This Shared Confocal Facility serves the Departments of Neuroscience and Psychiatry & Behavioral Sciences, as well as the faculty in the Charleston Alcohol Research and the Neurobiology of Addiction Research Centers. This facility provides users with access to a Carl Zeiss LSM 880 confocal microscopy system equipped with a fast module Airyscan super-resolution detector and three high-end workstations that run 3D image analysis and deconvolution software. For scheduling confocal access, please contact Dr. Patrick J. Mulholland.
The major features of this confocal microscope include:
• A 32-channel area Airyscan super-resolution detector where each detector functions as a single pinhole that collects all of the photons in an Airy pattern to surpass the diffraction limit. The Airyscan detector increases the resolution beyond the optical limitations of classical confocal point scanning microscopes, and allows resolution of 140 nm laterally and 400 nm axially even in thick tissue section (e.g., 150 microns).
• The system was upgraded to include the Fast Module for the Airyscan detector that improves speed by a factor of four while maintaining a 1.5x improvement in resolution.
• 2 GaAsP actively-cooled detectors that have significantly more sensitivity (i.e., quantum efficiency) than older PMTs, making them particularly useful for imaging weak signals in small structures, such as dendritic spines, due to major improvements in signal-to-noise ratio.
• Multi-laser lines (405, 458, 488, 514, 561, and 633 nm), including 405 nm, and 6 objectives (2.5x, 5x, 10x, 20x, 40x and 63x). The ELYRA 63x objective (1.4 NA) has a 190 micron working distance that is designed for Airyscan imaging of fine structures in the micron range, such as dendritic spines, presynaptic terminals, and axon initial segments.
• Versatile and user-friendly Zen software that allows frame sizes of 8192 x 8192 pixels with continuously variable hardware-zoom from x0.6 to x40 in 0.1 steps. The scan field also is freely rotatable in 0.1° steps for 360°. Zen software also allows for stitching of tiled images.
• The LSM 880 has an incubator that allows for long-term live cell imaging under stable environmental conditions, and single channel FCS is already possible and implemented with the system described here. Additionally, N&B analysis (number & brightness) is implemented, which allows detection and distinguishing of monomers, dimers and polymers.
The Shared Confocal Facility provides access to three high-end 64-bit workstations. One is fitted with an Intel Core i9-7980XE Extreme Edition (18-Core) 2.6 GHz processor, an Radeon Pro WX 9100 16GB graphics card, and 128 GB RAM running Windows 10. The second workstation is fitted with an AMD 8-core 4 GHz processor, an AMD R9 4 GB 256-bit graphics card, and 32 GB of RAM running Windows 10. The third workstation is fitted with dual Intel Xeon 4 GHz processors, an NVIDIA Quadro K2200 4GB 128-bit graphics card, and 128 GB of RAM running Windows 7. These computers are running ImarisXT 3D image analysis and AutoQuant X3 deconvolution software, and data are stored on MUSC servers. The ‘Filament’, ‘CoLoc’, 'Spots’, and ‘Surfaces’ modules in ImarisXT software in conjunction with a MATLAB runtime extension are used to identify and classify dendritic spines based on their morphological characteristics and reveal protein colocalization, microdomain expression patterns, and synapse-to-nuclear trafficking. Version 9 of ImarisXT now allows for faster rendering of large data sets (100+ GB) in their ‘Surfaces’ module. AutoQuant X3 is a deconvolution program that uses theoretical or calculated diffraction-limited point spread function to reduce blur, increase the signal-to-noise ratio, and improve resolution of fluorescent images. ImarisXT and AutoQuant X3 supports file extensions from our Carl Zeiss LSM system and a wide range of other extensions.