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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 our prominent position in this cutting-edge field. Integral to our TIRFM system is our high numerical aperture objectives: 100x/1.65NA Apochromat; 150x/1.45NA U Apochromat with correction collar for temperature and cover glass thickness; 60x/1.45NA Plan Apochromat with correction collar; and 100x/1.45NA Plan Apochromat. Our U Apochromat objective is the world's only 150x/1.45NA TIRFM objective with UV transmission down to 340nm making the lens excellent for imaging and single molecule fluorescence observation. TIRFM is a specialized optical technique, most commonly employed by cell biologists and neuroscientists, to observe molecular and cell membrane fluorescence.
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Olympus TIRFM Illuminator on IX2 Frame
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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.
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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.
Our 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 Apochromat 150x, 100x, 60x, and 100x Plan Apochromat Olympus objectives boasting benchmark numerical apertures of 1.65 and 1.45. 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.
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Our 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.
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Sanford M. Simon et. al.
The Rockefeller University
Laboratory of Cellular Biophysics
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Total Internal Reflection
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Epifluorescence Image
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Localization of a basolateral membrane protein (LDL receptor) and a v-SNARE (VAMP7) involved in apical targeting of proteins analyzed in HeLa cells using Total Internal Reflection Fluorescence Microscopy.
TIRFM allows enhanced imaging of cell-surface proteins by substantially reducing background fluorescence from inside the cell. TIRFM image (left shows only fluorophores in vicinity of plasma membrane while in epi-fluorescence (right) most of the fluorescence is from the Golgi. Overlapping vesicles in the epifluorescence image make it appear that some of the vesicles are positive for both LDL-receptor and VAMP7. However, using TIRFM it is clear that they do not co-localize.
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