DNA fragments in circulation released from apoptotic and necrotic cells were regarded as a novel prognostic or predictive biomarker for clinical diagnosis in recent years. However, DNA concentration in plasma ranged between 1 and 10 ng ml-1, which needed a single-molecule technology to analyze the base pair and concentration of DNA fragments. In this study, a series of different lengths of DNA fragments were studied, which showed that a good linear relationship between the DNA concentration and the molar concentration. The results suggested fluorescence correlation spectroscopy could access the nanomolar concentration of DNA labelled by SYBR Green I. Moreover, the relationship between the length of DNA fragment and the diffusion coefficient of DNA was scaled with the standard samples. The results demonstrates fluorescence correlation spectroscopy is a highly sensitive method for DNA detection.
The phosphatase and tensin homolog on chromosome 10 (PTEN) is one of important tumor suppressor proteins in ovarian cancer via negatively regulating the phosphatidylinositol 3-kinase–AKT signaling pathway and controlling genomic stability. Recent studies showed the physiological function of PTEN was closely related with its subcellular compartments. But only a few technologies could quantitatively measure the concentration of PTEN at different subcellular compartments in living cells. In this study, we used fluorescence correlation spectroscopy to measure the concentrations and dynamics of EGFP-PTEN in ovarian cancer cells HO-8910. Our results showed the increasing concentration of PTEN in the cytoplasm had an opposite trends with the nucleus after the oxidative stress stimulation which was induced by H2O2. Furthermore, the altered diffusion of PTEN at different subcellular compartments also illustrated the PTEN was trafficked from the cytoplasm to nucleus.
Biomechanics of cell plays an important role in the behavior and development of diseases, which has a profound influence on the health, structural integrity, and function of cells. In this study, we proposed a method to assess the biomechanical properties in single breast cancer cell line MCF-7 by combining structured illumination microscopy (SIM) with atomic force microscopy (AFM). High resolution optical image of actin in MCF-7 cell and its elastography were obtained. The result shows that the quantitative resolution was improved by SIM, with 490 nm of conventional fluorescence image and 285 nm of reconstructed SIM image, which could give a precise location for AFM measurement. The elasticity of actin is about in the range of 10~1000 kPa. The proposed methods will be helpful in the understanding and clinical diagnosis of diseases at single cell level.
Stimulated emission depletion (STED) optical nanoscopy can achieve super-resolution fluorescence imaging, by suppressing fluorescence on the peripheral of excitation center with a 0-2pi spiral phase plate. Previously, the STED intensity distribution at the focal plane and the derived expression of resolution are generally analytical described by vectorial integral. To overcome the complex and multifarious of the vectorial calculation, we proposed scalar integral method and used the Collins-Huygens integral to analytically describe the peak intensity, the central intensity of the doughnut spot and the resolution of STED nanoscopy. We verified our method by comparing our results with vector theory. And we found it agreed well with vectorial theory under the high STED power, which was commonly used experimental condition for high resolution. Our method provides a fast and convenient way to evaluate the performance of STED with spiral phase modulation.
The photophysical properties of a novel dendritic phthalocyanine di-{3,5-di-(4-methoxycarbonyl group benzyloxy)
benzyloxy) benzyloxy} axially substituted silicon (IV) phthalocyanine (DSiPc) were studied by UV/Vis, steady state and
time-resolved spectroscopic methods. The effect of dendritic structure on the photophysical properties and photoinduced
intermolecular electron transfer were investigated. The maximum absorption, fluorescence intensity, lifetime and
fluorescence quantum yield of DSiPc were greatly sensitized by the dendritic structure on the axially position of silicon
(IV) phthalocyanine. The photoinduced intermolecular electron transfer between this novel macromolecule and
benzoquinone (BQ) was studied. The results showed that the fluorescence emission of this dendritic phthalocyanine
could be quenched by BQ with KSV value of DSiPc is 52.84 dm3 mol-1. The cyclic voltammogram and square wave
voltammogram of DSiPc in DMF further evidenced the electron was transfer from DSiPc to BQ from thermodynamics.
Therefore, this novel dendritic phthalocyanine was an effective new electron donor and transmission complex could be
used as a potential artificial photosynthesis system.
Lipid rafts are cholesterol- and glycosphingolipids- enriched microdomains on plasma membrane surface of mammal cells, involved in a variety of cellular processes. Depleting cholesterol from the plasma membrane by drugs influences the trafficking of lipid raft markers. Optical imaging techniques are powerful tools to study lipid rafts in live cells due to its noninvasive feature. In this study, breast cancer cells MCF-7 were treated with different concentrations of MβCD to deplete cholesterol and an environmentally sensitive fluorescence probe, Laurdan was loaded to image lipid order by two-photon microscopy. The generalized polarization (GP) values were calculated to distinguish the lipid order and disorder phase. GP images and GP distributions of native and cholesterol-depleted MCF-7 cells were obtained. Our results suggest that even at low concentration (0.5 mM) of MβCD, the morphology of the MCF-7 cells changes. Small high GP areas (lipid order phase) decrease more rapidly than low GP areas (lipid disorder phase), indicating that lipid raft structure was altered more severely than nonraft domains. The data demonstrates that cholesterol dramatically affect raft coverage and plasma membrane fluidity in living cells.
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