Advances in multiphoton microscopy for imaging embryos

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Multiphoton imaging is a promising approach for addressing current issues in systems biology and high-content investigation of embryonic development. Recent advances in multiphoton microscopy, including light-sheet illumination, optimized laser scanning, adaptive and label-free strategies, open new opportunities for embryo imaging. However, the literature is often unclear about which microscopy technique is most adapted for achieving specific experimental goals. In this review, we describe and discuss the key concepts of imaging speed, imaging depth, photodamage, and nonlinear contrast mechanisms in the context of recent advances in live embryo imaging. We illustrate the potentials of these new imaging approaches with a selection of recent applications in developmental biology.

Introduction

From a microscopy perspective, live embryos present uniquely challenging characteristics compared to other biological samples. Embryos are smaller than 1 mm, at least during early developmental stages, making them accessible for three-dimensional (3D) imaging with light microscopy. However, they typically have an ellipsoidal shape and their inner structure is inhomogeneous and constantly changing. In addition, embryos are sensitive to manipulation and photodamage, and their labeling can be difficult. These properties challenge the performance of microscopy techniques in terms of imaging depth, imaging speed, photodamage and contrast. Since its introduction in 1990 [1], 2-photon excited fluorescence (2PEF) microscopy has proven to be the most effective approach for deep tissue fluorescence microscopy (Box 1). It has found many applications in neuroscience [2, 3] and more recently in other fields, such as in immunology [4]. Multiphoton (or nonlinear) imaging is attractive also for embryo imaging and in recent years has been applied to an increasing number of published studies in developmental biology using various model systems, such as fruit fly [5, 6, 7, 8], quail [9], zebrafish [10], or mouse embryos [11, 12]. Multiphoton imaging is also promising for addressing current issues in systems biology and high-content experimental investigation of embryonic development [13] requiring novel methods for faster and deeper imaging of embryos with better contrast and resolution. In this review we analyze the parameters limiting imaging speed and depth in the currently available imaging modalities, and we discuss promising recent advances in multiphoton microscopy of live embryos, including light-sheet excitation and label-free imaging.

Section snippets

Fast imaging of live embryos with multiphoton light-sheet microscopy

Imaging developmental processes often requires time-lapse 3D-image acquisitions (4D imaging). The imaging speed of a microscope can be defined by its pixel (or voxel) rate, that is, the number of pixels per unit time that can be obtained with sufficient signal and contrast. A high pixel rate permits capturing with adequate time resolution fast processes such as heart development (50–130 frames per second (fps) in Refs. [14, 15, 16••]), cilia beating (900 fps in Ref. [17]) or fluid flow in

Parameters governing imaging depth in tissue microscopy

The imaging depth corresponds to how deep into the tissue images can be recorded with sufficient quality (resolution, signal intensity, contrast). Imaging depth is limited by light scattering [25] and by sample-induced optical aberrations. Point-scanning 2p-microscopy is usually considered as the gold-standard in imaging depth for in vivo fluorescence imaging of tissues and embryos [26] (Figure 3). For instance, the large-scale dynamic analysis of the deepest mesoderm cells during Drosophila

Adaptive advantages of point-scanning for multiphoton imaging of embryos

A developing embryo is a dynamic and inhomogeneous biological system. Optical properties vary between species and tissues [26], and they also constantly evolve in time and space during embryonic development [27•]. As a consequence, embryo imaging would strongly benefit from the ability of microscope illumination and acquisition schemes to adapt to the changing properties of the developing tissue. In this context, point-scanning multiphoton techniques have a fundamental advantage compared to

Beyond fluorescence: other nonlinear contrast mechanisms (SHG, THG, CARS, and SRS)

Another advantage of nonlinear microscopy is that in addition to fluorescence, other multiphoton processes can be used as contrast mechanisms to provide complementary information. These include second-harmonic generation (SHG), third-harmonic generation (THG), coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS). These imaging modalities [35] share the benefits of point-scanning 2p-microscopy in terms of 3D resolution and penetration depth. However they rely on

Conclusion and perspectives

Recent advances in multiphoton microscopy, including light-sheet, adaptive and label-free strategies, open promising avenues for embryo imaging. However, the literature is often unclear about the comparative performances of microscopy modalities. Therefore, it is important to understand the principles, advantages and limitations of each microscope implementation. In this review, we provide keys to understand recent methodological developments in the perspective of their application to

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgments

We thank members of the Laboratory for Optics and Biosciences at Ecole Polytechnique (Palaiseau, France) and of Scott E. Fraser group at Caltech (Pasadena, CA, USA) for fruitful discussions. T.V.T. is supported by the NIH (U01 DE020063). This work was supported by ANR (ANR-2010-JCJC-1510-01) and the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 268379 (intotoMorphogenesis project) to W.S.

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