Confocal and Specialized Microscopy Shared Resource (HICCC)

The Confocal and Specialized Microscopy Shared Resource (CSMSR), managed by the Herbert Irving Comprehensive Cancer Center, provides advanced microscope systems for multidimensional optical imaging of living and fixed cells and tissues. The CSMSR offers a wide range of state-of-the-art microscopy technologies, including scanning confocal microscopy, two-photon and second-harmonic generation microscopy, spinning-disk confocal microscopy, wide-field fluorescence and color histology imaging,super-resolution microscopy, laser capture, and micro-irradiation. The full-time staff provide consultation, assistance, training, support for image processing, visualization, quantitative analysis, and technical support. The staff (directed by Dr. Liza Pon, and managed by Dr. Theresa Swayne) is trained in cell biology, biophysics, microbiology, and image analysis, with in-depth expertise in light microscopy, and can help with experimental design, image acquisition, and data interpretation.

The CSMSR is located in the Lasker Biomedical Research Building room 320. The room is equipped with blackout curtains that separate each of the 5 microscopes. The space also accommodates a computer workstation for image viewing and analysis, a bench with a sink for sample preparation, and office space for the Shared Resource staff.  The Confocal Shared Resource also has a 100 sf tissue culture room, housing a biosafety cabinet and tissue-culture incubator. A Windows desktop workstation with 32 GB of RAM is available for users to process and analyze image data. It is equipped with commercial software (Volocity and NIS-Elements) as well as commonly used free scientific software for image and data analysis (ImageJ/Fiji, CellProfiler, R, MATLAB).

Equipment

  • Nikon A1 Scanning Confocal Microscope 
  • Nikon A1R MP Spectral, Resonant-Scanning One- and Two-Photon Confocal 
  • Yokogawa CSU-X1 and Nikon N-STORM Spinning-Disk Confocal, STORM Super-resolution, and TIRF Imaging System 
  • Nikon N-SIM and A1 3D structured illumination super-resolution/scanning confocal microscope 
  • Zeiss MicroBeam IV on AxioObserver Z.1 MicroBeam IV microirradiation and laser capture imaging system 
  • Cytation5/BioStack Plate Reader/Imager
  • Cytation5/BioSpa Live-Cell Imager
  • BioTek Autoscratch wound assay apparatus  
  • EMD Millipore CellAsic Onix microfluidic platform 
     

Objective lenses

  • Two Nikon Apo-TIRF 100x/1.49, for SIM and STORM
  • Two Nikon Plan-Apo Lambda 100x/1.45, for spinning-disk and scanning confocal
  • Zeiss EC-Plan-NeoFluar 100x/1.3, for wide-field fluorescence and brightfield
  • Two Nikon Apo-TIRF 60x/1.49, for scanning confocal and spinning-disk)
  • Nikon Plan-Apo VC 60x/1.20 W, for high-resolution imaging of live cells
  • Nikon Apo LWD IR 25x/1.1 W, for long-working-distance water-immersion multiphoton imaging
     

The Confocal and Specialized Microscopy Shared Resource (CMSR) facility is comprisedof a central roomisolated from vibrational interference that issubdivided into satellite spaces that contain microscopy imaging equipment and supporting facilities, including an image analysis station. An adjoining space is designated as a specimen preparation area, and a cell culture incubator with CO2and a class II biosafety cabinet are available for users to maintain live samples before and during their microscope time, and carry out basic cell manipulations.CSMSR has two image processing workstations whichallow users to perform tasks such as 3D reconstruction and image analysis.  One new Windows desktop computer with 256 GB of RAM, and another with 32GB RAM, are equipped with commercial software (Volocity and NIS-Elements) as well as commonly used open-source scientific software for image and data analysis (ImageJ/Fiji, CellProfiler, R). The CSMSR also has a license for MATLAB. 

Major microscope systems include thescanning confocal microscope (Nikon A1 on Ti-E stand) obtained in 2012 using funds provided by the HICCC. This instrument is used for routine confocal imaging in up to 4 colors plus bright-field in fixed samples on slides, and for small-scale tile imaging (2–20 fields). The system has lasers at 405, 488, 561, and 640 nm and four PMT detectors with filters for standard blue, green, red, and far-red fluorophores. Focus stabilization (PFS) is available for tile scanning. 

The spectral, resonant-scanning one- and two-Photon confocal microscope system (Nikon A1R MP on Ti-E stand) was acquired in 2010 through an NIH Shared Instrumentation Grant headed by the CSMSR director, Dr. Pon, and upgraded to state-of-the-art using funds provided by the Herbert Irving Comprehensive Cancer Center (HICCC) in 2018. Live-cell imaging can be performed for up to 1-2 days with the stage-top incubator, gas mixer, and focus stabilization. Sensitive GaAsP detectors, added in 2016, allow imaging live cells with low laser power, and a stage-mounted piezoelectric focus drive enables fast 3D imaging. The spectral detector is used to capture and unmix overlapping fluorescent spectra. The 1024x1024 resonant scanner, upgraded in 2018, is 20x faster than the standard scanner, and is used for fast dynamic studies and large-volume imaging of cleared samples. Finally, a pulsed Chameleon Vision II laser, tunable from 680-1040 nm, together with 3 nondescanned detectors, allow multiphoton excitation and second-harmonic generation (SHG) imaging. New solid-state visible lasers for excitation at 405, 488, 561, and 640 nm, and a new computer, were obtained as part of the 2018 upgrades.

The spinning-disk confocal microscope, STORM super-resolution, and TIRF Imaging System (Yokogawa CSU-X1 and Nikon N-STORM on Ti-E stand),obtained in 2014 using institutional funds, offersthree distinct imaging technologies. First, a spinning-disk confocal head allows faster, less phototoxic imaging than scanning confocal instruments. As this method is well suited to live cells, the system is equipped with a stage-top incubator for environmental control. Fast imaging is enhanced by a stage-mounted piezo focus drive and laser triggering of the cameras. Solid-state lasers are available at 405, 488, 568, and 647 nm and a Sutter filter wheel provides emission filters for standard blue, green, red, and far-red fluorophores. Two cameras are available: for greater sensitivity, a 512 x 512 EMCCD (Photometrics Evolve); and for greater speed and spatial resolution, a 2048 x 2048 sCMOS (Andor Zyla 4.2). The second method available on this confocal microscope system is TIRF (total internal reflection fluorescence). This method drastically improves the signal:background ratio for structures at the basal plasma membrane (up to ~200 nm from the coverslip), and is well suited for studying membrane receptors and the cortical cytoskeleton. The third technique available is super-resolution localization microscopy, implemented as Stochastic Optical Reconstruction Microscopy (N-STORM) with astigmatic imaging for 3D resolution. 3D-STORM offers xy resolution < 50 nm and z resolution < 100 nm with specially prepared samples. 

The Nikon N-SIM 3D structured illumination super-resolution/scanning confocal microscope and A1 on Ti-E standwas installed in the CSMSR in 2014, using funds obtained from a Shared Instrumentation Grant headed by Dr. Pon. To increase confocal imaging capacity in the core, in 2018 this system was upgraded, using funds provided by the HICCC, with an A1 confocal scan head, and a A1 DUV-B 2-Channel GaAsP spectral detector. This upgrade allows routine confocal imaging as well as spectral unmixing. Structured illumination microscopy (SIM) achieves super-resolution by generating a finely spaced excitation pattern using a diffraction grating. By varying this known excitation pattern and reconstructing the image computationally, high-resolution details can be revealed, with an improvement of both lateral and axial resolution by about 2-fold over conventional wide-field and confocal techniques. Unlike STORM, SIM can be performed with most common fluorophores including fluorescent proteins and organic dyes. This system includes lasers for excitation at 405, 488, and 561 nm and supports both 3D-SIM (for imaging several micrometers deep into a sample) and 2D-SIM (for faster imaging in planes close to the coverslip). The SIM microscope is used by researchers seeking finer structural detail than can be achieved with confocal imaging. 

The MicroBeam IV microirradiation and laser capture imaging system (Zeiss MicroBeam IV on AxioObserver Z.1) was acquired by the CSMSR in 2009 using funds provided by the HICCC. This imaging/microirradiation/laser capture system is used for laser capture microdissection of histological sections or cell cultures. A 355 nm solid-state laser is used to dissect cells of interestand catapult material into collection tubes without contact, minimizing contamination. This UV laser can also be used to induce targeted DNA breaks in sensitized cells. A cooled monochrome CCD camera (Hamamatsu Orca ER), along with LED excitation (Colibri) and Zen software support fluorescence imaging of blue, green, red, and far-red fluorophores as well as CFP and YFP. A color camera is used for guiding LCM and for histology including tile images of large tissue sections.

The Cytation5/BioStack Plate Reader/Imager is an existing plate reader equipped with UV-Vis absorbance, fluorescence and luminescence detection and a microplate stacker. The system was obtained by CSMSR in 2019. The Cytation5/BioSpa Live-Cell Imager is a long-term live-cell imaging system.  The system consists of a temperature- and gas-controlled imaging platform for phase-contrast and wide-field fluorescence imaging (4x – 60x magnification), an incubator that can accommodate up to 8 different samples, and a robotic arm that transfers samples from the incubator to the imaging platform, enabling multiple live-cell experiments to be carried out in parallel. The CSMSR acquired the system in 2019 using funds provided by the HICCC.

The BioTek Autoscratch wound assay apparatus generates uniform scratch wounds in cell monolayers for cell migration assays in multiwell plates. A CellAsic Onix (EMD Millipore) microfluidic platform supports continuous low-volume perfusion, gradient generation, and drug treatment while imaging on any of our microscopes. In addition to standard research-grade objective lenses, several high-end objective lenses are available for microscopes in the CMSR.

Standards, Rigor, and Reproducibility
Quality control procedures recommended by the instrument manufacturer are in place to ensure proper instrument operation. To ensure rigor and reproducibility, all analytical services include standard operating protocols and are routinely validated using technical replicas.