Single-quantum dot imaging with a photon counting camera.

The expanding spectrum of applications of single-molecule fluorescence imaging ranges from fundamental in vitro studies of biomolecular activity to tracking of receptors in live cells. The success of these assays has relied on progress in organic and non-organic fluorescent probe developments as well as improvements in the sensitivity of light detectors. We describe a new type of detector developed with the specific goal of ultra-sensitive single-molecule imaging. It is a wide-field, photon-counting detector providing high temporal and high spatial resolution information for each incoming photon. It can be used as a standard low-light level camera, but also allows access to a lot more information, such as fluorescence lifetime and spatio-temporal correlations. We illustrate the single-molecule imaging performance of our current prototype using quantum dots and discuss on-going and future developments of this detector.

Expression of mitochondrial transcription factor A (TFAM) during porcine gametogenesis and preimplantation embryo development.

Mitochondrial transcription factor A (TFAM) is responsible for stability, maintenance, and transcriptional control of mitochondrial DNA (mtDNA). We have studied the expression and distribution of TFAM in the gametes and preimplantation embryos of the domestic pig (Sus scrofa). We hypothesized that TFAM is not present in the boar sperm mitochondria to reduce the possibility of paternal mtDNA propagation in the progeny. In contrast, we anticipated that Tfam gene is expressed in a developmental stage-dependent manner in porcine oocytes and embryos. The appropriate TFAM band of 25 kDa was detected by Western blotting in ejaculated boar spermatozoa, as well as in porcine oocytes and zygotes. Boar sperm extracts also displayed several bands >25 kDa suggestive of post-translational modification by ubiquitination, confirmed by affinity purification of ubiquitinated proteins. TFAM immunoreactivity was relegated to the sperm tail principal piece and sperm head in fully differentiated spermatozoa. The content of Tfam mRNA increased considerably from the germinal vesicle to blastocyst stage and also between in vitro fertilized and cultured blastocysts compared to in vivo-derived blastocysts. TFAM protein accumulated in the oocytes during maturation and was reduced by proteolysis after fertilization. This pattern was not mirrored in parthenogenetically activated oocytes and zygotes reconstructed by SCNT, suggesting deviant processing of TFAM protein and transcript after oocyte/embryo manipulation. Thus, TFAM may exert a critical role in porcine gametogenesis and preimplantation embryo development. Altogether, our data on the role of TFAM in mitochondrial function and inheritance have broad implications for cell physiology and evolutionary biology.