Jul 21 2007
Total Internal Reflection Fluorescence (TIRF) is an excellent tool for the functional examination of processes in living cells.
In this method, fluorophores are excited using an evanescent field generated by total reflection. The Wetzlar, Germany-based optics manufacturer has developed a scanner technology for TIRF that greatly simplifies this otherwise very complicated technique. With the Leica AM TIRF, users can concentrate completely on their research work, while leaving instrument operation up to a powerful software program. Vesicle transport examinations, analysis of interactions between molecules, applications in membrane research and even single molecule examinations can be tailored to specific needs using the Leica AM TIRF.
The software-controlled TIRF scanner automatically finds the correlation between the penetration depth of the evanescent field and the corresponding TIRF angles. With the Leica AM TIRF, complicated manual settings and complex calculations are unnecessary - the intelligent Leica AF6000 fluorescence software assumes these tasks, making the system convenient and easy to use. Furthermore, various penetration depths can be reproduced at any time, and the laser beam can be rotated within the inlet pupil of the objective in order to generate various directions of the evanescent field.
With the Leica AM TIRF, all contrast methods are possible, either simultaneously or sequentially. For example, transmitted light and fluorescence, or DIC and TIRF, can be combined easily with each other.The evanescent field penetrates the specimen only to a depth of approximately 100-300nm. All of the fluorophores at a greater depth in the specimen remain inactive. Thus the TIRF method allows a significantly better signal-to-noise ratio than widefield fluorescence microscopy. TIRF also achieves higher resolution in the z-axis. Normally, the axial resolution of a high-end objective is physically limited by the wavelength of the light being used to about 200-300nm. The highly accurate TIRF excitation of 50-300nm in the z-axis bypasses this physical limitation. Furthermore, the excitation of the fluorochrome using an evanescent field reduces bleaching on the specimen. The cells are not directly exposed to laser light.
The Leica TIRF module, which consists of a Leica HXC planapochromatic objective with a numerical aperture of 1.46 and the high-performance scanner that can be moved in three dimensions, can be adapted to various inverted microscopy systems from Leica Microsystems. Microscopes available in the widefield area, adapted to individual needs and requirements, are the Leica DMI6000 B fully automated research microscope, the semi-automated DMI4000 B and the Leica AF6000 LX multidimensional fluorescence workstation. Alternatively, Leica offers two confocal systems: The Leica TCS SPE entry-level system and the TCS SP5 high-end system.
http://www.leica-microsystems.com/