Microscopy and Imaging Shared Resource

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Olympus IX81 Total Internal Reflection Microscope

Olympus AH2Olympus IX81 Total Internal Reflection Microscope

The Total Internal Reflection Fluorescence microscope (TIRFM) utilizes an optical phenomenon called Total Internal Reflection (TIR), which occurs when light travels from a higher to a lower refractive index medium at an angle greater than the critical angle.  Beyond this angle of total reflection, the electromagnetic field of the incoming/ reflected light extends into the Z direction and is termed the evanescent field.  One property of an evanescent field is that the magnetic strength of the field decreases exponentionally with distance, and so its effects extend only a few hundred nanometers into the second medium.  The portion of the specimen within the evanescent field, usually the cell membrane, can be excited to emit fluorescence which consequently can be seen or recorded.  TIRFM allows enhanced imaging of cell-surface proteins by substantially reducing background fluorescence from inside the cell.


  • Metamorph Advanced Acquisition version 7.6


  • Plan Apo 60X OIL/ N.A. 1.45, WD 0.1 mm with correction collar

  • U Apo 150X OIL/ N.A. 1.45, WD 0.1 mm with correction collar for temperature and cover glass thickness

Other Capabilities:

  • Environmental Chamber

  • Biopteks objective heater

  • Interference reflection imaging (IRM) for visualization of cell contacts

  • Total internal reflection fluorescence (TIRF) or epifluorescence

  • Specialized dishes required (#1.5, special cleaning procedure, live imaging dishes)

  • DualView facilitating a rapid simultaneous TIRF imaging of green and red fluorescence labeled sample.

  • Sequential timelapse using TIRFM and IRM


  • Argon 457nm, 488nm, 514nm

  • Krypton 568nm, 633nm


  • Hamamatsu C9100-12; EM 512X512 back thinned CCD digital camera


  • Simultaneous imaging in two colors: CFP/ YFP or GFP/ RFP

PLEASE REMEMBER: For funding purposes, it is important to acknowledge the Microscopy & Imaging Shared Resource Facility in all publications that include data derived from the Facility. For the TIRF microscope, this should include the following statement:

"This work was supported in part by the Lombardi Comprehensive Cancer Center Microscopy and Imaging Shared Resource funded by U.S. Public Health Service Grant 2P30-CA-51008, 1S10 RR15768-01, 1 S10 RR019291-01A2, and the Carey Lackman Slease Fund."

If you have comments or suggestions, email Michael Johnson, director of MISR at Johnsom@georgetown.edu.