Microscopy Core

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 ranging from study design and method development to quantitative image analysis and figures and manuscripts. Instrument training and user support for scanning electron microscopy (SEM) and atomic force microscopy (AFM) are also available.

Widefield Microscopy

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, Nikon DS-Ri2 color cameras, and high-NA Plan Apochromat air and oil objectives up to 100x. Key differences are described below:

  • Eclipse Ti2-E – White Light: Lumencor SOLA white LED engine for fluorescence, and polarizer/analyzer set for polarized microscopy.
  • Eclipse Ti2-E – Multiline: Nikon D-LEDI multiline LED engine that emits discrete excitation wavelengths for the four major fluorescence 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 powerful analysis algorithms. A stage-top Tokai Hit environmental control chamber is available for live cell imaging.

Upright Zeiss Axio Scope.A1 and Axio Imager.M2 microscopes are also available for widefield imaging.

Confocal Microscopy

The core houses three inverted confocal microscopes for fluorescence imaging of thick samples and fine structures with higher axial resolution by optical sectioning.

  • Nikon AX R confocal: Both galvano (up to 8K) and high-speed resonant (up to 2K) scanning modes, with resonant mode allowing for much faster and gentler image acquisition than conventional galvano mode. Four laser lines (405, 488, 561, and 640 nm) and four high-sensitivity GaAsP detectors for simultaneous four-channel imaging. Two of the detectors are tunable and suitable for spectral unmixing. Software modules include AI-based imaging tools (Denoise.ai and Autosignal.ai) and automated custom acquisition routines and analysis algorithms.
  • Zeiss LSM 800 confocal: 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.  
  • Crest Optics X-Light V3 spinning disk confocal: Gentle and fast acquisition with a high-quantum efficiency and low-noise Hamamatsu ORCA Fusion camera. The system has a Lumencor Celesta laser launch with seven lines (405, 446, 488, 518, 545, 637, and 748 nm), high-NA Plan Apochromat objectives, and the fastest spinning disk speed and largest field of view available. Software modules include Denoise.ai and automated custom acquisition routines and analysis algorithms.

Additional capabilities on both point-scanning confocal instruments include high-speed line scanning, bleaching and stimulation, and FRET. Stage-top environmental control chambers for live cell imaging are available for all confocal systems.

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 field emission (FE) scanning electron microscope (SEM) and an Emitech sputter coater are available for imaging at magnifications ranging from 30x to 800,000x. The S-4800 offers upper, lower, and backscatter detectors for scanning electron microscopy, and is capable of operating in scanning transmission electron microscopy (STEM) mode to capture TEM-like images at lower accelerating voltages than traditional TEM.

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 Asylum AFM data analysis. Image processing and analysis capabilities include 2D and 3D deconvolution, 2D and 3D tracking, AI-based processing and segmentation tools, custom automated analysis algorithms, batch processing and analysis, and Airyscan processing. Expertise and training in custom image analysis is available through core staff.