Total internal reflection fluorescence microscopy (TIRFM) is a spatially limited imaging technique that is used primarily to visualize fluorescent molecules at or near the plasma membrane. Because the technique greatly minimizes out-of-focus, background fluorescence and uses a highly sensitive camera to detect low-level levels, it is possible to visualize and track the movement of fluorophores at the single-molecule level.
Over the last decade and a half, we have successfully used TIRFM toward understanding a wide range of signal transduction events that regulate immune cell biology. The facility has two TIRFM systems for live or fixed samples: a home-built rig using an Olympus IX 81 base and a Nikon N-STORM 4. Both systems can perform super-resolution STORM and PALM imaging experiments.
Olympus TIRF
Located in Fishers 5625, room 4S06E.
Features
- Computer-controlled Olympus IX 81 inverted microscope
- 60X, 100X, and 150X TIRFM lenses
- MetaMorph software operating system
- Liquid cooled 512 x 512 electron-multiplying CCD, (Evolve Delta, Photometrics)
- Four laser excitation lines (405, 488, 561, and 640 nm)
- Environmental control: heat, CO2, and humidity
- Auto-focus laser
- Automated X, Y, Z stage
Nikon N-STORM 4
Located in Fishers 5625, room 4S06E. This state-of-the-art system can perform up to three color, three dimensional, super-resolution STORM or PALM imaging.
Features
- Computer-controlled Nikon Eclipse Ti2
- 100XTIRFM lens
- Nikon Elements software operating system
- 512 x 512 electron-multiplying CCD cameras, (iXon Ultra, Andor)
- Four laser excitation lines (405, 488, 561, and 640 nm)
- Differential interference contrast (DIC)/Nomarski imaging
- LED Epi-illumination
- Interference reflection microscopy (IRM)
- Environmental control: heat, CO2, and humidity
- Auto-focus laser
- Automated X, Y, Z stage