Simultaneous two-photon excited fluorescence (TPF) and second-harmonic gene
ration (SHG) imaging is demonstrated using a single femtosecond laser and a
scanning microscope. This composite nonlinear microscopic technique was ap
plied to imaging DNA and chromosomes, and it was shown that the two differe
nt interaction mechanisms provide complementary information on the structur
e and nonlinear properties of these biological materials, beyond that achie
vable using either TPF or SHG imaging alone. The use of separate modes of d
etection, in reflection and transmission respectively, and the simultaneous
nature of the acquisition of the two images allows pure TPF and SHG images
in precise registration to be obtained. (C) 2001 Elsevier Science Ltd. All
rights reserved.