Similar strategies have been developed in linear (a-c) and nonlinear (d-f) microscopy. Both point-scanning (a and d) and multifocal (b and e) approaches use a collinear geometry: the illumination and the detection paths are collinear. Light-sheet microscopy (c and f) uses an orthogonal geometry: the illumination path is orthogonal to the detection path. Pixel rate range typically from 10⁵–10⁶ pixels.s⁻¹ in point-scanning microscopy to 10⁷–10⁸ pixels.s⁻¹ in light-sheet microscopy. While nonlinear microscopy generally provides deeper imaging than linear microscopy, differences exist in imaging depth performance between the different implementations of nonlinear microscopy, as discussed in the text.

This figure illustrates various recent applications of multiphoton microscopy in developmental biology. (a) Long term imaging of Drosophila embryos using light-sheet 2p-microscopy (reprinted from [16], scale bar is 50 μm). (b) Standard raster scanning in multiphoton microscopy produces inhomogeneous signal levels across the embryo. Conformal scanning adapted to the embryo shape provide homogeneous signal for the entire image (image adapted from [30] and reprinted with permission from AAAS, scale bar is 100 μm). (c) 3D rendering of point-scanning 2p-microscopy imaging of a mouse embryo before and after correction of sample-induced aberrations showing improvement in both signal intensity and spatial resolution (adapted from [33] and reprinted with permission from OSA). (d) Simultaneous 2PEF, SHG and THG imaging of zebrafish embryos allows detection of histones, mitotic spindles and cell contours, respectively. Such label-free imaging with SHG and THG signals has been used for 3D cell segmentation and tracking and for reconstructing the cell lineage of early zebrafish development (image adapted from [30] and reprinted with permission from AAAS, scale bars are 200 μm (top) and 20 μm (bottom)). (e) Maturation of axons and dendrites is probed with SHG in mouse hippocampal slices (adapted from [49], copyright (2008) National Academy of Sciences, USA). (f) 3D reconstruction of combined 2PEF (from Bodipy TR dye in red) and SHG (from muscle endogenous signal and BaTiO3 nanoparticle in blue) signals recorded in zebrafish embryos: the strong SHG signal from the nanoprobe (yellow arrow) is detected twice deeper than the endogeneous SHG from muscles (image adapted from [51] and reprinted with permission from authors, scale bar is 50 μm).

The principles governing imaging depth are analyzed in three steps: sample illumination (a-d), fluorescence excitation (e-h) and fluorescent detection (i-l). 2p-microscopy uses longer excitation wavelengths (red in a and c) than 1p-microscopy (blue in b and d), resulting in reduced scattering inside biological tissues, preserving focus quality in depth with less out-of-focus sample illumination (dashed area in a-d). Compared to confocal and point-scanning 2p-microscopy, the light-sheet illumination has the advantage of using low-NA illumination focusing, which is less sensitive to sample-induced optical aberrations (c-d). The linear fluorescence excitation in 1p-microscopy, results in a direct equivalence between illumination (blue in b and d) and fluorescence excitation volumes (green in f and h). It results in a high sensitivity to illumination light scattering and a poor confinement of fluorescence excitation. In light-sheet 1p-microscopy, this poor confinement results in lower axial sectioning and degraded axial resolution due to the scattering-induced thickening of the light-sheet at high sample depth (h). The nonlinearity of fluorescence excitation in 2p-microscopy confines the excitation to the region with highest illumination intensity, resulting in an excitation volume (green in e and g) smaller than the illumination volume (red in a and c). This robust confinement of the fluorescence excitation makes 2p-microscopy less sensitive to scattering of the illumination light and allows preserving spatial resolution deeper into biological tissues (e and g). Finally, concerning fluorescence signal detection, point-scanning 2p-microscopy has a key advantage compared to other techniques. Namely, the 3D-confinement of the fluorescence excitation (green in i) guarantees the spatial origin of emitted photons, which allows collecting both scattered and non-scattered photons to build the fluorescence signal. Hence, point-scanning 2p-microscopy has the most efficient signal collection using scattered emitted photons as part of the signal. In any other techniques of microscopy, these scattered photons are either rejected (using a pinhole such as in confocal microscopy, (j)) or degrade the image quality. For instance, in light-sheet microscopy, scattering of the fluorescence on its way to the camera results in cross talk between adjacent pixels and image blurring (k and l): only ballistic photons contribute to the signal while scattered photons cause contrast and resolution degradation.