Multiphoton Multicolor FISH

摘要

We describe a novel method of three dimensional imaging of specific regions of DNA in interphase nuclei and tissues based on multiphoton microscopy and multicolor fluorescence in situ hybridization (M-FISH). Multiphoton Multicolor FISH (MM-FISH) combines the advantages of (i) using a single NIR excitation wavelength for the simultaneous excitation of multiple FISH fluorophores, (ii) absence of fading in out-of-focus regions, (iii) intrinsic 3D imaging capability and (iv) high light penetration depth. Detection of chromosomal aberrations in amniocytes and tumor cells as well as imaging of FISH fluorophores in biopsies using femtosecond laser pulses at 780 nm and 800 nm are described. First two-photon excited fluorescence decay curves of FISH fluorophores are presented. The fluorophores have been excited via non-resonant two-photon excitation with 150 fs pulses of 0.1 to 8 mW mean laser power of a frequency doubled ultracompact 50 MHz fiber laser and with 80 fs pulses of a compact 80 MHz Ti:sapphire laser. MM-FISH may become an interesting tool in preimplantation diagnosis and molecular pathology.