ABSTRACT
National Institute of Optics - National Research Council (INO-CNR), Largo E. Fermi 6, Florence,
Italy; European Laboratory for Nonlinear Spectroscopy (LENS), University of Florence, Via N.
Carrara 1, Sesto Fiorentino, Italy
Francesco S. Pavone
European Laboratory for Nonlinear Spectroscopy (LENS), University of Florence, Via N. Carrara
1, 50019 Sesto Fiorentino, Italy; Department of Physics, University of Florence, Via G. Sansone 1,
50019 Sesto Fiorentino, Italy
4.1 Introduction to Nonlinear Optical Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
4.2 Theoretical Background of TPF and SHG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
4.3 Spatial Resolution and Image Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
4.4 Technical Features of a Nonlinear Microscope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
4.5 Biological Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
Nonlinear microscopy has undergone impressive growth in the last 20 years, making it one of the
most used optical tools in biological and biomedical imaging of cells and tissues. The main fea-
tures offered by this microscopy technique consist in providing an intrinsic optical sectioning, an
increased penetration depth in optically turbid samples, and a higher spatial resolution with respect
to other laser scanning imaging modalities. The two most used imaging techniques based on non-
linear optical effects are two-photon fluorescence (TPF) and second-harmonic generation (SHG)
microscopy. Both TPF and SHG can be used in biological and biomedical imaging by means of
signal enhancers such as exogenous fluorophores and harmonophores or by taking advantage of the
endogenous tissue components able to emit fluorescence or to generate second-harmonic light.
