A major challenge for fluorescence imaging of living mammalian cells is maintaining viability following prolonged exposure to excitation illumination. We have monitored the dynamics of mitochondrial distribution in hamster embryos at frequent intervals over 24 h using two-photon microscopy (1,047 nm) while maintaining blastocyst, and even fetal, developmental competence. In contrast, confocal imaging for only 8 h inhibits development, even without fluorophore excitation. Photo-induced production of H 2 O 2 may account, in part, for this inhibition. Thus, twophoton microscopy, but not confocal microscopy, has permitted long-term fluorescence observations of the dynamics of three-dimensional cytoarchitecture in highly photosensitive specimens such as mammalian embryos.Keywords two-photon microscopy; laser scanning confocal microscopy; live cell fluorescence imaging; embryo; mitochondrial dynamics; mammal; hamster The detection of specific cellular components by imaging techniques such as wide-field epifluorescence or laser scanning confocal microscopy (LSCM) requires exposure to high intensity light that can cause cellular damage 1 . Consequently, the quantity or quality of images that can be collected is limited or, even worse, the reliability of the images may be compromised. This is a particular problem when imaging events that occur over periods of time ranging from hours to days, such as embryonic development. For this reason, much of our current understanding of subcellular morphological changes during mammalian embryonic development is based on images of fixed or static specimens at different developmental stages [2][3][4][5][6] . Thus, it can be difficult to interpret dynamic processes accurately, because the continuity of events must be inferred. The establishment of long-term fluorescence imaging methods that maintain the viability of live specimens is critical for advancing our understanding of cell biology and embryonic development in areas such as ion dynamics 7 , cytoplasmic reorganization, compaction and blastocoel formation, embryonic development in exotic species (where specimens are heterogeneous and difficult * Corresponding author (jsquirre@students.wisc.edu). to obtain), use of fluorescent tags for the preselection of embryos for subsequent embryo transfer 8 , and studies of protein expression in living cells using green fluorescent protein 9 . HHS Public AccessEmbryos of some mammals, particular hamsters, are very sensitive to culture conditions 10 . Furthermore, studies suggest that mammalian oocytes and embryos are adversely affected by exposure to visible light [11][12][13] . Because of this sensitivity, mammalian embryos are ideal to test live-cell imaging techniques. In addition, there are obvious morphological changes associated with differentiation, namely compaction and blastocoel formation, which can be used to assess viability. The embryo must undergo cell division during and after imaging as well as maintain a level of developmental competence that allows it to initiate dif...

In many organisms, cortical granules undergo exocytosis following fertilization, releasing cargo proteins that modify the extracellular covering of the zygote. We identified cortical granules in Caenorhabditis elegans and have found that degranulation occurs in a wave that initiates in the vicinity of the meiotic spindle during anaphase I. Previous studies identified genes that confer an embryonic osmotic sensitivity phenotype,thought to result from abnormal eggshell formation. Many of these genes are components of the cell cycle machinery. When we suppressed expression of several of these genes by RNAi, we observed that cortical granule trafficking was disrupted and the eggshell did not form properly. We conclude that osmotic sensitivity phenotypes occur because of defects in trafficking of cortical granules and the subsequent formation of an impermeable eggshell. We identified separase as a key cell cycle component that is required for degranulation. Separase localized to cortically located filamentous structures in prometaphase I upon oocyte maturation. After fertilization, separase disappeared from these structures and appeared on cortical granules by anaphase I. RNAi of sep-1 inhibited degranulation in addition to causing extensive chromosomal segregation failures. Although the temperature-sensitive sep-1(e2406) allele exhibited similar inhibition of degranulation, it had minimal effects on chromosome segregation. These observations lead us to speculate that SEP-1 has two separable yet coordinated functions: to regulate cortical granule exocytosis and to mediate chromosome separation.

Summary Several studies have reported reprogramming of fibroblasts to induced cardiomyocytes; however, reprogramming to proliferative induced cardiac progenitor cells (iCPCs) remains to be accomplished. Here we report that a combination of eleven or five cardiac factors along with canonical Wnt and JAK/STAT signaling reprogrammed adult cardiac, lung and tail-tip fibroblasts into iCPCs. The iCPCs were cardiac mesoderm-restricted progenitors, which could be extensively expanded while maintaining multipotency to differentiate into cardiomyocytes, smooth muscle cells and endothelial cells in vitro. Moreover, iCPCs injected into the cardiac crescent of mouse embryos differentiated into cardiomyocytes. iCPCs transplanted into the post-myocardial infarction mouse heart improved survival and differentiated into cardiomyocytes, smooth muscle cells and endothelial cells. Lineage reprogramming of adult somatic cells into iCPCs provides a scalable cell source for drug discovery, disease modeling, and cardiac regenerative therapy.

The endoplasmic reticulum (ER) is the major intracellular membrane system. The ER is essential for protein and lipid biosynthesis, transport of proteins along the secretory pathway, and calcium storage. Here, we describe our investigations into the dynamics and regulation of the ER in the early Caenorhabditis elegans embryo. Using a GFP fusion to the ER-resident signal peptidase SP12, we observed the morphological transitions of the ER through fertilization and the early cell-cycles in living embryos. These transitions were tightly coordinated with the division cycle: upon onset of mitosis, the ER formed structured sheets that redispersed at the initiation of cleavage. Although microtubules were not required for the transition of the ER between these different states, the actin cytoskeleton facilitated the dispersal of the ER at the end of mitosis. The ER had an asymmetric distribution in the early embryo, which was dependent on the establishment of polarity by the PAR proteins. The small GTPase ARF-1 played an essential role in the ER dynamics, although this function appeared to be unrelated to the role of ARF-1 in vesicular traffic. In addition, the ER-resident heat shock protein BiP and a homologue of the AAA ATPase Cdc48/p97 were found to be crucial for the ER transitions. Both proteins have been implicated in homotypic ER membrane fusion. We provide evidence that homotypic membrane fusion is required to form the sheet structure in the early embryo.

This study characterizes the morphologic features and the endogenous fluorescence in the stratified squamous epithelia of the 7,12-dimethylbenz(a)anthracene-treated hamster cheek pouch model of carcinogenesis using multiphoton laser scanning microscopy (MPLSM). MPLSM allows highresolution, three-dimensional image data to be collected deeper within thick tissue samples with reduced phototoxicity compared with single-photon imaging. Three-dimensional image stacks of normal (n = 13), precancerous (dysplasia, n = 12; carcinoma in situ, n = 9) and cancerous tissue [nonpapillary squamous cell carcinoma (SCC), n = 10, and papillary SCC, n = 7] sites in the hamster cheek pouch were collected in viable, unsectioned tissue biopsies at a twophoton excitation wavelength of 780 nm. Five features were quantified from the MPLSM images. These included nuclear density versus depth, keratin layer thickness, epithelial thickness, and the fluorescence per voxel in the keratin and epithelial layers. Statistically significant differences in all five features were found between normal and both precancerous and cancerous tissues. The only exception to this was a lack of statistically significant differences in the keratin fluorescence between normal tissues and papillary SCCs. Statistically significant differences were also observed in the epithelial thickness of dysplasia and carcinoma in situ, and in the keratin layer thickness of dysplasia and SCCs (both nonpapillary and papillary). This work clearly shows that three-dimensional images from MPLSM of endogenous tissue fluorescence can effectively distinguish between normal, precancerous, and cancerous epithelial tissues. This study provides the groundwork for further exploration into the application of multiphoton fluorescence endoscopy in a clinical setting. (Cancer Res 2005; 65(4): 1180-6)