Phase subtraction cell counting method

#Present imaging techniques for living cells and tissues are unable to produce accurate cell counts in cell clusters such as tissue samples and developing embryos past about eight cells in an aggregate, or past the eight cell stage of a living embryo non-invasively. Accurate cell counts of living embryos is important in the process of in vitro fertilization (IVF). In order to increase the success rate of IVF, doctors transfer multiple embryos back into the mother, with the transfer of three embryos having long been the standard. However, studies have shown that the transfer of two embryos does not decrease the overall ongoing pregnancy rate so there is a slow change toward reducing the standard. Even with the transfer of two embryos many IVF clinics report an overall pregnancy rate of only 30-40% (CDC, 2004; Gerris et al., 2002), with 20-40% of these successful births resulting in multiple pregnancies (Gerris et al., 2002; De Neubourg et al., 2002; ESHRE, 2001; Martin & Park, 1999). Multiple pregnancies increase the risks of cerebral palsy, preterm delivery, low birth weight, congenital malformations, and infant death (Gardner et al., 1995; Guyer et al., 1997; Kiely et al., 1998; Kinzler et al., 2000; Pharoah et al., 1996; Senat et al., 1998; Spellacy et al., 1990; Stromberg et al., 2002). Therefore, there is a worldwide effort to identify one healthy embryo for transfer back to the mother (Gurgan & Demirol, 2004).

#One reason for the low success rate of IVF is the uncertainty of knowing whether an embryo is healthy enough to produce a successful pregnancy. To assess the quality of the embryo during the preimplantation period, the number of cells and the overall morphology of the embryo are recorded over the observation period (De Neubourg et al., 2002). Developments in embryo culture systems have allowed for viable embryos to develop in vitro into the blastocyst stage, consisting of more than 30 cells in a mouse embryo, on day 5 or 6 (Gardner & Lane, 1997). With the extended observation time, better decisions can be made on the quality of the embryo, and slightly better percentages of pregnancies have been recorded.

#The rate of cell development during the one to eight cell stage is an important criterion for choosing a high quality embryo. However, available imaging technology makes obtaining this information non-invasively impossible past the eight cell stage. Fertility centers presently use differential interference contrast (DIC) microscopy to image 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. Some cell edges can be detected when only a couple cells overlap because embryos are optically transparent, allowing two layers of four cells to be imaged. However, cell boundaries cannot be detected for multiple layers of cells, making it impossible to produce accurate cell counts past the eight cell stage with DIC. As a result of insufficient imaging capabilities, the criteria for a high quality embryo at the blastocyst stage are primarily based on the morphology of the sample and the rate of development. Therefore, there is a need for an instrument that can non-invasively count the number of cells during embryo development up to the blastocyst stage in order to help physicians make better determinations regarding embryo quality. The availability of such an instrument would increase the success of single embryo transfer and reduce the motivation for multiple embryo transfers.

#FIG. 1 shows a flow chart depicting a method of cell counting according to the invention.

#FIG. 2 shows a Mach-Zehnder interferometer configuration for optical quadrature microscopy.

#FIG. 3 shows the selection of points along the perimeter of a mouse embryo used to register the DIC image to the optical quadrature image. FIG. 3A (left) shows the optical quadrature image. FIG. 3B (right) shows the DIC image.

#An accurate and non-invasive method and device for counting cells in a tissue sample or a cluster of cells, such as a living embryo, has been developed. The method involves a combination of a first imaging modality providing three-dimensional structural information of an unstained tissue specimen, such as optical quadrature microscopy (OQM), and a second imaging modality that provides easily detectable cell boundaries, such as differential interference contrast (DIC) microscopy. The method produces accurate cell counts of live embryos at the blastomere or morula stage ranging from 2 to at least 30 cells in a mouse embryo, and therefore is suitable for use in fertility clinics or livestock production facilities practicing in vitro fertilization. The individual steps of the cell counting method can be partially or fully automated, such that minimal or no operator input is required.

#A preferred first imaging modality is OQM. Alternatives to OQM as the first imaging modality include a variety of full-field phase imaging techniques, such as polarization interferometry (A. A. Lebedeff et al., “Polarization interferometer and its applications,” Rev. Opt. 9, p. 385, 1930), digital holography (see, e.g., E. Cuche et al., “Digital holography for quantitative phase-contrast imaging,” Optics Letters 24, pp. 291-293, 1999; F. Charriere et al., “Cell refractive index tomography by digital holographic microscopy,” Optics Letters 31, pp. 178-180, 2006; C. J. Mann, et al. “High-resolution quantitative phase-contrast microscopy by digital holography”, Optics Express, 13, pp. 8693-8698, 2005; P. Ferraro, et al. “Quantitative phase-contrast microscopy by a lateral shear approach to digital holographic image reconstruction”, Optics Letters, 31, pp. 1405-1407, 2006), Fourier phase microscopy (G. Popescu et al., “Fourier phase microscopy for investigation of biological structures and dynamics,” Optics Letters 29, pp. 2503-2505, 2004), Hilbert phase microscopy (T. Ikeda et al., “Hilbert phase microscopy for investigating fast dynamics in transparent systems,” Optics Letters 30, pp. 1165-1167, 2005), phase shifting interferometry (Guo & Devaney, 2004; I. Yamaguchi et al., “Phase-shifting digital holography,” Optics Letters 22, pp. 1268-1270, 1997), quantitative phase microscopy (A. Dubois et al., “Sinusoidally phase-modulated interference microscope for high-speed high-resolution tomographic imagery,” Optics Letters 26, pp. 1873-1875, 2001), Jamin-Lebedeff interferometry (Francon & Mallick, 1971; Spencer, 1982; Slayter & Slayter, 1992), and other interferometry techniques (see, e.g., C. J. Schwarz, et al. “Imaging interferometric microscopy”, Optics Letters, 28, pp 1424-1426, 2003; G. Popesku, et al., “Diffraction phase microscopy for quantifying cell structure and dynamics”, Optics Letters, 31, pp. 775-777, 2006; and M. Shribak and S. Inoue, “Orientation-independent differential interference contrast microscopy,” Appl. Opt. 45, 460-469, 2006).

#OQM is an interferometric imaging modality that measures the amplitude and phase of a light beam that travels through a cell or tissue sample, such as an embryo. The phase is transformed into an image of optical path difference, or optical path length deviation (OPD), which is based on differences in refractive index caused by cellular components. Thus, an OPD image is a map of a cell or tissue based largely on differences in refractive index. An OPD image provides three-dimensional information concerning the structures within a cell and/or the arrangement of cells within a cell cluster, such as a piece of tissue or an embryo.