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Advances in whole-embryo imaging: a quantitative transition is underway

Nature Reviews Molecular Cell Biology volume 15, pages 327–339 (2014)Cite this article

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Key Points

  • Over the past decade, advances in genetically encoded fluorescent probes have enabled quantitative studies that have resulted in a deeper knowledge and understanding of the dynamic processes of embryonic differentiation, patterning and morphogenesis.

  • However, the currently available fluorescent imaging approaches have limitations, and new developments, such as the advent of engineered nanoprobes, should positively affect the field of quantitative whole-animal imaging.

  • Innovations in multiphoton and light-sheet microscopy have enabled researchers to obtain deeper and faster in vivo whole-animal images with decreased photodamage in various applications.

  • Bottlenecks in image processing and analysis, including visualization, data registration and feature computation, currently limit imaging complexity. Addressing these issues will ultimately enable truly multidimensional and multiscale investigations of embryonic development.

Abstract

With the advent of imaging probes and live microscopy, developmental biologists have markedly extended our understanding of the molecular and cellular details of embryonic development. To fully comprehend the complex mechanistic framework that forms the developing organism, quantitative studies with high fidelity in space and time are now required. We discuss how integrating established, newly introduced and future imaging tools with quantitative analysis will ensure that imaging can fulfil its promise to elucidate how new life begins.

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Figure 1: Imaging probes and microscopy techniques for quantitative whole-embryo imaging.
Figure 2: Quantitative whole-embryo imaging: a selection flowchart for specific applications in cellular and molecular dynamics.

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Acknowledgements

The authors thank former and present members of their laboratories for discussions and feedback during the preparation of this Review. The authors apologize to those whose work could not be discussed owing to space limitations.

Author information

Authors and Affiliations

  1. Department of Biosystems Science and Engineering (D-BSSE), Eidgenössische Technische Hochschule (ETH) Zurich, Basel, 4058, Switzerland

    Periklis Pantazis

  2. Laboratory for Optics and Biosciences, École Polytechnique, Centre National de la Recherche Scientifique (CNRS) UMR7645, and Institut National de la Santé et de la Recherche Médicale (INSERM) U696, Palaiseau, 91120, France

    Willy Supatto

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  1. Periklis Pantazis
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Correspondence to Periklis Pantazis or Willy Supatto.

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Competing interests

Work mentioned in the Review is the subject of patent applications filed by the Swiss Federal Institute of Technology (ETH), Zurich, Switzerland, the California Institute of Technology (Caltech), Pasadena, California, USA, the Centre National de la Recherche Scientifique (CNRS), Paris, France, and the École Polytechnique, Palaiseau, France.

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FURTHER INFORMATION

BioEmergences

Core for Life

OpenSPIM

PowerPoint slides

Glossary

Confocal microscopy

An optical imaging technique that uses point-scanning illumination and a spatial pinhole to obtain optical sectioning and to eliminate out-of-focus signals in tissue.

Two-photon microscopy

An optical imaging technique that uses point-scanning illumination, a near-infrared spectrum femtosecond laser and two-photon absorption to obtain optical sectioning and improved imaging depth compared with confocal microscopy.

Light-sheet microscopy

An optical imaging technique whereby the specimen is illuminated with a sheet of light perpendicular to the detection direction, which provides excellent sectioning capabilities, fast imaging and low levels of photodamage.

Fluorescence recovery after photobleaching

(FRAP). An imaging assay that can determine the diffusion properties of labelled molecules in a tissue by photobleaching a small defined fluorescent region, which is followed by measuring the rate and extent of fluorescence recovery emanating from neighbouring regions.

Fluorescence correlation spectroscopy

(FCS). An imaging assay that can determine the diffusion properties of labelled molecules in a tissue by measuring fluorescence fluctuations in a small volume.

Morphogens

Signalling molecules that are typically present in gradients and that determine where specific cell types form in developing tissues.

PhOTO zebrafish

(Photoconvertible optical tracking of zebrafish). A transgenic zebrafish line with life-long fluorescent labelling of nuclear or plasma membrane proteins using the photoconvertible protein Dendra2. Taking advantage of the instantaneous photoconversion of the Dendra2 fusion protein, the non-invasive, targeted and high-contrast selection of any cells of interest can be accomplished, which greatly simplifies cell segmentation and tracking in time and space.

Multiphoton

All photonic processes depend nonlinearly on light intensity. Multiphoton microscopy takes advantage of such nonlinear processes as a source of contrast.

Fluorescence decay after photoactivation or photoconversion

(FDAP). An imaging assay that is similar to traditional fluorescence recovery after photobleaching (FRAP) but that uses photoactivatable (or photoconvertible) proteins as labelling molecules. The added benefit of visualizing only a limited population of photoactivated or photoconverted molecules is that this avoids the very high laser powers that are necessary to carry out FRAP experiments. It also enables the analysis of the diffusion behaviour and turn-over of photoactivated or photoconverted molecules independently of other proteins that are newly synthesized.

Blinking

Large intensity fluctuation between a bright state (On) and a dark state (Off) of an imaging probe that is under continuous excitation.

Raman reporter molecules

Nitrogen-containing cationic dyes, sulphur-containing dyes or thio-small molecules, which can inelastically scatter a fraction of the absorbed light into a series of different wavelengths that are indicative of the vibrational transitions in the molecules.

Bioorthogonal labelling

Labelling reactions that can occur inside living embryos without interfering with the native biological system.

Harmonic generation

A coherent contrast mechanism that relies on nonlinear scattering of light. Unlike fluorescence, second-harmonic generation (SHG) and third-harmonic generation (THG) do not involve light absorption.

Optical parametric oscillator

An optical device that converts an input laser wave into an output wave of a lower frequency (or greater wavelength). The one used in multiphoton microscopy is pumped by a standard titanium-sapphire laser in the 750–900 nm range, which enables output wavelengths to reach the 1,000–1,500 nm range.

Conformal scanning

Unlike raster scanning, which is used in most point-scanning microscopes, conformal scanning uses a laser scanning pattern that is adapted to the sample shape.

Dynamic aberration correction

An experimental procedure to correct for changing optical aberrations that are induced by embryonic tissues during development using adaptive optics and real-time corrections.

Gaussian beam

The intensity profile of a laser beam after focusing it with a standard microscope objective.

Bessel beam

A specific laser beam profile with elongated axial and short lateral extensions of the focal volume.

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Pantazis, P., Supatto, W. Advances in whole-embryo imaging: a quantitative transition is underway. Nat Rev Mol Cell Biol 15, 327–339 (2014). https://doi.org/10.1038/nrm3786

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