Viewing early life without labels: optical approaches for imaging the early embryo†

Abstract

Embryo quality is an important determinant of successful implantation and a resultant live birth. Current clinical approaches for evaluating embryo quality rely on subjective morphology assessments or an invasive biopsy for genetic testing. However, both approaches can be inherently inaccurate and crucially, fail to improve the live birth rate following the transfer of in vitro produced embryos. Optical imaging offers a potential non-invasive and accurate avenue for assessing embryo viability. Recent advances in various label-free optical imaging approaches have garnered increased interest in the field of reproductive biology due to their ability to rapidly capture images at high resolution, delivering both morphological and molecular information. This burgeoning field holds immense potential for further development, with profound implications for clinical translation. Here, our review aims to: (1) describe the principles of various imaging systems, distinguishing between approaches that capture morphological and molecular information, (2) highlight the recent application of these technologies in the field of reproductive biology, and (3) assess their respective merits and limitations concerning the capacity to evaluate embryo quality. Additionally, the review summarizes challenges in the translation of optical imaging systems into routine clinical practice, providing recommendations for their future development. Finally, we identify suitable imaging approaches for interrogating the mechanisms underpinning successful embryo development.

Keywords: autofluorescence; embryo assessment; fluorescent lifetime microscopy; hyperspectral imaging; label-free imaging; light sheet microscopy; metabolic imaging; non-invasive; optical imaging.

Graphical Abstract

Figure 1

Optical coherence tomography records backscattered light from different cell/tissue layers to form a 3D morphological image. It can work in the temporal or Fourier domain. Image on the right shows ovulated murine cumulus oocyte complexes within the oviduct. Schematic was created using BioRender.com. Image on the right adapted from [157] [CC-BY-NC-ND 4.0 License].

Figure 2

Digital holographic microscopy can take a phase image of the object. The phase is recorded by interfering a beam passing through the sample with a reference beam.

Figure 3

Refractive index profile of mouse embryos cultured in low- or high-lipid-containing culture media during preimplantation development. The refractive index was determined through digital holographic microscopy. Digital holographic microscopy was able to detect a higher refractive index in embryos cultured in high-lipid-containing media, particularly from the two-cell to the morula stage of development. These changes in refractive index were reflective of intracellular lipid abundance. Scale bar = 30 μm. Color coding indicates the determined refractive index. Figure adapted from [50] [CC-BY 4.0 License].

Figure 4

The principle of Raman scattering (left). Some light from the laser pulse is inelastically scattered following its interaction with molecules. The change in energy manifests itself as a change in the wavelength of the scattered light leading to a spectrum [denoted as the Raman shift, measured in wavenumbers (cm⁻¹)]. The peaks of the Raman signal can be attributed to specific proteins, nucleic acids, etc., in the sample. The spectra on the right show characteristic Raman bands for a living cell from a two-cell stage embryo. The position of the bands and relative intensities differ depending on the location within the cell investigated [top spectra from a lipid-rich (dark cytoplasm) area; middle spectra taken from a (light) cytoplasm region; and the bottom from within the nucleus]. Schematic was generated using BioRender.com. Figures adapted from [79] [CC-BY 4.0 License].

Figure 5

(A) shows the principle of epifluorescence microscopy, specifically the case for confocal microscopy. A laser is focused to a point on a sample. The resultant fluorescence from the focal point is captured by the very same microscope objective, passes through a pinhole, and is collected on a camera. An image in 3D can be captured in this way by point scanning the beam. At each point the light field passes through the sample above and below the plane of interest (compare to light sheet imaging). (B) Light sheet imaging illuminates a given plane of a sample with scattered or fluorescent light collected by a second microscope objective (detection objection) placed orthogonally to the one used for illumination. Light only illuminates the (focal) plane of interest [contrast this with confocal microscopy in (A)]. As such, the light dose is much lower than that for epifluorescence/confocal microscopy. The sample or illumination beam may be scanned to generate a 3D image. Representative autofluorescence images of murine blastocyst-stage embryos taken at an illumination wavelength of 405 nm are shown on the right and are false-colored for clarity. Schematic was generated using BioRender.com.

Figure 6

Fluorescence lifetime microscopy. Following excitation with a laser pulse the decay of the signal is measured to yield a fluorophore lifetime. The decay constant (lifetime) is dependent on the environment of the fluorophore and/or its conformational state. Importantly, is not dependent on the absolute intensity of the signal, and complements intensity-based imaging approaches. Images on the right are of a human blastocyst-stage embryo showing abundance of NADH and FAD following FLIM. ICM = inner cell mass, TE = trophectoderm. Scale bar = 40 μm. Schematic was generated using BioRender.com. Images adapted with permission from [98].

Figure 7

Hyperspectral and multispectral microscopy. The graphs on the left show the principal of multispectral and hyperspectral imaging. In both, spectral information is recorded. In multispectral imaging, several discrete wavelengths are captured, but in hyperspectral imaging, this is performed in a continuous fashion. On the right, hyperspectral light sheet microscopy was used to record cell autofluorescence from a murine blastocyst-stage embryo. From this we can create metabolic maps across the embryo and generate a 3D image. Schematic was generated using BioRender.com. Images adapted from [135] [CC-BY 4.0 License].

Figure 8

Autofluorescence profile of preimplantation mouse embryos as detected by hyperspectral light-sheet microscopy. Hyperspectral light sheet microscopy was able to detect dynamic changes in metabolism during preimplantation embryo development and the distribution of highly metabolically active sites within embryos. Scale bar = 20 μm. Images adapted from [135] [CC-BY 4.0 License].