IX2 Total Internal Reflection Fluorescence Microscopy (TIRFM)

Brochure

Leeds and Olympus proudly presents the third generation of the Total Internal Reflection Fluorescence Microscopy illuminator. Following the release in 1998 of the first commercial TIRFM illuminator the latest model further strengthens Olympus’ prominent position in this cutting-edge field. Integral to Olympus TIRFM system is high numerical aperture 100x/1.65NA Apochromat, 60x/1.45 Plan Apochromat and 100x/1.45NA Plan Apochromat Olympus objectives. The 60x/1.45 Plan Apochromat and 100/1.45NA Plan Apochromat use standard cover glass and immersion oil, while the 100x/1.65NA Apochromat uses special cover glass and immersion media. TIRFM is a specialized optical technique, most commonly employed by cell biologists and neuroscientists, to observe molecular and cell membrane fluorescence.

TIRFM relies on the extremely shallow evanescent wave produced when incident light rays strike the interface between two optical media of differing refractive indices. This effect occurs when the incident light strikes the optical interface at an angle greater than the critical angle for total internal reflection. A residual evanescent wave extends into the medium of lower refractive index, decreasing exponentially in intensity. The evanescent wave's shallow penetration can be used to selectively illuminate and excite fluorochromes within 100nm of the optical interface, thereby eliminating autofluorescence produced by the cell body and drastically improving the signal-to-noise ratio compared to conventional widefield microscopy techniques.

Olympus was the first company to offer a commercially available TIRFM system. The latest generation of our TIRFM illuminator takes several steps forward by offering multi-line illumination at three different wavelengths simultaneously through a single mode optical fiber. Installation of the illuminator requires no alterations to the IX2 microscope frame. A micrometer is built into the TIRFM illuminator to facilitate laser alignment. 

Olympus objective-based TIRFM system enables conventional reflected and transmitted widefield techniques such as bright field, darkfield, phase contrast and differential interference contrast to be employed with the TIRFM illuminator simultaneously in place. An objective-based TIRFM system eliminates the need for costly prisms mounted above the specimen that prohibit transmitted light techniques and risk disturbing the specimen. Central to this lens-based technique are unique TIRFM objectives, an Apochromatic 100x/1.65, Plan Apochromatic 100x/1.45 and Plan Apochromatic 60x/1.45.  With the 100x/1.65N.A. Fluorescence microscopy gains a 28% increase in brightness and the Raleigh Limit on resolution is extended from 0.240mm to 0.203mm over conventional 100xoil/1.4N.A. objectives. The TIRFM objectives' high numerical apertures also maximize the effective working area for alignment and adjustments to the incident light angle. Furthermore, the objectives double as perfectly aligned high numerical aperture condensers for TIRFM and other reflected light techniques.

Olympus highly innovative objective-based TIRFM system is part of a cutting edge-effort to advance the capabilities of optical microscopy by enabling fluorescence imaging at the molecular level. To learn more about TIRFM and other cutting-edge techniques, please visit the Olympus Microscopy Resource Center at www.olympusmicro.com.