TIRFM Microscopy Shows Cell Processes

A new family of objectives has been specifically designed for total internal reflection fluorescent microscopy (TIRFM), which selectively visualizes processes and structures of cell membranes. The objectives have magnifications between 60x and 150x, and high numerical apertures (NAs) including an NA of 1.65.

High NAs allow laser light to attain the critical angle required for total internal reflection at the cover slip-medium interface. This induces an evanescent field that excites only fluorophores at the interface. Unlike standard wide-field microscopy, light does not travel through the specimens, photo-damage is reduced, and cells are able to live longer. A higher NA enables a shallower laser light angle and reduces penetration depth of the evanescent field.

The series of TIRFM objectives was developed by Olympus (Hamburg, Germany) for live-cell surface observations. The high NA of the Apo 100xOHR reduces the penetration depth to below 100 nm, providing a very fine depth of focus for precise observations of cell surface and membrane-associated processes. This is achieved through a very high signal-to-background ratio and a z-resolution that is not even achieved by confocal microscopy. There is a total absence of any out-of-focus fluorescence, as excitation occurs only in the plane of observation directly adjacent to the cover-slip medium interface.

Other new Olympic objectives are the Plapon 60xO (NA 1.45) and the Plapo 100xO (NA 1.45), developed for conventional TIRFM applications. The Uapo 150xO (NA 1.45) objective, with its high magnification, was developed for single molecule applications. Like the Plapon 60xO objective, it also features a compensation collar for varying temperature and cover slip thickness.

The modular approach to TIRFM imaging stations that encompasses all necessary components enables Olympus to provide completely integrated systems. The series of TIRFM objectives, a range of lasers, and several multi-port illuminators enable users to tailor their systems to their own applications.




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