chapter  8
14 Pages

Multiphoton Microscopy

Conventional confocal microscopes provide fluorescence excitation in the visible range only. Many fluorochromes used by cell biologists are excited by light in the near-ultraviolet range, including popular calcium indicators (Fura, Indo) and DNA labels (DAPI, Hoechst). To cater to this, a few years ago some makers offered confocal microscopes equipped with ultraviolet lasers. These systems suffered from many disadvantages. The lasers then available (high-powered argon) were bulky and troublesome, and not compatible with the optical fibers commonly used to deliver light to the scanning head. The ultraviolet light is toxic to living cells and causes severe bleaching of fluorochromes above and below the plane of focus, limiting the ability of these microscopes to collect three-dimensional and time-course data sets. Furthermore, the lines available were not well suited to calcium ratiometric dyes. Alignment of the (invisible) beam was also tricky and potentially hazardous.