Multicolor nonlinear microscopy of living tissue using two-and three-photon-excited intrinsic fluorescence combined with second harmonic generation by supermolecular structures produces images with the resolution and detail of standard histology without the use of exogenous stains. Imaging of intrinsic indicators within tissue, such as nicotinamide adenine dinucleotide, retinol, indoleamines, and collagen provides crucial information for physiology and pathology. The efficient application of multiphoton microscopy to intrinsic imaging requires knowledge of the nonlinear optical properties of specific cell and tissue components. Here we compile and demonstrate applications involving a range of intrinsic molecules and molecular assemblies that enable direct visualization of tissue morphology, cell metabolism, and disease states such as Alzheimer's disease and cancer.M ultiphoton microscopy (MPM) (1, 2) is well suited for high-resolution imaging of intrinsic molecular signals in living specimens. It provides convenient excitation of the characteristic UV absorption bands of intrinsic fluorophores using IR illumination, leaving a broad uninterrupted spectral region for efficient multicolor fluorescence collection. The ability of MPM to produce images deep in optically thick preparations is crucial for intravital tissue microscopy. In addition, second harmonic generation (SHG) enables direct imaging (3) of anisotropic biological structures possessing large hyperpolarizabilities, such as collagen (4, 5). These imaging modalities are easy to implement simultaneously and differ only in optical filter selection and detector placement.To date, most biological MPM has depended on labeling with conventional fluorophores or fluorescent proteins such as the GFPs; however, a few studies have used two-photon excitation (2PE) of intrinsic molecules such as NAD(P)H (6-8) and flavins (9), three-photon excitation (3PE) of serotonin (10-12), and SHG of collagen, skeletal muscle, and microtubules (2, 13). The combination of intrinsic and extrinsic signals is particularly powerful. For example, the process of tumor cell migration along collagen fibers can be observed by using GFP-labeled tumor cells and intrinsic collagen SHG (14). 2PE fluorescence spectra currently exist for NAD(P)H and some flavins (9, 15), and 3PE spectra exist for serotonin, tryptophan, and dopamine (10). Here we report the SHG efficiency spectrum for various collagens and 2PE cross sections of a ''basis set'' of tissue 2PE fluorophores. We demonstrate m-resolution multiphoton imaging of normal tissue structure and of disease states such as Alzheimer's disease (AD) and cancer. Intrinsic emission MPM in living specimens yields detail that may ultimately prove useful to clinical diagnostics as well as to basic biological research. Materials and MethodsInstrumentation and the associated methodologies used in these investigations are described in detail in Supporting Materials and Methods, which is published as supporting information on the PNAS web site, www.pnas.org...

MicroRNAs (miRNA) are a recently discovered class of noncoding RNAs that negatively regulate gene expression. Recent evidence indicates that miRNAs may play an important role in cancer. However, the mechanism of their deregulation in neoplastic transformation has only begun to be understood. To elucidate the role of tumor suppressor p53 in regulation of miRNAs, we have analyzed changes in miRNA microarray expression profile immediately after conditional inactivation of p53 in primary mouse ovarian surface epithelium cells. Among the most significantly affected miRNAs were miR-34b and miR-34c, which were down-regulated 12-fold according to quantitative reverse transcription-PCR analysis. Computational promoter analysis of the mir-34b/mir-34c locus identified the presence of evolutionarily conserved p53 binding sites f3 kb upstream of the miRNA coding sequence. Consistent with evolutionary conservation, mir-34b/mir-34c were also down-regulated in p53 -null human ovarian carcinoma cells. Furthermore, as expected from p53 binding to the mir34b/c promoter, doxorubicin treatment of wild-type, but not p53-deficient, cells resulted in an increase of mir-34b/ mir-34c expression. Importantly, miR-34b and miR-34c cooperate in suppressing proliferation and soft-agar colony formation of neoplastic epithelial ovarian cells, in agreement with the partially overlapping spectrum of their predicted targets. Taken together, these results show the existence of a novel mechanism by which p53 suppresses such critical components of neoplastic growth as cell proliferation and adhesion-independent colony formation. [Cancer Res 2007; 67(18):8433-8]

Epithelial ovarian cancer (EOC) is the fifth-leading cause of cancer death among women in the United States, but its pathogenesis is poorly understood 1-3. Some epithelial cancers are known to occur in transitional zones between two types of epithelium, while others have been shown to originate in epithelial tissue stem cells 4-6. The stem cell niche of the ovarian surface epithelium (OSE), which is ruptured and regenerates during ovulation, has not yet been unequivocally defined. Here we identify the hilum region of the mouse ovary, the transitional/junction area between OSE, mesothelium and tubal (oviductal) epithelium as a previously unrecognized stem cell niche of the OSE. We find that cells of the hilum OSE are slowly-cycling and express stem/progenitor cell markers ALDH1, Lgr5, Lef1, CD133, and CK6b. These cells display long-term stem cell properties ex vivo and in vivo, as shown by our serial sphere generation and by long-term lineage tracing assays. Importantly, the hilum cells exhibit increased transformation potential after inactivation of tumour suppressor genes Trp53 and Rb1, whose pathways are frequently altered in the most aggressive and common type of human EOC, high-grade serous adenocarcinoma 7,8. Our study experimentally supports the notion that susceptibility of transitional zones to malignant transformation may be explained by the presence of stem cell niches in those areas. Identification of a stem cell niche for the OSE may have important implications for understanding EOC pathogenesis.