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High spatial resolution Raman maps of fixed cells in an aqueous environment are reported. These maps were obtained by collecting individual Raman spectra via a Raman microspectrometer in a raster pattern on a 0.5-μm grid and assembling pseudocolor maps from the spectral hypercubes by multivariate methods. The Raman maps show the nucleus and the nucleoli of cells as well as subcellular organization in the cytoplasm. In particular, the distribution of mitochondria in the perinuclear region could be demonstrated by correlating distinct areas of the Raman maps with corresponding areas of fluorescence maps of the same cells after staining with mitochondria-specific labels. To the best of our knowledge, this is the first report of label-free detection of mitochondria inside a somatic mammalian cell using Raman microspectroscopy.

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 eight-cell 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.

The mouse preimplantation embryo development (Ped) gene product, Qa-2, which is the homolog of human HLA-G, influences the rate of preimplantation embryonic development and overall reproductive success. The sex ratio in preimplantation embryos from Ped gene congenic mice was examined in order to determine whether embryo sex is a confounding factor in the control of the rate of preimplantation development. B6.K1 (Ped slow) and B6.K2 (Ped fast) congenic mice differ only in the absence (B6.K1) or presence (B6.K2) of the genes encoding Qa-2 protein. We analyzed the sex of B6.K1 (n=221) and B6.K2 (n=260) preimplantation embryos by using Real-Time PCR with primers specific for the X and Y chromosomes. We found that there was no statistically significant difference in the ratio of male to female preimplantation embryos in either strain. We conclude that the sex of the embryos is not a confounding factor that affects the Ped gene control of the rate of preimplantation development. Therefore, the Ped gene is entirely responsible for mediating the faster development of B6.K2 embryos compared to B6.K1 embryos.

[...]she's dressed like an old-time matriarch of another era, from her sensible shoes to the French roll in her snow-white hair.

The goal of this research was to study the role of mitochondrial activity and apoptosis in pluripotent stem (iPS) cell growth and differentiation. To accomplish this goal we defined four specific aims: (1) create and characterize an embryonic stem (ES) cell line from mtGFP-tg mice that have their mitochondria endogenously labeled with GFP, and then use the mtGFP-tg ES cell line to evaluate mitochondrial localization; (2) compare cell division, pluripotency markers, and differentiation markers in undifferentiated and differentiating mouse pluripotent stem cells by comparing the mtGFP-tg ES cell line, a conventional C57BL/6 ES cell line, and an iPS cell line; (3) analyze mitochondrial activity and localization in undifferentiated and differentiating mouse pluripotent stem cells by comparing the mtGFP-tg ES cell line, a conventional C57BL/6 ES cell line, and an iPS cell line; (4) analyze early, intermediate, and late stage apoptosis markers in undifferentiated and differentiating mouse pluripotent stem cells by comparing a conventional C57BL/6 ES cell line and an iPS cell line. We successfully created an ES cell line from mtGFP-tg mice. The mtGFP-tg ES cells had a normal karyotype, and they demonstrated characteristics of pluripotency and differentiation capabilities comparable to conventional ES cells. Their mitochondrial GFP fluorescence was extremely bright and stable, allowing us to image, for the first time, mitochondria in undifferentiated and differentiating cells without the use of externally applied mitochondrial stains. Our imaging data represent the first 3D sectioning through an EB, made possible by using endogenous mitochondrial fluorescence. Using our mtGFP-tg ES cell line in conjunction with a C57BL/6 ES and an iPS cell line, we confirmed that all three pluripotent stem cell lines had high levels of pluripotency markers in their undifferentiated state. Upon differentiation, the pluripotency markers decreased whereas markers for the three developing germ layers increased throughout differentiation. These data confirm the known characteristics of robust pluripotent stem cells. We analyzed mitochondrial activity in all three pluripotent stem cell lines by using JC-1 and TMRE staining. We found that pluripotent stem cells had high levels of mitochondrial activity in their undifferentiated state. Mitochondrial activity was confined to the perimeters of undifferentiated pluripotent stem cell colonies, an observation which has not been published previously. Upon differentiation, mitochondrial activity significantly decreased. Early, intermediate, and late apoptosis were assessed in C57BL/6 ES and iPS cells. The two pluripotent stem cell lines showed low levels of apoptosis in their undifferentiated state. Upon differentiation, apoptosis significantly increased. These data represent the first full analysis of mitochondrial activity and apoptosis throughout the differentiation process. Taken together, these results support a role for mitochondrial activity and apoptosis in pluripotent stem cell growth and differentiation. In particular, high mitochondrial activity is associated with pluripotency whereas apoptosis is associated with loss of pluripotency. To the best of our knowledge, these data provide the first analyses of mitochondrial activity and apoptosis in iPS cells. iPS cells were equivalent to conventional C57BL/6 ES cells in terms of pluripotency, mitochondrial activity, and apoptosis in their undifferentiated state. However, upon differentiation or long-term culture we found several significant differences in pluripotency, mitochondrial activity, and apoptosis thus bringing into question the complete equivalency of iPS cells to conventional ES cells. All together, we conclude from our analyses that there are significant differences in pluripotency, mitochondrial activity, and apoptosis during differentiation of pluripotent stem cells and that there are several significant differences among the mtGFP-tg, C57BL/6, and iPS pluripotent stem cell lines. These data increase our knowledge of the pluripotency and differentiation capabilities of pluripotent stem cells, which may impact the development of stem cell therapy for the treatment of human diseases. (Abstract shortened by UMI.)

More info muny.org; 314-534-1111 Judith Newmark is the Post-Dispatch's theater critic.

May 17--Lana Pepper, who directed a wry production of A.R. Gurney's \"Sylvia\" at Stray Dog Theatre in March, has again created theater for grown-ups.