Light Imaging Core Facility

National Institute Of Neurological Disorders And Stroke

Investigators

Linked publications & trials

Abstract

Over the past several years, the LIF has acquired new confocal microscopes that feature enhanced detectors and capabilities that were unavailable on older microscopes. 1. Our Zeiss LSM880 AiryScan has a unique detector that provides nearly a two-fold improvement in resolution and four-fold improvement in signal-to-noise ratio over conventional confocal detectors. The sensitivity of the detector makes it possible to collect superb images even with very low levels of laser illumination. The enhanced resolution is advantageous for studying small cellular structures such as the dendritic spines of neurons or intracellular structures and organelles. The system has a heated stage for temperature control in living samples. 2. The Zeiss LSM880 Spectral confocal microscope has a highly sensitive 32-channel detector that allows the user to define optimal windows for detection of different fluorophores. The ability to simultaneously collect and discriminate signal from multiple fluorephores speeds image acquisition, an advantage for viewing rapidly moving structures in live specimens. The microscope has a stage-top incubator for controlling temperature and CO2 concentration. The spectral detector also is useful for multiplex imaging with up to up to five different fluorophores. 3. The Zeiss LSM800 confocal microscope has two highly sensitive detectors that can be configured to capture signals from up to four different fluorophores (blue, green red and far-red), the most popular combinations of fluorophores. The unique feature of this instrument is its highly versatile stage controller, which allows efficient capture of tiled images from brain slices. 4. The Leica STED3X confocal microscope provides super-resolution imaging-approximately four-fold improvement relative to a conventional confocal microscope. The gated STED technique implemented in STED3x requires use of relatively intense laser illumination, making it challenging to apply to living specimens. In 2020, the system was upgraded with components to enable fluorescence lifetime imaging and to perform a new type of STED super-resolution imaging that can be achieved with much lower intensity laser illumination than the gated STED technique. This new technology is helpful for experiments that require capturing images at multiple focal planes and/or time points. The LIF has two Zeiss LSM510 confocal microscopes (one in the Porter Center and one in Building 49). Although these instruments lack the sensitivity and versatility of the newer systems, they are satisfactory for routine work, such as visualizing multi-color fluorescent signals in fixed samples. The Facility maintains imaging workstations with software for Volocity 3D Image Analysis, Huygens deconvolution, Zeiss LSM and Zen, Leica LAS, Metamorph and SyGlass virtual reality 3D viewer. LIF users have access to a workstation with Imaris software for 3D image analysis located in the NICHD Microscopy Core. *Training, maintenance, research One measure of the success of the LIF over the 28 years of its existence is the number and quality of the publications resulting from work accomplished. Publications resulting from work during the past year are listed below. The success of the LIF also is evident in the productive scientific careers of many of the hundreds of trainees that learned light microscopic imaging techniques while working in the LIF. Arpino, G., Somasundaram, A., Shin, W., Ge, L., Villareal, S., Chan, C. Y., Ashery, U., Shupliakov, O., Taraska, J. W., & Wu, L. G. (2022b). Clathrin-mediated endocytosis cooperates with bulk endocytosis to generate vesicles. IScience, 25(2). https://doi.org/10.1016/J.ISCI.2022.103809 Ballesteros, A., & Swartz, K. J. (2022). Regulation of membrane homeostasis by TMC1 mechanoelectrical transduction channels is essential for hearing. Science Advances, 8(31), 5550. https://doi.org/10.1126/SCIADV.ABM5550 Ge, L., Shin, W., Arpino, G., Wei, L., Chan, C. Y., Bleck, C. K. E., Zhao, W., & Wu, L. G. (2022a). Sequential compound fusion and kiss-and-run mediate exo- and endocytosis in excitable cells. Science Advances, 8(24). https://doi.org/10.1126/sciadv.abm6049 Gu, Q., Duan, K., Petralia, R. S., Wang, Y. X., & Li, Z. (2022a). BAX regulates dendritic spine development via mitochondrial fusion. Neuroscience Research, 182, 2531. https://doi.org/10.1016/J.NEURES.2022.06.002 Guo, X., Han, S., Wei, L., Arpino, G., Shin, W., Wang, X., & Wu, L.-G. (2022). Real-time visualization of exo- and endocytosis membrane dynamics with confocal and super-resolution microscopy. STAR Protocols, 3(2), 101404. https://doi.org/10.1016/J.XPRO.2022.101404 Isonaka, R., Sullivan, P., & Goldstein, D. S. (2022). Pathophysiological significance of increased -synuclein deposition in sympathetic nerves in Parkinsons disease: a post-mortem observational study. Translational Neurodegeneration, 11(1). https://doi.org/10.1186/S40035-022-00289-Y Kramer, P. F., Brill-Weil, S. G., Cummins, A. C., Zhang, R., Camacho-Hernandez, G. A., Newman, A. H., Eldridge, M. A. G., Averbeck, B. B., & Khaliq, Z. M. (2022). Synaptic-like axo-axonal transmission from striatal cholinergic interneurons onto dopaminergic fibers. Neuron, 110(18), 2949-2960.e4. https://doi.org/10.1016/J.NEURON.2022.07.011 Le Guerrou, F., Bunker, E. N., Rosencrans, W. M., Nguyen, J. T., Basar, M. A., Werner, A., Chou, T. F., Wang, C., & Youle, R. J. (2023). TNIP1 inhibits selective autophagy via bipartite interaction with LC3/GABARAP and TAX1BP1. Molecular Cell, 83(6), 927-941.e8. https://doi.org/10.1016/j.molcel.2023.02.023 Roney, J. C., Cheng, X. T., & Sheng, Z. H. (2022). Neuronal endolysosomal transport and lysosomal functionality in maintaining axonostasis. The Journal of Cell Biology, 221(3). https://doi.org/10.1083/JCB.202111077 Sarraf, S. A., Shah, H. V., Kanfer, G., Pickrell, A. M., Holtzclaw, L. A., Ward, M. E., & Youle, R. J. (2022). Loss of TAX1BP1-Directed Autophagy Results in Protein Aggregate Accumulation in the Brain. Molecular Cell, 82(7), 13831385. https://doi.org/10.1016/J.MOLCEL.2022.03.020 Shi, B., Wu, X. S., Cordero, N. P., Moreira, S. L., & Wu, L. G. (2022). Light and electron microscopic imaging of synaptic vesicle endocytosis at mouse hippocampal cultures. STAR Protocols, 3(3). https://doi.org/10.1016/J.XPRO.2022.101495 Shin, W., Zucker, B., Kundu, N., Lee, S. H., Shi, B., Chan, C. Y., Guo, X., Harrison, J. T., Turechek, J. M., Hinshaw, J. E., Kozlov, M. M., & Wu, L.-G. (2022). Molecular mechanics underlying flat-to-round membrane budding in live secretory cells. Nature Communications, 13(1), 3697. https://doi.org/10.1038/S41467-022-31286-4