Multiplex FISH and three-dimensional DNA imaging with near infrared femtosecond laser pulses

We report on a novel technology for multicolor gene and chromosome detection as well as for three-dimensional (3D) DNA imaging by multiphoton excitation of multiple FISH fluorophores and DNA stains. Near infrared femtosecond laser pulses at 770 nm were used to simultaneously excite the visible fluorescence of a wide range of FISH fluorophores, such as FITC, DAC, Cy3, Cy5, Cy5.5, rhodamine, spectrum aqua, spectrum green, spectrum orange, Jenfluor, and Texas red as well as of DNA/chromosome stains, for example Hoechst 33342, DAPI, SYBR green, propidium iodide, ethidium homodimer, and Giemsa. In addition to the advantage of using only one excitation wavelength for a variety of fluorophores, multiphoton excitation provided the intrinsic possibility of 3D fluorescence imaging. The technology has been used in human genetics for the diagnosis of numerical chromosome aberrations and microdeletions. In particular, multicolor 3D images of the intranuclear localization of FISH-labeled chromosome territories in interphase nuclei of amniotic fluid cells have been obtained. Using the high light penetration depth at 770 nm, optical sectioning of Hoechst 33342-labeled DNA within living culture cells and within tissue of living tumor-bearing mice was performed.