Abstract
Third-harmonic generation (THG) imaging of thick samples or large organisms requires TH light to be epicollected through the focusing objective. In this study we first estimate the amount of backward-to-forward TH radiation created by an isolated object as a function of size and spatial frequencies in the object. Theory and model experiments indicate that no significant signal can be epidetected from a (biological) dielectric structure embedded in a transparent medium. In contrast, backward emission is observed from metal nanoparticles where THG is partly a surface effect. We then address the case of an object embedded in a turbid medium. Experiments and Monte Carlo simulations show that epidetection is possible when the absorption mean free path of harmonic light in the medium exceeds its reduced scattering length, and that epicollection efficiency critically depends on the microscope field-of-view even at shallow depths, because backscattered light is essentially diffusive. These observations provide guidelines for optimizing epidetection in third-harmonic, second-harmonic, or CARS imaging of thick tissues.
Highlights
One principal application of nonlinear microscopy in the life sciences is its use as a minimally invasive tool to study thick samples such as intact organs or small organisms at the micrometer scale [1]
The situation is different in the case of coherent nonlinear imaging (i.e. based on contrast mechanisms such as coherent anti-Stokes Raman scattering (CARS), second, or third-harmonic generation (SHG, Third-harmonic generation (THG)))
We focus on THG microscopy [13], a technique recently demonstrated for imaging biological samples with three-dimensional resolution [9, 14,15,16,17,18,19,20] and providing information complementary to two-photon excited fluorescence (2PEF) and SHG microscopies
Summary
One principal application of nonlinear microscopy in the life sciences is its use as a minimally invasive tool to study thick samples such as intact organs or small organisms at the micrometer scale [1]. Examples include in situ imaging of neuronal and vascular activity in intact brain [2,3,4], physiological studies in immunology [5, 6] and cancer [7], or long-term imaging of developing embryos [8, 9] For many of these applications, epicollection of the signal through the focusing objective is required due to sample thickness. We discuss the incidence of axial spatial frequencies in the object [23] on coherent backward TH emission These calculations and corresponding experiments in model samples indicate that THG from detectable heterogeneities is largely forward-directed, except in the case of surface-enhanced emission and in some periodic structures. We investigate the impact of experimental parameters such as objective field-of-view and numerical aperture on TH epicollection efficiency, which turns out to differ from fluorescence epidetection We corroborate this analysis by experiments on model and biological samples
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
