Biomedical Engineering Reference
In-Depth Information
(a)
(b)
(c)
hν
Excitation
THG
3
hν
THG
hν
hν
400
800
1200 (nm)
FIgurE 3.1
Schematic view of the THG process. (a) Three photons of energy
h
ν are coherently scattered to
produce a harmonic photon of energy 3
h
ν. (b) Virtual energy levels of the molecule involved in the THG process.
(c) Wavelength representation.
The first experimental demonstrations of THG in calcite [1,2], gases [3,4], and liquids [5,6] were per-
formed shortly after the demonstration of SHG, and an accurate theory of THG with focused Gaussian
beams was proposed shortly after [3,4]. Tsang et al. [7,8] later reported efficient THG at interfaces
between two dielectric volumes.
THG microscopy was initially demonstrated by two groups at the end of the 1990s: Barad, Eisenberg,
Horowitz, and Silberberg at the Weizmann Institute [9,10]; and Squier, Müller, Brakenhoff, and Wilson
at UCSD (University of California, San Diego) [11,12]. The authors have shown that THG can be used as
a contrast mechanism and allows structural imaging of several samples (root tips, algae, neurons, yeast
cells, etc.) with micrometer resolution. Although the contrast mechanisms were not entirely character-
ized at this point, these pioneering studies showed that because of the phase shift experienced by focused
beams, THG signals mainly originate from interfaces and sub-micron-sized structures, which were in
good agreement with both the experimental demonstration of THG at interfaces [7] and the theoretical
analysis performed by Ward and New [4] who had shown that there is no THG from a homogeneous
isotropic medium and a maximum signal from structures approximately half the Rayleigh range of the
focused excitation beam.
From these early studies, one can already point out important differences between THG and SHG
microscopies:
1. THG is observed only at interfaces or inclusions, that is to say where the sample is heterogeneous
at the scale of the wavelength.
2. However, THG is possible at the interface between two homogeneous isotropic media, and is not
limited to organized structures like SHG is.
Therefore, the two modalities provide complementary information about the sample, which makes
THG/SHG microscopy an interesting combination.
3.1.2 chapter outline
In this chapter, we will concentrate on the principles of THG microscopy. We will start by discussing
the contrast mechanisms in the case of isotropic media, and present applications of combined THG/
SHG imaging in developmental biology illustrating the complementarity between the two signals. In a
second part, we will briefly discuss the mechanisms of THG from organized media, and illustrate this
analysis with the case of THG/SHG imaging of the human cornea.
3.2 tHG Microscopy of isotropic Media
3.2.1 introduction
The coherent nature of signal generation in THG/SHG microscopy is one fundamental difference with
fluorescence microscopy, as it makes the geometrical structure of the sample become an important
