Abstract

Present imaging techniques used in in vitro fertilization (IVF) clinics are unable to produce accurate cell counts in developing embryos past the eight cell stage. We have developed a method that has produced accurate cell counts in live mouse embryos ranging from 8 – 26 cells by combining Differential Interference Contrast (DIC) and Optical Quadrature Microscopy (OQM). OQM is an interferometric imaging modality that measures the amplitude and phase of the signal beam that travels through the embryo. The phase is transformed into an image of optical path length difference, which is used to determine the maximum optical path length deviation of a single cell. DIC microscopy gives distinct cell boundaries for cells within the focal plane when other cells do not lie in the path to the objective. Fitting an ellipse to the boundary of a single cell in the DIC image and combining it with the maximum optical path length deviation of a single cell creates an ellipsoidal cell model of optical path length deviation. Subtracting the model cell from the Optical Quadrature image will either show the optical path length deviation of the culture medium or reveal another cell underneath. Once all the boundaries are used in the DIC image, the subtracted Optical Quadrature image is used to determine the cell boundaries of the remaining cells. The final cell count is produced when no more cells can be subtracted. We have produced exact cell counts on 10 samples, which have been validated by Epi-Fluorescence images of Hoechst stained nuclei.

Accurate Cell Counts in Live Mouse Embryos using Optical Quadrature and Differential Interference

Contrast Microscopy William C. Warger II1-3, Judith A. Newmark1,4, Carol M. Warner1,4, Charles A. DiMarzio1-3

1The Center for Subsurface Sensing and Imaging Systems (CenSSIS)2Optical Science Laboratory, 3Dept. of ECE, 4Dept. of Biology, Northeastern University, Boston, MA

Acknowledgement: This work was supported in part by CenSSIS, the Center for Subsurface Sensing and Imaging Systems, under the Engineering Research Centers Program of the National Science Foundation (Award Number EEC 9986821).

References

Preliminary Results

State of the Art

Since 1978 the use of in vitro fertilization (IVF) procedures has resulted in the birth of over two million babies1,2, yet the records show that the IVF procedure has a successful pregnancy rate of only 30-40%3,4. Of concern is the fact that, of these successes, 20-40% result in multiple pregnancies3-7, which are directly attributable to the transfer of multiple embryos8 by the clinician to increase the probability of including a single, healthy embryo. The predominantly accepted viability markers for embryos created by IVF are 1) the rate of cell division and 2) the overall morphology of the embryo3,7,9-13. Because non-toxic imaging technologies cannot accurately determine the number of cells beyond the eight-cell stage, the clinician is forced to decide on the viability of the embryo solely on its overall morphology for Day 5 (blastocyst) transfers.

Centers presently use Differential Interference Contrast (DIC) microscopy to image the embryos non-invasively. This method shows distinct cell boundaries within the focal plane when multiple cells do not lie in the path to the microscope objective. Since an embryo is optically transparent, some cell edges can be seen when a couple of cells are in the path to the objective, allowing two layers of four cells to be imaged. For this reason accurate cell counts cannot be produced past the eight cell stage and therefore cannot be a decisive factor for a high quality embryo after the eight cell stage.

Hoechst Stain This Method

Sample 1 8 cells, 1 polar body 8 cells, 2 polar bodiesb

Sample 2 12 cells, 1 polar body 12 cells, 1 polar bodyb

Sample 3 13 cells 13 cellsk

Sample 4 14 cells 14 cellsk

Sample 5 15 cells, 2 polar bodies 15 cellsb

Sample 6 16 cells 16 cellsk

Sample 7 17 cells 17 cells, 1 polar bodyk

Sample 8 21 cells, 2 polar bodies 21 cells, 2 polar bodiesb

Sample 9 25 cells 25 cellsk

Sample 10 26 cells 26 cells, 1 polar bodyb

b Blind Countk Number of cells known before the cell count was completed.

Figure 2: (a) Line selected to plot on Optical Quadrature image. (b) Plotted optical path length deviation from selected line

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Optical Path Length Deviation (m)

0.40.60.811.21.41.61.822.22.4

Cell Overlap

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Culture Medium

Zona Pellucida

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Figure 1: (a) Optical Quadrature and (b) DIC Images

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Figure 3: (a) Elliptical boundary plotted on the DIC image by selecting points for the center point, minimum radius, and maximum radius. (b) Ellipsoidal model cell created by

combining the elliptical boundary with the optical path length deviation of a single cell.

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Figure 4: (a) Optical Quadrature image after subtraction of the model cell. (b) Optical Quadrature image after all cells have been subtracted.

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