The implementation of a fast fluorescence life‐time imaging method in a confocal laser scanning microscope is described. The set up utilizes a low‐power continuous wave (CW) argon ion laser equipped with an electro‐optic chopper producing nanosecond pulses with a repetition rate up to 25 MHz. A time‐gated detection technique enables the measurement of the lifetime of a pixel in 40 μs. The first confocal fluorescence lifetime contrast images are presented. Application of fluorescence lifetime imaging in multilabelling experiments for discrimination between different labels with overlapping emission bands, for probing the local environment of a fluorescent molecule, and for quantitative fluorescence are discussed.
A confocal laser‐scanning microscope (CLSM) differs from a conventional microscope by affording an extreme depth discrimination, as well as a slightly improved resolution. These features afford improved imaging, and make possible new imaging techniques. The CLSM developed at TNO has standard video‐rate imaging, and is capable of working in reflection and in fluorescence mode simultaneously. Nonconfocally the laser‐scanning microscope can also be used in transmission mode. In addition to the evident advantages of a fast system when searching objects or studying living objects, the time needed to produce an image of extended depth of focus and high resolution is very short. Furthermore, the high‐speed averaging of many images at low laser‐power levels, and the short dwelling time of the focused laser beam (60 ns) obviate quenching effects in fluorescence microscopy and prevent damage to the object. In this article the TNO‐CLSM system is outlined. The most important specifications are summarized, and some representative micrographs obtained with the system are shown. Furthermore, the performance of the system is illustrated by some experimental results.
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