Olympus has developed a new family of objectives specifically for Total Internal Reflection Fluorescence Microscopy (TIRFM) which selectively visualises processes and structures of the cell membrane. Researchers can now choose from four different specially designed TIRFM objectives with magnifications between 60x and 150x, and extremely high numerical apertures (NA) - including a world record NA of 1.65.
Objectives with high NA are required for TIRFM. High NAs allow the laser light to attain the critical angle required for total internal reflection at the cover slip-medium interface. This induces an evanescent field which only excites fluorophores at the interface. Unlike standard widefield microscopy, the light does not travel through specimens, reducing photo-damage and enabling cells to live longer. A higher NA enables a shallower laser light angle and reduces penetration depth of the evanescent field.
The APO 100xOHR holds the world record for light efficiency and resolution due to its unparalleled NA of 1.65. With this objective, the penetration depth of the evanescent field can be reduced below 100nm, providing an exceptionally fine depth of focus for highly precise observation of the 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. This is possible due to the total absence of any out-of-focus fluorescence, as excitation only occurs in the plane of observation directly adjacent to the cover slip-medium interface.
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