Determination of the Number of Cells in Preimplantation Embryos by Using Noninvasive Optical Quadrature Microscopy in Conjunction with Differential Interference Contrast Microscopy

Abstract

The number of cells in a preimplantation embryo is directly correlated to the health and viability of the embryo. There are currently no methods to count the number of cells in late-stage preimplantation embryos noninvasively. We assessed the ability of optical quadrature microscopy (OQM) to count the number of cells in mouse preimplantation embryos noninvasively. First, to test for possible light toxicity, we exposed two-cell mouse embryos to OQM and differential interference contrast (DIC) microscopy and assessed their ability to develop to the blastocyst stage. We found no inhibition of development from either mode of microscopy for up to 2 h of light exposure. We also imaged eightcell to morula stage mouse preimplantation embryos by OQM nd developed two methods for counting the number of cells. The contour signature method (CSM) used OQM images alone and the phase subtraction method (PSM) used both OQM and DIC images. We compared both methods to standard cell counting techniques and found that the PSM was superior to all other noninvasive cell counting methods. Our work on mouse embryos should be applicable to human embryos. The ability to correctly count the number of cells in human preimplantation embryos could lead to the transfer of fewer embryos in in vitro fertilization (IVF)clinics and consequently a lower rate of high-risk multiple-infant births.

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“…OQM has been used to reliably count blastomeres compared to traditional staining/counting approaches. Images were adapted from [114] and [118] with permission.…”

“…Optical quadratic microscopy (OQM) measures both the amplitude and phase of light as it passes through an object [114] and noninvasively reconstructs them through use of an algorithm to produce a quantitative image. OQM was shown to be safe for mouse embryos, permitting development from the 2-cell to blastocyst stage [114].…”

“…OQM was shown to be safe for mouse embryos, permitting development from the 2-cell to blastocyst stage [114]. Briefly, OQM using a 15 mW, 633 nm red-light laser resulting in a peak irradiance of 6.6 3 10 À4 W/cm 2 , a 5-fold lower exposure of light intensity compared to differential interference contrast (DIC), was used to image cells.…”

“…Briefly, OQM using a 15 mW, 633 nm red-light laser resulting in a peak irradiance of 6.6 3 10 À4 W/cm 2 , a 5-fold lower exposure of light intensity compared to differential interference contrast (DIC), was used to image cells. Exposing 2-cell embryos for 0-120 min did not compromise development compared to no light exposure of DIC-exposed controls [114]. Subsequently, OQM was used to count cells in early preimplantation embryos (Fig.…”

“…6). However, accuracy was not ideal [114]. While used alone, OQM does not offer insight into morphologic features used to grade embryos and is not overly informative.…”