Simultaneous multichannel nonlinear imaging: combined two-photon excited fluorescence and second-harmonic generation microscopy

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.