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Cellular Imaging using NEw Microscopy Approaches (CINEMA Lab)

Purpose

The Cinema Lab was created as a state-of-the-art light microscope imaging center. The facility provides cutting edge technologies to address complex cell biological questions that cannot be addressed with standard methodologies.

For instance, Total Internal Reflection Fluorescent Microscopy (TIRFM) allows imaging of a very thin plane (<100 nm) so events that occur near the cell surface (e.g. membrane trafficking, cytoskeleton) can be observed with exquisite clarity. With this approach detection of single molecules are possible. A 4D (3D + time) multicolor spinning disk confocal microscopy (SDCM) allows for fast live cell imaging of fast moving dim objects that traverse several focal planes.

The Cinema Lab imaging center is equipped with an automated Evos FL2 wide-field fluorescent microscope with a CO2 and humidity chamber that allows over-night imaging acquisition of tissue culture cells.

In addition to these live cell imaging instruments, the Cinema Lab imaging center can produce super-resolution fluorescent microscopy images with an 8-fold increase in resolution (lateral resolution of 100-125nm and an axial resolution of 250nm) by imaging fixed samples at the Structured Illumination microscopy (SIM).

Site and On-site Operations Capacities

The CINEMA Lab imaging center is located in SHM IE17/19/21 and is adjacent to the CCMI facility and EM facilities (IE-wing, basement of SHM). The CINEMA Lab facility is equipped for 6 advanced microscope setups. The lab can accommodate water-cooled lasers (20-70 Amps), multiple air-cooled lasers and has ceiling equipment racks and blackout curtains. The space is designed for use with research grade lasers in safety: the room is electronically card coded, has emergency cut-off switches and warning signs, curtained partitions, and water leak detection.

The lab has an adjacent tissue cell culture room with CO2 incubators, two class II biosafety hoods, refrigerator/freezer, and centrifuges. The tissue culture facility is also certified for lenti-virus production and infection. Researchers can easily prepare samples in advance without damaging cells in transit and use sterile techniques when preparing cells or working with viruses. A ‘wet-lab’ bench is provided for researchers to easily manipulate samples before or during microscopy sessions.

The facility has an analysis computer station and server available free of charge for the users to visualize and analyze their data. Users will be granted access to the Cinema Lab microscopes after two training sessions (two-hours each) by the Cinema lab manager or by a colleague from their lab that is already a Cinema lab user. When training is complete the user will gain access to the Cinema Lab and calendar web account to reserve microscopy sessions. Users will be charged $55 dollars per hour per session. The reserved session may be cancelled within 24 hours prior free of charge or can be modified the same day if the user completes work earlier than scheduled. For same-day changes the user will be charged a minimum of one hour. To schedule a training session or to discuss best use of the facility for your experiments please contact Dr. Felix E. Rivera-Molina by email at felix.rivera-molina@yale.edu.

Staff

Equipment

Evos FL2 wide-field microscope

  • LED for 405, 488, 568, 647nm
  • 10X, 20X, 40X and 60X
  • Temperature, CO2, humidity control
  • Mono and color cameras.
  • Automated multi-point imaging

Ultraview spinning disc confocal

  • Lasers 405, 440, 488, 514, 568, 647nm
  • 40X and 60X
  • Temperature, control, multi-point
  • FRAP unit
  • EMCCD camera

OMX-SIM and TIRF microscope

  • Lasers 488, 568, and 647nm
  • 60X and 60X TIRF objective
  • Three CCD cameras. Simultaneous imaging
  • FRAP in TIRF mode

Two custom-built TIRF microscopes

  • RingTIRF
    • Lasers 488, 568 and 647nm
    • Two sCMOS Zyla cameras for simultaneous imaging
    • Temperature control
    • Multi-point imaging
  • GalvoTIRF
    • Lasers 405, 488 and 568nm
    • EMCCD camera
    • Temperature control
    • FRAP

Analysis computer station and server

  • Volocity visualization and analysis
  • Fiji
  • 80Tb for data storage
  • Matlab

Tissue culture facility certified for lenti-virus production and infection