes to discrete cellular membranes is fundamental to cell biology and human health. Many proteins associated with trafficking pathways are linked to serious and crippling human diseases, especially neurological diseases and disorders. Although many trafficking proteins and some pathways are well characterized, we still do not understand other trafficking pathways that we infer must exist between membranes. This constitutes an enormous gap in our understanding of fundamental cell biology. Our goal is to elucidate the molecular structures and functions of important coat protein complexes that initiate trafficking pathways by forming coats around vesicles or tubules. Coat proteins recognize and package relevant cargoes, and they promote efficient assembly of additional required protein components, like accessory proteins and SNAREs. Clathrin coats have long served as an important paradigm, but increasing evidence demonstrates how other coats use distinct mechanisms. We investigate the retromer and Assembly Polypeptide (AP) family of coat complexes (COPI, AP-4, AP-5) by using a variety of tools to ascertain molecular mechanisms of coat assembly and regulation. Biochemical approaches allow us to identify and test new interactions in coat complexes, including how accessory and regulatory proteins drive function. Integrated structural methods including X-ray crystallography, cryo-electron microscopy (cryo-EM), and cryo-electron tomography (cryo-ET) provide detailed evidence for how coats interact with key partners and allow us to generate specific hypotheses to test function. Biophysical techniques enable us to quantify binding affinities and to probe interfaces identified in structural models. With collaborators, we use molecular data to design experiments in cultured cell lines or in model organisms to explore how protein-protein interactions drive phenotypes at the cellular or organismal levels. Ultimately, we hope to gain a molecular understanding of how coats assemble at distinct membranes to drive different trafficking pathways. We anticipate this work will reveal new mechanisms of coat assembly and regulation and will provide fundamental insights into the protein networks that underlie key cellular events on membranes.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY/ABSTRACT This proposal requests funds for acquisition of an AKTA pure 25 fast protein liquid chromatography (FPLC) system for biomolecule purification at Eastern Washington University (EWU), a primarily undergraduate institute. This instrument will greatly enhance our research and education capacities. At EWU we strive to provide students with hands-on biomedical research experiences through our classes and research labs. The AKTA pure will provide a much needed high quality protein purification system for our biomedical research community. Currently our protein purification projects mainly use gravity flow purification methods with poor yield, and purification quality. With the AKTA pure, we will be able to make use of advanced purification methods, and perform chromatography based biochemical assays, considerably expanding our experimentation capabilities. The instrument will enable us to offer a new class focused on protein purification using the FPLC. We will also incorporate FPLC skills into several current upper-level classes. It will support a diverse range of ongoing research projects, including two NIH funded projects, and allow researchers to gather data for new grant applications. Furthermore, it will enable users to explore new avenues of research, such as small RNA purification and analytical chromatography techniques for protein characterization that we are currently not able to perform. The classes and research projects supported by the AKTA pure will provide a large number of EWU students the opportunity to gain training in a state of the art instrument widely used in biomedical research. Students will gain valuable research experience, and proficiency in using the FPLC will give them a career-ready skill for the biomedical field. The AKTA pure FPLC system will be an invaluable tool for advancing research and education at EWU.

NIGMS - National Institute of General Medical Sciences

Project Summary Project Title: Integrated Mixing and Inkjet Deposition for Time Resolved cryo-EM Sample Preparation Company Name: Hummingbird Precision Machine Co., dba Hummingbird Scientific Principal Investigator: Norman Salmon Summary: This project aims to develop, for the first time, an integrated mixing, inkjet deposition, and gas- assisted rapid freezing system for time-resolved cryogenic electron microscopy (cryo-EM) sample preparation, enabling the capture of short-lived biomolecular states essential for understanding biomolecular dynamics. Cryo-EM provides atomic-resolution structures of biomolecules in their native hydrated states, but current sample preparation methods take several seconds from sample application to freezing—too slow to capture short-lived intermediates that exist only on the millisecond timescales. Accessing these intermediates is essential for elucidating disease mechanisms, advancing therapeutics, and designing more efficient enzymes. To address this gap, this new system will combine microfluidic mixing with our patent pending methods for precision inkjet dispensing and rapid cold-gas assisted freezing into a compact sample preparation platform compatible with standard cryo-EM workflows. The system features an inkjet printhead with an integrated micromixer for on-demand mixing, followed by programmable picolitre droplet deposition in a defined pattern with millisecond-scale temporal control. The deposited samples are rapidly vitrified using a cold gas flow, eliminating the variability and inconsistencies of conventional blotting and plunge freezing methods that utilize liquid cryogens. This approach enables preparation of cryo-EM grids at precisely defined reaction time points, allowing structural characterization of dynamic molecular transitions and direct visualization of short-lived intermediates that are otherwise inaccessible with current methods. The project’s long-term goal is to transform structural biology by providing researchers with a tool to capture biomolecules in motion on the millisecond timescale. To achieve this, the specific aims include optimizing micromixer design and integration, refining piezoelectric dispensing electronics and control software, integrating sample freezing hardware, and validating system performance through cryo-EM imaging of test samples. By enabling routine capture of short-lived intermediates, this technology will support NIH’s mission to advance biomedical research and therapeutic development, with broad applications in drug discovery, disease mechanism elucidation, and enzyme engineering.

NIGMS - National Institute of General Medical Sciences

Project Summary Selective autophagy pathways use cargo receptors to degrade organelles, organelle subdomains, and misfolded proteins that fail to be degraded by the proteosome. Endoplasmic reticulum (ER) autophagy (also called ER-phagy) is a selective autophagy pathway that acts in ER quality control. ER-phagy cargo receptors connect an ER domain to the autophagosome biogenesis machinery via their ability to bind Atg8 family members (LC3 or GABARAP in mammals). The studies in this proposal are aimed at addressing two important unanswered questions in the field. First, we will ask how the conserved yeast ER-phagy cargo receptor, Atg40, fragments domains of the ER that it targets for degradation. Second, we will ask how Atg40 loads ER domains into autophagosomes, sealed double-membrane structures that are delivered to vacuoles (yeast) or lysosomes (mammals) for degradation. We have found that a non-canonical form of the yeast COPII coat subcomplex, Sec23-Lst1, works with Atg40 to package ER domains into autophagosomes during ER-phagy. The COPII coat is a multi-subunit coat complex that is known for its role in sorting ER proteins into transport carriers that traffic on the secretory pathway. We identified a novel role for Sec23-Lst1 in ER-phagy that is independent of its role in secretion. While the reticulon homology domain of Atg40 has been implicated in ER fission, it is unclear if Atg40 requires Sec23-Lst1 to fragment ER domains. An in vitro approach is needed to unambiguously answer this question. We have found that a variation of the COPII coat in vitro vesicle budding assay can be used to assess the requirements for Atg40-mediated ER fission. The COPII coat is formed by the sequential interactions of the Sar1 GTPase and cytoplasmic coat subcomplexes. We will use this in vitro assay to address if Atg40, Sec23- Lst1 and Atg8 are all needed for fission. Additionally, we will determine if purified Sar1, and other purified cytoplasmic COPII subcomplexes are also required. A long-term goal of these studies is to develop a similar in vitro ER fragmentation assay with mammalian COPII coat subcomplexes. To address how Atg40 sequesters membrane domains into autophagosomes, we will take advantage of an unusual phenotype we observed in lipid droplet (LD) deficient cells. Lipid droplets are ER-derived organelles that contain a neutral lipid core, triacylglycerides (TAG) and sterol esters (SE), surrounded by a phospholipid monolayer. When yeast cells are devoid of LD, resident ER membrane proteins fail to be delivered to the vacuole via ER-phagy. This defect appears to be due to the inability of the cargo receptor, Atg40, to sequester ER domains into autophagosomes. We will perform biochemical, genetic and localization studies to ask how LD are needed to couple Atg40 to its cargo. A long-term goal of these studies will be to address the role of LD in mammalian ER-phagy. These studies will be relevant to variety of metabolic disorders in humans, including diabetes and obesity.

NIGMS - National Institute of General Medical Sciences

Abstract The Department of Anesthesiology and Perioperative Medicine (DAPM) at UCLA has a long tradition of academic productivity and excellence as reflected by our consistent ranking among top 10 Anesthesiology departments in the country. The department has trained many leaders in the fields of Anesthesiology, Critical Care and Pain Medicine and has a long history of excellence in both basic and clinical research. The main goal of our T32 training program is to provide training and mentoring to anesthesiology residents/fellows/junior faculty early in their careers in basic, translational, and preclinical research in the department. Our T32 research training program has four main research themes: 1) Perioperative Organ Protection, 2) Cardiovascular, 3) Neurosciences and Brain Health, and 4) Biocomputing/Bioengineering and Health Informatics. The department has strong leaders in each of these fields. We have recruited 27 exceptional faculty mentors (13 PhD scientists and 14 physician-scientists) from the Department of Anesthesiology and other departments across UCLA, including the Departments of Medicine, Bioengineering, Physiology, Surgery, Pathology, Molecular, Cell, and Developmental Biology, Psychiatry and Biobehavioral Sciences, Computer Science, and Human Genetics. Our faculty have expertise in a wide range of research areas broadly related to the anesthesiology specialty. Eleven of these faculty are from the Department of Anesthesiology. We have also recruited eight junior anesthesiologist scientists from our department as “Up and Coming Faculty”. All of these junior faculty are on track to becoming independent physician-scientists and are dedicated to training the next generation anesthesiologist-scientists. We request two trainee slots for the first year and three slots thereafter. We will focus our recruitment efforts towards outstanding MD and MD/PhD candidates from the pool of our residents (mainly from Research Scholars Track, which is a five-year program that includes almost two years of protected research time junior faculty, or Research Pathway that includes up to 11 months of research during residency) as well as our fellows and junior faculty. We will require a minimum two-year commitment from our T32 trainees but will extend the training to three years if the additional year is beneficial to the individual upon approval from the T32 Executive Committee. Our T32 training program is specifically designed to train the next generation of academic anesthesiologists to become independent physician- scientists in the field of Anesthesiology and Perioperative Medicine.

NIGMS - National Institute of General Medical Sciences

Project Summary The University of Nebraska Medical Center (UNMC) requests funds to purchase a Rigaku HyPix-Arc 100° Curved Hybrid Photon Counting (HPC) Detector to provide state-of-the-art crystallographic X-ray diffraction data collection for the three-dimensional solution of medically important biological macromolecules and their complexes. This instrument for macromolecular crystallography is not currently available at UNMC, any nearby university or in the region. The HyPix-Arc 100° is a unique, curved detector using HPC technology. To provide the best data quality it is essential to measure accurate data with minimal correction. With a curved detector arrangement, diffracted beams arrive at the detector as close to perpendicular to the surface as possible. This prevents unwanted reflection enlargement and minimizes the associated corrections. Additionally, the curvature allows the detector to see a higher theta range than larger flat detectors. This means more reflections collected at the same time under the same conditions for truer measurement with better scaling and data quality whilst also increasing measurement speed for sensitive or unstable samples. This instrument will dramatically improve the quality of X-ray diffraction data we collect to solve crystal structures for basic science and drug development projects. The proposed project is viable because of strong investigator support illustrated by the 15 NIH- funded researchers and their projects on nucleosome assembly, ribonucleoproteins, enzyme catalysis, redox biology, cancer therapeutics, neurodegenerative and infectious disease. Participating investigators are from several departments at UNMC, as well as nearby institutions, such as the University of Nebraska-Lincoln, and Creighton University. Each project will benefit directly from the use of the HyPix-Arc 100° detector: enabling sophisticated experiments and expediting progress on NIH-funded science. These projects enjoy strong technical support in the Eppley Structural Biology Facility (ESBF) that includes well-experienced staff that have worked together with the director for over 23 years promoting all aspects of structural biology research in Nebraska and surrounding states. The proposal also has strong institutional support as evidenced by salary and service agreement support by the Vice Chancellor for Research (Dr. Bayles) and the Director of the Eppley Institute for Research in Cancer (Dr. Sweasy). The detector will be housed in the ESBF and installed on the right port of our Rigaku FRE+ ultrahigh-intensity rotating anode X-ray generator. The ESBF is the only structural biology facility in the region and gives easy access for data collection to all interested researchers. The combination of strong user interest, significant research projects, technical expertise, administrative experience, and a solid long-term plan will ensure successful implementation and extensive use of the HyPix-Arc 100° detector.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY/ABSTRACT The University of Puerto Rico at Aguadilla (UPR-Aguadilla) requests funding to acquire a Nikon AX Galvo Confocal Laser Scanning Microscope (LSCM) with a TI2-E inverted platform and a four- laser configuration (405/488/561/640 nm) to establish transformative imaging capabilities at our resource-limited institution serving 96% Pell Grant recipients. This state-of-the-art instrument addresses a critical infrastructure gap, enabling high-resolution fluorescence imaging, live-cell microscopy, and quantitative analysis essential for competitive biomedical research and undergraduate education. The LSCM will directly support four active research projects spanning parasitology (monogenean host-specificity studies), plant pathology (coffee biocontrol development), environmental chemistry (metalloprotein biomarkers), and neuroscience (astrocyte dysfunction in diabetic epilepsy) while integrating into core laboratory courses including Immunology (BIOL 4009) and Undergraduate research courses (BIOL 3108 and QUIM 4999). Our multidisciplinary faculty, in partnership with the Neuroimaging and Electrophysiology Facility (NIEF) Excellence Imaging Center, offers expertise in confocal microscopy, encompassing advanced imaging and specialized sample preparation techniques. This collaboration ensures effective implementation of the technology, sustained technical support, and high-quality training programs that will enhance research productivity and broaden educational impact. The broad, long-term objective is to transform UPR-Aguadilla from a primarily teaching institution into a research-active campus capable of producing graduate-school-ready students equipped with cutting-edge technical skills. Access to advanced confocal microscopy will stimulate new research collaborations, enhance faculty productivity, and provide 30-40 students annually with hands-on experience in modern imaging technologies currently absent from our curriculum. The instrument will strengthen our partnership with the emerging Natural History Museum of Puerto Rico for specimen digitization and support comprehensive outreach programs targeting 25-50 high school students annually through "Seeing Science Up Close" workshops. Expected outcomes include 1- 2 peer-reviewed publications within three years, establishment of 1-2 new institutional collaborations, and measurable enhancement of biomedical research capacity. This investment will significantly advance STEM education and research opportunities at UPR-Aguadilla while expanding access to cutting-edge scientific instrumentation for students pursuing biomedical careers and contributing to the development of skilled researchers in the biomedical sciences.

NIGMS - National Institute of General Medical Sciences

Project Summary/Abstract Development of ClusterBuild to Synthesize Biosynthetic Gene Clusters Opportunity Number: PA-24-245 PIs: Christopher L. Warren and Mary S. Ozers The ability to synthesize large biosynthetic gene clusters (BGCs) is essential for unlocking the therapeutic potential of natural products, including next-generation antibiotics. Many promising BGCs are derived from unculturable microbes, making direct access to their DNA challenging. Existing methods for assembling large constructs, such as PCR assembly, Golden Gate cloning, and solid-phase synthesis, are prohibitively expensive, labor-intensive, and slow when scaled beyond 10 kb, presenting a critical bottleneck in natural product discovery. These limitations hinder broader participation in synthetic biology and delay translation from genomic data to biomedical applications. This project proposes ClusterBuild, a novel platform that enables cost-effective, high-fidelity synthesis of large DNA constructs (up to 100 kb) by combining DNA microarrays with peptide nucleic acid (PNA)-based error correction and isothermal assembly. PNAs can be used to correct DNA synthesis errors due to their high binding affinity and specificity for complementary DNA sequences, enabling them to selectively hybridize to mismatched regions and facilitate targeted removal of erroneous DNA strands. ClusterBuild leverages high-density photolithographic microarrays to generate massive oligo libraries, while complementary PNA arrays are used to selectively purify error-free sequences. Aim 1 will demonstrate that PNA microarrays can purify high-fidelity oligonucleotides generated from a commercial DNA microarray using sequence-specific melting temperature (Tm) elution. Aim 2 will verify that purified DNA oligos can be efficiently assembled into a 12 kb biosynthetic gene cluster using barcoded, overlapping oligonucleotides and multi-stage isothermal assembly. Aim 3 will confirm functional expression of the assembled cluster in E. coli to produce a class II lanthipeptide, a promising compound with low micromolar activity against the pathogen Klebsiella pneumoniae. In Phase II, the full end-to-end workflow will be implemented using a digital micromirror device (DMD)-based microarray synthesizer to scale synthesis up to 100 megabases. The ClusterBuild technology will offer three key advantages over commercially available options by achieving more than 100-fold cost savings, a 50-fold increase in construct length, and fewer errors of one per 10 kb length. This proposal supports the "Build" step in the Design-Build-Test-Learn cycle, unlocking broader participation in synthetic biology and natural product drug discovery.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY/ABSTRACT This project requests funds to purchase a state-of-the-art electron paramagnetic resonance (EPR) spectrometer. EPR spectroscopy is an important technique that provides detailed insight into the structure of function of biomedically relevant proteins. The resulting data are essential for understanding the molecular basis of life and of disease and for providing the knowledge base necessary for developing new drugs. This instrument will advance the research of several NIH-funded projects that investigate metalloenzymes and their mechanism of action and study conformational flexibility of proteins and their functional relevance.

NIGMS - National Institute of General Medical Sciences

Project Summary Monod Bio’s Fast-Track proposal aims to advance the NovoLISA™ technology, transforming biosensing methods in Life Sciences research. Traditional biosensing approaches, such as ELISAs, face significant limitations, including prolonged time-to-results (90–270 minutes), complex procedures, extensive antibody development times, and the risk of cross-reactivity due to insufficient antibody validation. In contrast, Monod Bio’s NovoLISA assay offers a rapid (<15 minutes), single-step, no-wash solution that utilizes computationally designed proteins (NovoBodies) as reagents. NovoBodies serve as effective alternatives to traditional antibodies, developed in just 4-5 weeks, and are used in assays powered by Monod Bio’s proprietary split luciferase system, LuxSit Pro™. This system enables quick, accurate biomarker detection via luminescence using standard plate readers, which are widely available in research labs. Monod Bio has already developed a sensitive NovoLISA assay for C-reactive protein (CRP) and is working on additional assays for key biomarkers, including IL-6, TNF-α, and PSA. Phase I of this project focuses on enhancing the sensitivity and adaptability of these assays for a broader range of laboratory environments, with specific goals to improve performance metrics such as limit of detection (LOD), time-to-result, and specificity. Phase II will expand the scope of research by developing assays for three new biomarkers—IL- 2, TSH, and VEGF—and optimizing them for use in complex biological matrices. Additionally, the project will include a pilot manufacturing study for quality control and will engage external laboratories for testing to ensure robustness and reproducibility across diverse research settings. At the conclusion of this proposal, Monod Bio will have developed up to six commercially ready assay kits that will significantly reduce assay times for researchers. Monod Bio will continue to expand the NovoLISA platform catalog of biomarker quantification kits. The goal of this Fast-Track proposal is to provide the scientific community—particularly immunology, oncology, and infectious disease research—with powerful, rapid, and sensitive tools for biomarker detection. Our NovoLISA technology has the potential to replace traditional biosensing methods like ELISA in research-use-only (RUO) settings, offering unparalleled efficiency, scalability, and precision. While this proposal focuses on (RUO) applications, we envision the NovoBody and NovoLISA platforms ultimately expanding into the In Vitro Diagnostics (IVD) space. As a testament of the anticipated impact, Monod Bio was recently awarded a $1.5 million grant from the Gates Foundation to develop next-generation tuberculosis diagnostics using the NovoBody platform, as well as a $750,000 grant to develop preeclampsia diagnostics leveraging the NovoLISA platform. This Fast-Track SBIR grant will enable us to demonstrate the impact of our NovoLISA platform in the RUO space, supporting a wide range of basic and clinical research efforts across academia and industry.

NIGMS - National Institute of General Medical Sciences

Project Summary / Abstract The University of Chicago Integrated Light Microscopy Facility (ILMF) requests funds to purchase a high-end laser scanning confocal microscope. The ILMF currently serves 420 users in 80 labs from across the University. Sixty-seven of those labs use laser scanning confocal microscopy, and 78% of those labs have NIH funding. Usage hours have increased as the ILMF’s microscope capacity has decreased. Two of our confocal microscopes, both Leica SP5 models, are over 14 years old. Leica has designated them end-of-life, meaning they are no longer manufacturing parts for these systems and replacements are not guaranteed. We have already experienced failure of the 488nm Argon and 592nm depletion lasers on one, and failure of the Mai Tai multiphoton excitation laser on the other, with no possibility of replacing any of these components. We expect to decommission at least one SP5 within the next year, making users hesitant to start new projects on those systems. This has stressed our two newer laser scanning confocal systems (purchased with institutional funds in 2016 and 2020), pushing them to use levels averaging 91% of AUT, defined as 3640 hours per year. The system proposed here is the Evident (formerly Olympus) Fluoview 4000 (FV4000), released in 2024. The system will increase the capacity and functionality of laser scanning confocal microscopes in the ILMF, allowing users to collect high-quality data more readily. Several features of the FV4000 will be new to the ILMF, and satisfy a number of outstanding investigator needs. Features include: state-of-the-art, patented, fast signal processing silicon photomultiplier (SiPM, Evident SilVIRTM) detectors, to significantly improve signal-to-noise levels, enhancing detection of Golgi cisternae and other organelle sub-structures; four high magnification, long working distance silicone immersion objectives for detailed, multi-color, 3-dimentional imaging of organoids, thick tissues and tumor samples; and three near-infrared wavelength lasers for excitation of fluorophores beyond the current imaging spectrum, allowing for investigation of a larger number of molecules of interest in a single sample. The FV4000 will also feature full environmental control, allowing users to take advantage of faster imaging speeds to image live samples. This will make it possible to image longer sessions at higher frame rates with less photodamage, resulting in more robust and reliable data from live samples than currently possible. Finally, the FV4000 base is modular in design, allowing for field upgrades with Evident or third-party resources (e.g. a single molecule localization module) as users’ experimental needs grow. In summary, adding an Evident FV4000 laser scanning confocal microscope to the ILMF will make it possible for users to gather information from samples that are currently challenging but valuable research models.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY This application seeks funds to purchase a MINFLUX 3D Microscope. This instrument would support nine NIH-funded users in five departments and four colleges within the Texas A&M University (TAMU) community and a group of four NIH-funded investigators at UT Southwestern Medical Center. The MINFLUX microscope would be the second major instrument in a recently established shared user facility, the Joint Microscopy Laboratory (JML), which is focused on single molecule fluorescence applications. The JML includes significant wet-lab and tissue culture space to encourage use by more distant laboratories both on campus and external to the university. MINFLUX is a relatively new state-of-the-art pointillistic imaging and particle tracking strategy that is extremely thrifty with the use of photons, requiring ~10-fold less photons than the common PALM/STORM- type pointillistic super-resolution approaches. Consequently, MINFLUX can achieve precision levels of a few nanometers on a sub-millisecond timescale within functionally active cellular systems and long single molecule trajectories in three-dimensions (3D) can be obtained using single fluorophore tags. The requested MINFLUX 3D system will enable numerous multi-color strategies combining both static imaging and molecular tracking approaches. Users will examine well-controlled in vitro systems as well as stabilized and complex cellular systems (fixed and permeabilized cells), many with an eye towards live cell investigations. The Major Users will examine fundamental and diverse cell biological and mechanistic biochemistry questions focused on nucleocytoplasmic transport, condensates, bacterial pili, nuclear mechanical stress and synapses. The Minor Users projects include structure, function and biophysical studies of mitochondrial RNA editing and kinase signaling, chemotaxis, endosomal escape, endocytic recycling, antibiotic biosynthesis, phage infection, and additional projects on condensates and synapses. The full-time technician needed to run the MINFLUX microscope will be supported by a combination of user fees and ongoing contributions from departments, colleges, Texas A&M Health, and the Vice President for Research, emphasizing the widespread importance of the new microscope capabilities to advance the capabilities and growth of current research programs. The instrument will be housed in the College of Medicine by the Department of Cell Biology and Genetics, which has donated substantial equipment and space for the nascent JML microscope facility. Altogether, the identified users have planned new research directions that are expected to ultimately require > 90% of the total accessible user time, indicating the substantial demand for both existing and newly developing projects. In total, the requested MINFLUX 3D microscope will provide substantial and fundamental infrastructural support for a wide range of projects important for understanding and improving human health.

NIGMS - National Institute of General Medical Sciences

Title: A Fully Integrated Platform for Reproducible and Scalable Single-Cell Sequencing in Clinical Research Abstract: Single-cell sequencing (SC-seq) is transforming biomedical research by enabling high-resolution profiling of cellular heterogeneity, immune responses, and disease mechanisms. However, current SC-seq platforms suffer from major workflow limitations that restrict their use in clinical and translational settings. Chief among these are poor reproducibility due to variable sample preparation, limited cell retention that constrains throughput, and complex, labor-intensive workflows that are difficult to standardize across operators and sites. These challenges are particularly problematic for applications requiring detection of rare cell populations, such as immunotherapy development, precision diagnostics, and minimal residual disease detection. Sansimeon is developing a turnkey platform that integrates high-efficiency, unbiased sample preparation with scalable, deterministic cell encapsulation to support high-throughput single-cell sequencing from limited clinical specimens. Our approach unifies red blood cell and platelet removal, cell enrichment, multiplexed tagging, and CITE-seq labeling into a streamlined, one-step workflow—eliminating the need for centrifugation and minimizing hands-on time. The platform also incorporates a high-speed deterministic encapsulation system capable of processing up to 100 million cells per run with significantly improved cell-bead pairing efficiency. In Phase I, we will develop and validate a unified one-step incubation protocol for SC-seq multiplexing, CITE-seq, and cell enrichment in peripheral whole blood. In Phase II, we will build an automated benchtop instrument and an injection-molded microfluidic cartridge to enable standardized, reproducible sample preparation. We will validate the integrated system across multiple donors, operators, and timepoints, using commercial and in-house SC-seq platforms. Successful completion of this project will deliver a clinically relevant, scalable single-cell sequencing solution that improves reproducibility and expands access to single-cell technologies across immunotherapy, precision medicine, and drug discovery applications.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY/ABSTRACT Clemson University’s rapid growth in externally funded research has propelled it to R1 status, reflecting its expanding impact in advanced scientific inquiry. Five of Clemson’s nine academic colleges are STEM-fo- cused, encompassing over 30 academic units. In the last decade, Clemson’s NIH-funded portfolio has grown from $5.5 million in 2014 to $26 million in 2024, currently spanning 85 awards across 57 investigators. This growth is fueled by the university’s strategic investments in research infrastructure, equipment, and facilities. These actions foster a resource-rich environment capable of attracting and retaining top research talent. Three major NIH Centers of Biomedical Research Excellence (COBRE) underscore Clemson’s priorities: the Eukaryotic Pathogens Innovation Center (EPIC), the South Carolina COBRE for Translational Research Improving Muscu- loskeletal Health (SC-TRIMH), and the Clemson University Center for Human Genetics. To further strengthen Clemson’s research capabilities, we seek to acquire a Refeyn mass photometer, a transformative technology that enables single-particle mass measurements. Unlike bulk methods such as dynamic light scattering (DLS), mass photometry quantifies molecular mass, providing an unparalleled view of heterogeneity, oligomerization states, and binding interactions at the single-molecule level. This technology is valuable across disciplines and research foci on campus, from probing protein-ligand and protein-protein inter- actions that occur in eukaryotic pathogens (EPIC) to evaluating biomolecular assembly and integrity in mus- culoskeletal research (SC-TRIMH). Investigators in human genetics can rapidly assess protein-DNA/RNA interactions and more complex heterogenous multi protein-protein-DNA/RNA complexes. The Refeyn mass photometer offers remarkable advantages: (1) Minimal sample requirement—only a few microliters of material are needed; (2) Low per-sample cost, approximately $2, making it accessible to both established laboratories and student trainees; (3) Ease of operation, a simple pipetting step onto a glass slide significantly reduces technical barriers; and (4) Broad applicability, it excels in characterizing oligomeric states, monitoring antibody binding, and confirming molecular compositions in diverse sample types. By installing this mass photometer at Clemson, we will foster interdisciplinary collaboration, enrich hands- on learning opportunities, and expand the capabilities of ongoing NIH-funded projects. Moreover, this cutting- edge instrument will position Clemson researchers at the forefront of next-generation single-particle analytics, supporting a range of studies—from early discovery to late-stage translational research. Although it also inte- grates seamlessly into cryo-EM workflows, the mass photometer’s utility extends well beyond structural biology. Ultimately, this S10 instrumentation request will allow Clemson to elevate its research enterprise, amplify productivity, accelerate innovation, and strengthen the university as a leader in biomedical and research.

NIGMS - National Institute of General Medical Sciences

Project Summary The broad, long-term objectives of this application are to characterize mechanisms that allow a competitive germline stem cell (GSC) and its descendants to dominate the GSC population and cause super-Mendelian inheritance. The proposal will determine how a GSC in the Drosophila testis remodels its niche and causes the selective loss of WT neighbor GSCs. To accomplish this, the proposal will utilize immunofluorescence, genetics, RNA interference, extended ex vivo live-cell imaging, transcriptomics, chromatin labelling, and innovative assays of GSC competition and allele inheritance in F1 offspring. We will capitalize upon the powerful genetics available in Drosophila, as well as the ability to unequivocally identify the niche, GSCs, differentiating germline cells, and somatic stem cells (CySCs) and their lineage in the Drosophila testes. This proposal is supported by our published results demonstrating that (1) loss of the transcription factor Chinmo in a GSC causes the ectopic secretion of the extracellular matrix (ECM) protein Perlecan (Pcan), (2) this Pcan accumulates around the endogenous niche resulting in an ectopic ECM termed the moat within the testis lumen; (3) the moat causes the selective loss of WT neighbor GSCs, which no longer have strong adhesion with niche cells; (4) chinmo-/- GSCs remain in the resculpted niche because they upregulate ECM-binding proteins. This proposal is also supported by our unpublished results showing that Chinmo protein expression is promoted by an RNA-binding protein (RBP) in GSCs and that a ZAD-ZNF protein likely acts as a Chinmo co-factor in GSCs. In the first goal, we will determine whether clonal loss of the RBP that promotes Chinmo expression imparts that GSC with a competitive advantage. We will also determine what regulates that RBP in GSCs and test whether loss of any regulators of the RBP imparts a competitive advantage to a mutant GSC. In the second goal, we will determine whether Chinmo and the ZAD-ZNF protein work together to repress Pcan by recruiting histone methyltransferases. We will also determine how niche cells promote the ectopic Pcan produced by chinmo-/- GSCs. In the third goal, we will test the role of somatic stem cells (CySCs) in GSC competition and assess whether they push out WT neighbors GSCs. We will also use live-cell imaging to determine the types of GSC division that occur in chinmo- /- GSCs. The studies in this proposal will increase the knowledge base about GSC competition and will foster new avenues of research into mechanisms and possible treatments for human paternal age effect disorders caused by competitive spermatogonial stem cells and for tumor cells which remodel their microenvironment to benefit themselves and disadvantage WT neighboring cells.

NIGMS - National Institute of General Medical Sciences

This proposal requests funding for a Liquid Chromatography-Mass Spectrometry (LCMS) system to support biomedical research and educational initiatives across East Tennessee State University's Academic Health Science Center. The instrument will primarily serve researchers and students in the Colleges of Medicine and Pharmacy, while remaining accessible to investigators from the Colleges of Public Health and Arts and Sciences. The acquisition of this LCMS system addresses a critical infrastructure gap that emerged in 2022 when the university's previous LCMS instrument became non-functional. From 2009-2022, LCMS capabilities at ETSU facilitated significant research productivity across multiple investigative teams, resulting in over 40 peer-reviewed publications. This research spans an array of biomedical applications including: pharmacokinetic studies of therapeutic drugs and substances of abuse; development of novel drug delivery systems; quantification of endogenous biomarkers in disease states; stability studies of compounded pharmaceuticals; analysis of environmental contaminants; and investigation of lipid mediators in cardiovascular disease. This productivity has been severely hampered by the lack of this essential analytical capability since 2022. Beyond supporting faculty research programs, this instrument will provide exceptional educational opportunities for a broad spectrum of students, including PharmD, MD, PhD, MS, and undergraduate trainees. Hands-on training with sophisticated LCMS technology will equip these learners with specialized analytical skills highly valued in both academic and industrial research settings. This training represents an uncommon opportunity, particularly for undergraduate science students, enhancing their competitiveness for advanced educational programs and future employment. The strategic placement of this instrument within our shared research infrastructure will maximize its impact, supporting ongoing NIH-funded investigations in areas including pharmacokinetics, drug metabolism, natural product chemistry, biomarker discovery, and neonatal abstinence syndrome research. Additionally, the instrument will enable new collaborative research directions that align with institutional priorities in addiction science, infectious disease, and rural health disparities. In summary, this LCMS system will rejuvenate research capabilities that previously flourished at ETSU, while simultaneously enriching the educational experience of our student population in the biomedical sciences.

NIGMS - National Institute of General Medical Sciences

Project Summary/Abstract The Center for Structural Biology, located in the Life Sciences Institute, is a comprehensive structural biology resource for researchers at University of Michigan (U-M) whose mission is to provide access to state-of-the-art facilities as well as expertise and training on all aspects of protein production and characterization through structure determination and analysis. We are requesting funds to purchase an Octet-RH16 system employing bio-layer interferometry (BLI) technology to calculate the rate of association (ka), rate of dissociation (kd) and affinity constants (KD) for biomolecular interactions in real-time. To run a successful drug development program, of which U-M is a leader having contributed to the most FDA approved drugs of any University in the US (Patridge et al., Drug Discovery Today 20, 1182-1187, 2015), access to technology that measures the kinetics of biomolecular interactions of drug-like molecules and biologics to their targets to prove target engagement and rank order the compounds/biologics is essential to the optimization of leads for clinical trials. This system will replace our current failing Octet-Red system, which is a high-demand instrument and whose end of service life was July 2019. Currently there are no other BLI instruments on campus available to our center users. Our current users are developing therapeutics to treat or prevent a broad range of diseases. They are developing new vaccines, new antibiotics, new therapies to combat neurodevelopmental disorders such as autism, new therapeutics against blood cancers such as myeloproliferative neoplasm and acute myeloid leukemia, new therapeutics against castration-resistant prostate cancer, and systemic and ocular toxoplasmosis in vulnerable populations. They are also engineering biosynthetic pathways to develop new drugs or improve existing ones. The new Octet-RH16 will double the number of samples that we can measure in parallel (16) and increase the number of samples we can measure in a single experiment by 8-fold, thus increasing our throughput by 700%. In addition, the required sample volumes will decrease by 5-fold. The new regeneration and re-racking standard feature will be cost saving as it allows the reuse of biosensors. This increase in productivity and reliability along with the decrease in operating cost will open new avenues of research for the U-M research community. It will provide an opportunity to directly screen targets against small drug-like chemical libraries housed at the U-M Center for Chemical Genomics, for example the Prestwick Library, where 95% of the compounds are drugs approved by the FDA-, EMA-, or other agencies.

NIGMS - National Institute of General Medical Sciences

Project Summary/Abstract: We are requesting funds through the NIH Shared Instrumentation Grant (SIG) program to acquire a FUJIFILM VisualSonics Vevo F2 LT high-resolution ultrasound imaging system. This instrument will establish new, centralized ultrasound imaging capabilities, where no small-animal ultrasound system currently exists. The addition of the Vevo F2 LT system will significantly expand the research capacity of our campus, enabling innovative investigations that are not currently feasible due to lack of access to real-time, non-invasive in vivo cardiac and tumor imaging. The Vevo F2 LT will support the research of 10 investigators across multiple departments and two universities. Major users are leading research projects focused on heart disease, cancer, metabolic dysfunction, and regenerative medicine—fields that require precise, longitudinal measurement of tissue structure, function, perfusion, fibrosis, and vascular health in small animal models. Minor users and early-career investigators will also benefit through training, pilot data generation, and increased competitiveness for NIH and other extramural funding. The Vevo F2 LT ultrasound offers a unique combination of high spatial and temporal resolution that allows for contrast-enhanced imaging and cardiac function assessment in animals ranging in size from the rat to zebrafish — previously unavailable on our campus. Institutional support includes dedicated space within our vivarium’s centralized imaging core, personnel for training and operations, and a user structure to ensure efficient access to the instrument. This investment will strengthen cross-disciplinary collaborations and foster a broader scope of research for all users. The addition of the Vevo F2 LT system will address a major gap in our research infrastructure, catalyzing new lines of inquiry and accelerating progress on major public health challenges, while advancing biomedical research at our institution.

NIGMS - National Institute of General Medical Sciences

Modified Project Summary/Abstract Section This proposal requests funding for a Wyatt DAWN 8 Multi-Angle Light Scattering (MALS) system coupled with Size Exclusion Chromatography (SEC), referred to as SEC-MALS. The SEC-MALS system enables the measurements of molar mass, hydrodynamic radius, aggregation state, and sample heterogeneity of biomolecules and nanoparticles. The SEC-MALS system will support research with a biomedical focus in multiple departments, including Chemistry, Biology, Pharmacy, and the School of Medicine. In addition to supporting research, the instrument will enhance undergraduate training in biochemistry, organic, and polymer chemistry labs, aiding student training for careers in medicine and human related health fields. The long-term goal of this proposal is to build research and educational capacity at UT Tyler by enabling in-house access to advanced characterization tools. The SEC-MALS will eliminate reliance on external facilities, accelerate research, and improve hands-on student learning in STEM and biomedical fields. Plans are in place to extend access to researchers at nearby institutions, maximizing the instrument’s regional impact across East Texas.

NIGMS - National Institute of General Medical Sciences

ABSTRACT Alternative splicing of pre-mRNA enables a gene to give rise to multiple distinct mRNA transcripts, yielding protein isoforms with different, even opposing, functions. The long-term goal of our research is to understand the molecular mechanisms and function of alternative splicing on biological activities. The focus of this proposal is to investigate the regulation of alternative splicing in epithelial-mesenchymal transition (EMT). EMT is an essential developmental process that allows cells to change from a tightly packed cobble-stone-like epithelial cellular state to a motile and spindle-shaped mesenchymal cellular state. When abnormally activated, EMT promotes many types of diseases, including tissue fibrosis and cancer metastasis. Through working at the intersection of RNA splicing and cell biology, my lab has made several important findings connecting RNA regulation and EMT. Our work revealed, for the first time, that splice isoform switching of the CD44 gene causally controls EMT. By manipulating CD44 alternative splicing, we were able to convert cells between the epithelial and mesenchymal states. We have also identified the RNA-binding protein (RBP) hnRNPM as a critical splicing factor that promotes EMT through the regulation of alternative splicing. These results suggest that RNA splicing regulation could serve as an important mechanism that provides cellular plasticity. By shifting the programs of alternative splicing, cells are able to convert between the epithelial and mesenchymal cellular states. This capacity of reversing phenotypes is important for normal developmental EMT, as well as for cancer metastasis. Major new discoveries are necessary to fully understand this phenomenon and its underlying mechanisms. Our proposed research program is focused on (1) determining whether splice isoform switching acts as a prevalent mechanism that drives EMT or, if it is largely a phenomenon of byproducts; (2) understanding how RBPs precisely control alternative splicing during EMT; and (3) dissecting how signaling cascades elicit signals to RBPs and trigger alternative splicing changes during EMT. We have made major efforts in the past several years to build up experimental systems and gain expertise to help carry out our proposed studies. These efforts include large-scale RNA profiling of alternative splicing in multiple EMT systems, bioinformatics and experimental analysis of RBPs, as well as our recently completed kinase screen for EMT-associated alternative splicing alterations. Accomplishing the proposed work will provide new insights into our understanding of the regulatory mechanisms of alternative splicing, thus contributing to biological relevance in normal development and diseases.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY We request funds for a Zeiss Lightsheet 7 microscope for the Microscopy and Flow Cytometry (MFC) Facility at the University of Texas at Austin. The MFC is a centralized core facility used annually by over 500 researchers in 130 labs across 24 departments and 5 colleges. While the UT Austin campus offers several advanced microscopes, there is no shared light sheet microscope. With this microscope, exciting research approaches will be enabled for 17 investigators with 29 NIH-sponsored projects. Light sheet microscopes acquire large volumes quickly. A thin sheet of light is created by illumination objective(s) that are decoupled from the detection objective(s). A lower numerical aperture illumination lens creates the thin excitation sheet across the entire focal plane, and a higher numerical aperture objective resolves small features. Use of a camera allows fast capture with the inherent optical sectioning. Other microscopes risk sample damage from laser exposure, loss of fluorescence from photobleaching, and scattering artifacts deeper into a sample. When high resolution is needed or to image thin samples, our researchers have access to a suite of confocal microscopes, multiphoton microscopes, spinning disk, TIRF, and super resolution microscopes. But for rapid, 3D imaging, we do not have the right tool, a light sheet microscope. Developmental biology and neuroscience researchers, in particular, need light sheet microscopy. Many of our researchers use small, living organisms to investigate eye disease and injury, scoliosis and spinal development, spina bifida and other birth defects, and mitochondrial disorders. Others use mouse models to study brain disorders and will use a variety of clearing techniques to make large tissues transparent for even deeper light penetration. These users study strokes, motor control, Parkinson’s, substance abuse, and bipolar disorder. The third cohort of our group uses 3D cell constructs and organoids to model physiological tissues and study therapeutics in vitro. For all these samples, imaging is currently time-consuming, arduous, or impossible. The Zeiss Lightsheet 7 was designed for living samples, most of our imaging needs, but with large chambers and long working distance objectives, it can also image cleared organisms and tissues. To serve a world-class, multidisciplinary research institution, we chose the Zeiss because of its versatility and image quality. Our faculty have long wanted this technology, and our institution has committed funds to purchase service contracts, providing long-term sustainability. With this level of support, the need and enthusiasm of our users, and the care of the MFC, the Zeiss Lightsheet 7 will have transformative impact on current and future biomedical research at UT Austin.