Tuesday, May 21, 2024
5/21/2024
Project Summary This request is for a Nikon CSU-W1 SoRa super resolution (SoRa-SR) spinning disk confocal microscope with photo-stimulation and photo-ablation capabilities. The long-term goal is to increase our research productivity by obtaining higher quality and temporally resolved images at larger scales and high content (HC). Our diverse projects, all funded by NIH, include identifying mitotic errors and genome instability, studying DNA damage and repair signaling, investigating lysosome and mitochondria trafficking, examining chromatin assembling and remodeling, understanding pain sensitization and hypersensitivity at the dendritic level, exploring cytoskeleton dynamics involved in regeneration, cell extrusions, dendritic morphology and synapses formation, and investigating reproductive tract, kidney and lung development. All these projects have high impact and relevance to understand how cancer cells function and progress. The photo-stimulation module is required for Fluorescence Recovery After Photo-Bleaching (FRAP) to study the dynamics of cytoskeleton and proteins associated with promyelocytic leukemia protein nuclear bodies. The photoablation capability is required to study linkage-specific ubiquitin conjugation at DNA damages sites, measure cellular tension by studying recoiling of actin-myosin during pulsatile contractions, create gaps within the Mullerian duct to study reproductive tract development, and to induce cell-cell contact damage to follow actin filament polymerization at forming adherens junctions between nephron progenitor cells. The SoRa-SR is required to reveal synaptonemal complex nanostructure inside chromatin, to visualize the apical and basal actin-myosin layers, which are important for understating cellular extrusion, and to understand the spatial-localization of proteins involved in dendritic arborization and synapse formation. The SoRa-SR will be also used in combination with HC-imaging to study mitotic errors that lead to chromosomal instability and it will be needed by many other projects. The need for live-imaging (short or long term) with optical sectioning combined with the ability to image larger fields of view in 3D with low photo-bleaching and low photo-toxicity is essential to all these projects. Some projects need to image large montages in 3D with HC. Other projects need to use multi-well formats to study cells under various conditions (e. g. chemotherapeutic drugs). Our current spinning disk microscope system (CSU-X1), which is 13 years old, is currently limiting our projects. The proposed system would greatly benefit our research due to the ability to image a wide range of sample thickness and the needs within our user group (11 Major & 4 Minor). It would allow us to perform FRAP, live ratiometric FRET and ABLATION, besides performing high-resolution live imaging, including thicker specimens. During a 10-day demonstration of the SoRa-SR system in our department, our user’s needs were achieved and in some cases they observed events they had not previously seen. The versatility of the proposed system will make a significant impact on our research, bringing our core up to date with new technologies and allowing us to excel in our research at MD Anderson Cancer Center.
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