The Microscopy Core provides training, consultation and advanced support for optical microscopy and image analysis to investigators throughout Nationwide Children’s Hospital. Expertise and assistance are available for all steps of the process from study design to preparation of figures and manuscripts. Instrument training and user support for scanning electron microscopy (SEM), atomic force microscopy (AFM) and laser capture microdissection (LCM) are also available.
The core has two inverted Nikon Eclipse Ti2-E widefield light microscopes equipped with fully motorized major components, high-sensitivity Hamamatsu ORCA Fusion CMOS cameras, and high-NA Plan Apochromat air and oil objectives. One widefield microscope has a white light episcopic LED engine for fluorescence, and a diascopic LED lamp and color Nikon DS-Ri2 camera for brightfield and polarized microscopy. The second widefield microscope has a multiline LED engine that emits discrete excitation wavelengths for the four major channels with optional high-speed emission cleanup for up to 6 times faster multichannel acquisition. These microscopes are controlled using NIS-Elements software that includes modules for automated custom acquisition routines and extensive analysis algorithms. A stage-top environmental control chamber is available for live cell imaging.
The core houses two point-scanning confocal microscopes for fluorescence imaging of thick samples and fine structures with higher axial resolution by optical sectioning.
The Nikon AX R confocal offers both galvano and high-speed resonant scanning modes, with resonant mode allowing for much faster and gentler image acquisition than conventional galvano mode. The AX R has four laser lines (405, 488, 561, and 640 nm) and four high-sensitivity GaAsP detectors for truly simultaneous four-channel imaging. Two of the detectors are tunable and allow for spectral unmixing. The AX R Software modules include AI-based imaging tools (Denoise.ai and Autosignal.ai) and automated custom acquisition routines and analysis algorithms.
The Zeiss LSM 800 is a galvano scanner with four laser lines (405, 488, 561, and 640 nm), one high-resolution high-sensitivity Airyscan GaAsP detector, and two multialkali PMT detectors. The Airyscan module enables axial and lateral resolution beyond the diffraction limit by using a specialized detector with 32 GaAsP elements, allowing for reconstruction of a higher-resolution image using information from discrete elements.
Additional capabilities on both point-scanning confocal instruments include high-speed line scanning, bleaching and stimulation, FRET, and spectral unmixing. Stage-top environmental control chambers for live cell imaging are available for both confocal systems.
Laser Capture Microdissection
Microdissection of tissues on slides is available using a Zeiss PALM MicroBeam laser capture microscope (LCM) with a pulsed UV laser coupled to a Zeiss Axio Observer microscope. The laser is capable of micro-dissection and micro-ablation of small particles with sub-micrometer precision.
Atomic Force Microscopy
The core has an Asylum MFP-3D-BIO atomic force microscope (AFM) on an inverted Zeiss Axio Observer Z1 microscope. The microscope includes a Zeiss Apotome 2 structured illumination module to allow for better optical sectioning with image reconstruction. The MFP-3D-BIO AFM allows investigators to measure picoNewton forces and resolve the smallest features of a sample. Fast Force mapping including automated adhesion and elastic modulus analysis are available. The Asylum MFP-3D-Infinity-BIO atomic force microscope was purchased with federal funds from an NIH S10 grant awarded to Aaron J. Trask, PhD, FAHA.
Scanning Electron Microscopy
A Hitachi S-4800 high resolution scanning electron microscope (SEM) and a sputter coater are available for imaging at magnifications ranging from 30x to 800,000x.
Image Processing and Analysis
Four image analysis workstations are available to core instrument users: two with Nikon NIS-Elements software, one with Zeiss ZEN software, and one exclusively for AFM data analysis. Image processing and analysis capabilities include deconvolution, AI-based processing and segmentation tools, custom automated analysis algorithms, batch processing and analysis, and Airyscan reconstruction.