Total Internal Reflection Fluorescence Microscopy (TIRFM) provides an unrivalled technique for the study of single molecules and membrane related cell function, including adhesion, motility, endo- and exo-cytosis.
In TIRFM the electromagnetic field of the TIR light extends into the sample beyond the interface by 100-200nm into the medium of lower refractive index. Since the field decreases exponentially along the z-axis, only a very thin section of the specimen undergoes fluorescence excitation.
TIRFM produces extremely high contrast, low background images, ideal for detection with our iXonEM+ EMCCD cameras and is a valuable tool in the study of e.g. cellular membrane traffic, cytoskeleton dynamics and single molecule studies.
Andor Revolution components provide:
The Revolution® laser combiner (ALC) offers a small physical, electrical and power footprint, based on state-of-the art solid state laser technology; the unit has been designed for multi-modal imaging. As well as wide range of laser wavelengths (405-640 nm) and powers (25-100 mW), ALC has a highly efficient AOTF integrated into the system for laser wavelength and intensity control and offers microsecond switching and blanking (shuttering) with isolation between channels of 120 dB. ALC blanking can be controlled by the camera exposure signal, minimizing specimen photo-toxicity and bleaching.
In order to use the same laser lines for multiple imaging modes, we have developed a proprietary multi-port switch (MPS), which delivers 100% of laser power to one of three fiber delivery ports. MPS uses a fast galvanometer to switch outputs in approximately 1 ms, with external TTL control. Stability is remarkably good, showing in the order of 0.5% over a 12 hour period. This includes SS laser long term intensity fluctuations. The rapid performance of MPS ensures that switching between imaging mode is not limited by the ALC, but other factors in the system. For best speed of switching dichroics for example (required for TIRF-CSU switching), we have found the TILL linear filter cube controller provides a switching time of <100 ms, while in more conventional microscopes filter turret rotations can take >1 second.
iXonEM and iXonEM+ EMCCDs have long been the camera of choice in single molecule studies and increasingly their benefits are seen in low background applications such as TIRFM. EM Gain can be used to best effect in low background imaging and supports high frame rate imaging, which is important in the study of real time motion, signaling and trafficking events.
Many users now obtain their entire detection, laser and data system (iQ workstation) from Andor to couple to the preferred microscope and TIRF illuminator. We have successfully integrated our Revolution® TIRF systems to many Nikon TE2000E and Olympus IX81 with corresponding TIRF illuminator. These systems accept single mode optical fiber inputs with FC connectors, which match our standard fiber optics.
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