Confocal laser scanning microscopy (CLSM) is commonly used to observe molecules of biological relevance in their native environment, the live cell, and study their spatial distribution and interactions nondestructively. CLSM can be easily extended to measure the lifetime of the excited state, the concentration and the diffusion properties of fluorescently labeled molecules, using fluorescence lifetime imaging microscopy (FLIM) and fluorescence correlation spectroscopy (FCS), respectively, in order to provide information about the local environment and the kinetics of molecular interaction in live cells. However, these parameters cannot be measured simultaneously using conventional CLSM due to damaging effects that are associated with strong illumination, including phototoxicity, photobleaching, and saturation of the fluorescence signal. To overcome these limitations, we have developed a new camera consisting of 1024 single-photon avalanche diodes that is optimized for multifocal microscopy, FLIM and FCS. We show proof-of-principle measurements of fluorescence intensity distribution and lifetime of the enhanced green fluorescent protein expressed in live cells and measurement of quantum dot diffusion in solution by FCS using the same detector.

Remodelling of collagen fibers has been described during every phase of cancer genesis and progression. Changes in morphology and organization of collagen fibers contribute to the formation of microenvironment that favors cancer progression and development of metastasis. However, there are only few data about remodelling of collagen fibers in healthy looking mucosa distant from the cancer. Using SHG imaging, electron microscopy and specialized softwares (CT-FIRE, CurveAlign and FiberFit), we objectively visualized and quantified changes in morphology and organization of collagen fibers and investigated possible causes of collagen remodelling (change in syntheses, degradation and collagen cross-linking) in the colon mucosa 10 cm and 20 cm away from the cancer in comparison with healthy mucosa. We showed that in the lamina propria this far from the colon cancer, there were changes in collagen architecture (width, straightness, alignment of collagen fibers and collagen molecules inside fibers), increased representation of myofibroblasts and increase expression of collagen-remodelling enzymes (LOX and MMP2). Thus, the changes in organization of collagen fibers, which were already described in the cancer microenvironment, also exist in the mucosa far from the cancer, but smaller in magnitude. Extracellular matrix (ECM) is no longer considered as an inert substrate, a three-dimensional network which only "fills the spaces" between cells and provide mechanical support 1,2. Today, ECM is known to be a complex and dynamic structure, whose chemical and biophysical properties affect cell adhesion 3 , proliferation 4 morphology 5 , migration 6 , regulate tissue morphogenesis 7,8 and fluid volume in tissues 9. The most abundant component of ECM in the lamina propria of the colon mucosa is type I collagen. Remodelling of collagen fibers has been described in almost every solid cancer, including colorectal cancer. During tumor formation and progression, collagen remodelling is constantly carried out: degradation, synthesis, cross-linking of fibers, change of fiber orientation, and interaction of cells of the innate and acquired immune system with collagen fibers 10,11. Changes in morphology, representation, and organization of collagen fibers contribute to the formation of the microenvironment that favors tumor progression, primarily through its effect on cell migration and polarization 12. Also, remodelling of collagen fibers on premetastatic sites is of great importance in determination of survival and growth of disseminated cancer cells, and thus, formation of metastasis 13,14. Remodelling of collagen fibers may be a result of changes in synthesis, degradation or cross-linking. Main cells responsible for synthesis of collagen in colon mucosa are fibroblasts and myofibroblasts. The most important enzymes for degradation of collagen fibers are matrix metalloproteinases (MMPs). It has been shown that expression of MMP2 and MMP9 is increased in colorectal cancer and influences its progression and

We present the Hanle EIT resonances obtained from the various segments of the Gaussian laser beam cross-section, selected by moving the small aperture (placed in front of the detector) radially along the laser beam. Significant differences in the Hanle lineshapes are observed depending on whether the central or outer parts of the Gaussian laser beam are detected. The line narrowing and two counter-sign peaks occur at outer, less intense parts of the beam. The theoretical model suggests that the EIT lineshapes in the laser wings are result of the interference of the laser light and coherently prepared atoms coming from the central part of the beam. By blocking the central part of the laser beam in front of the detector, narrower, and for high laser intensities, even more contrasted Hanle resonances are obtained.

We performed a study of the nonlinear optical properties of chemically purified chitin and insect cuticle using two-photon excited autofluorescence (TPEF) and second-harmonic generation (SHG) microscopy. Excitation spectrum, fluorescence time, polarization sensitivity, and bleaching speed were measured. We have found that the maximum autofluorescence signal requires an excitation wavelength below 850 nm. At longer wavelengths, we were able to penetrate more than 150-um deep into the sample through the chitinous structures. The excitation power was kept below 10 mW (at the sample) in order to diminish bleaching. The SHG from the purified chitin was confirmed by spectral- and time-resolved measurements. Two cave-dwelling, depigmented, insect species were analyzed and three-dimensional images of the cuticular structures were obtained.