Simultaneous multichannel nonlinear imaging: combined two-photon excited fluorescence and second-harmonic generation microscopy.
Simultaneous two-photon excited fluorescence (TPF) and second-harmonic generation (SHG) imaging is demonstrated using a single femtosecond laser and a scanning microscope. This composite nonlinear microscopic technique was applied to imaging DNA and chromosomes, and it was shown that the two different interaction mechanisms provide complementary information on the structure and nonlinear properties of these biological materials, beyond that achievable using either TPF or SHG imaging alone. The use of separate modes of detection, 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.
Gauderon
R
R
Department of Physical Optics, School of Physics A28, University of Sydney, NSW 2006, Sydney, Australia.
Lukins
P B
PB
Sheppard
C J
CJ
D000818:Animals; D002875:Chromosomes / Q000648:ultrastructure*; D004247:DNA / Q000648:ultrastructure*; D004330:Drosophila; D005399:Fishes; D005456:Fluorescent Dyes / Q000378:metabolism; D007211:Indoles / Q000378:metabolism; D008297:Male; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D019697:Onions; D018517:Plant Roots / Q000648:ultrastructure; D013094:Spermatozoa
D005456:Fluorescent Dyes; D007211:Indoles; C007293:DAPI; D004247:DNA