Investigation of refractive index dynamics during in vitro embryo development using off-axis digital holographic microscopy

Abstract

Embryo quality is a crucial factor affecting live birth outcomes. However, an accurate diagnostic for embryo quality remains elusive in the in vitro fertilization clinic. Determining physical parameters of the embryo may offer key information for this purpose. Here, we demonstrate that digital holographic microscopy (DHM) can rapidly and non-invasively assess the refractive index of mouse embryos. Murine embryos were cultured in either low- or high-lipid containing media and digital holograms recorded at various stages of development. The phase of the recorded hologram was numerically retrieved, from which the refractive index of the embryo was calculated. We showed that DHM can detect spatio-temporal changes in refractive index during embryo development that are reflective of its lipid content. As accumulation of intracellular lipid is known to compromise embryo health, DHM may prove beneficial in developing an accurate, non-invasive, multimodal diagnostic.

Label-free, high-throughput holographic imaging to evaluate mammalian gametes and embryos

“… Major milestones in holographic (IPM/QPI) gamete and embryo analysis. The instance of the use of each methodology is indicated, i.e., DPM of fixed sperm [ 45 ], DPM of live sperm [ 46 ], lens-free HI for sperm tracking [ 47 ], MCHI for biovolume estimation [ 48 ], DHM for automated sperm tracking [ 49 ], IPM [ 50 ], IPM with automated sperm analysis [ 35 ], GLIM for bovine embryo evaluation [ 30 ], Br-OCT for mouse embryo evaluation [ 70 ], q-cLEDscope for motility analysis [ 51 ], SLIM for sperm [ 29 ], IPM with microfluidics to isolate individual sperm [ 72 ], SLIM of sperm DNA-liposomes [ 53 ], DHM with AI to screen for oxidative stress [ 56 ], SLIM bovine oocyte evaluation [ 5 ], PICS [ 36 ], PSC-DHM with AI [ 57 ], PTLS-DHM for motility analysis [ 58 ], BDHSM for metabolic evaluation of oocytes and embryos [ 65 ], OCM for mouse oocyte evaluation [ 66 ], SSP-QPM to assess oocyte sub-cellular structures [ 69 ], LC-SICA with AI for DNA fragmentation scoring [ 43 ], and DHM for mouse embryo evaluation [ 71 ]. …”

“…Based on the findings from various studies cited in this review, it appears that holographic microscopy is superior to currently used BFM-based techniques for evaluation of sperm [ 29 , 36 , 53 , 54 , 58 , 72 , 73 ], oocytes [ 5 , 65 , 66 ], and embryos [ 30 , 70 , 71 , 74 ]. Although the advantages of QPI for sperm are relatively straightforward, the data regarding its value to image oocytes and embryos is not as abundant.…”

“…used Brillouin microscopy in conjunction with optical coherence tomography to identify embryonic structures in an E8.5 developing mouse embryo [ 70 ], and Dwapanyin et al. used DHM to investigate the refractive index of developing embryos [ 71 ] ( Table 3 ).…”

Digital in-line holographic microscopy for label-free identification and tracking of biological cells

“…A band-pass filter is commonly employed to separate real, twin, and zero-order images in the frequency domain of a recorded hologram. The off-axis DHM systems have been utilized for quantitative phase imaging [ 208 – 210 ], holographic tomography [ 211 , 212 ], and dynamic analysis of biological samples [ 213 – 216 ]. On the contrary, obtaining clear real images from holograms recorded by DIHM systems is challenging due to the twin-image problem.…”

Noninvasive time-lapse 3D subcellular analysis of embryo development for machine learning-enabled prediction of blastocyst formation

“…QPI has been applied across a wide range of fields, including cell biology [13][14][15] , microbiology [16][17][18] , immunology [19][20][21] , and biophysics 22,23 . Among these, holotomography (HT), a 3D QPI technique, has proven effective for analyzing thick samples, such as 3D cell-culture models [24][25][26] , thick tissues [27][28][29] , and embryos [30][31][32][33][34] from various species including C.elegans 33 , Bovine 31 , pig 32,35 , and mouse embryos 34,36 . Despite HT's evident potential in developmental biology, previous studies have yet to delve into the 3D subcellular intricacies of early embryo development.…”