Beyond monochromatic light: three-dimensional confocal laser scanning microscopy using a supercontinuum source

Confocal laser scanning microscopy (CLSM) has rapidly become an essential tool in the life sciences laboratory, enabling high-resolution and minimally intrusive optical sectioning of fluorescent samples. Most commercially available CLSM systems employ a gas laser, e.g. a Kr/Ar laser, to provide the excitation radiation. However, such lasers have several shortcomings, including the maintenance requirements, short lifetimes and high noise levels. To overcome these limitations, a light source for CLSM that is based on supercontinuum generation in photonic crystal fiber has been developed. This source provides the necessary wavelength range required to excite the widest possible variety of fluorophores. A novel method of extracting the desired wavelengths from the supercontinuum source using a digital micro-mirror device (DMD) is also described.