ABSTRACT
Conventional confocal microscopes provide ªuorescence excitation in the visible range only. Many ªuorochromes 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, some 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 Žbers commonly used to deliver light to the scanning head. The ultraviolet light was toxic to living cells and caused severe bleaching of ªuorochromes 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.
