| Since W. Denk et al. introduced the two-photon excitation method into fluorescence microscopy in 1990,there has been increasing interest in two-photon excitation fluorescence. Two-photon fluorescence microscopic imaging became the first widely used the two-photon technology in practice. Compared to traditional single-photon microscopic imaging, two-photon fluorescence imaging has many advantages.First of all, it uses near infrared light as the excitation source,on the one hand, which can increase the penetration depth of tissue, to solve the problem of depth imaging in biological tissue. On the other hand, it can reduce the tissue absorption of excitation light, which can help to obtain stronger fluorescence. Secondly, dark field imaging can be realized because of two-photon fluorescence wavelength away from the excitation wavelength. Thirdly, two-photon fluorescence can effectively avoid the photobleaching and photodamage. Fourthly, two-photon transition has highly selective excitation, which is beneficial for the research of imaging of specific substances in the biological tissue. Fifthly, two-photon imaging has high transverse and longitudinal resolutions. Because of its high spatial selectivity, it can also get high-definition three- dimensional images without the use of confocal pinhole. All of these make the design and the operation of the two-photon confocal laser scanning microscope easier .Sixthly, due to the tissue auto-fluorescence excitation wavelength over the 350-560nm range the interference of tissue auto-fluorescence can be avoided by expanding the excitation source can avoid. At last, it can avoid UV damage to the samples and the restriction of the ultraviolet components, and extend the observation time of biological samples in vivo .The advantages of two-photon fluorescence imaging promote the development of bio-imaging technology, and lead people to the passion of further research and exploration, for example, two-photon fluorescence probes, two-photon excitation photodynamic therapy and so on. But there is a difficult problem that we still lack of efficient two-photon absorption properties dyes. At present, most of dyes are still traditional single-photon absorption fluorescent material, their two-photon absorption cross section is small, fluorescence quantum efficiency is usually low, and they easily quench in water. Although the mark used in cells has been succeed, It is not suitable for real-time monitoring and observation to obtain the fluorescence imaging with high sensitivity in the living tissues or cells, and it is also not suitable for application of two-photon technology to achieve the desired therapeutic effect for certain diseases ,such as cancer, because of its low aggregation concentration in the cell and the weak fluorescence. Therefore,the lagging of two-photon materials has affected the competitiveness of the two-photon fluorescence microscopy over the confocal laser scanning microscope. And this hinders the development in depth such as the study of two-photon photodynamic therapy,which is stil at the inaitial stage. Now, we need urgently to develop a kind of fluorescent markers,which has larger two-photon absorption cross section, higher dye packing density with light stability and high sensitivity.Nanoparticles can enrich the dye with nano-size,so dye density of per unit volume with nanoparticles in target cell or tissue is much greater than the dye density with molecular. Mark with nanoparticle can obtain the high sensitivity and high quality fluorescent imaging. Based on this, we modify the structure of original classic two-photon materials DPA-DSB, and design a new organic molecular system DPA-TSB which possesses a large two-photon absorption cross section.Then, water-dispersed two-photon functional organic nanoparticles was prepared by reprecipitation method, aiming to increase active dye loading density and enhance sensitive bioimaging in the microscopic level. Taking the advantage of imaging depth in living cells with confocal laser scanning microscope, we made two-photon nanoparticles as a new type of fluorescent probes to observe its uptake by tumor cells and fluorescence imaging in tumor cells, to detect its cytotoxicity, and to invent a new method of early diagnosis and targeting treatment of malignant tumors.The results are as following:(1) Using reprecipitation method, we successfully prepared water-dispersed nanoparticles DPA-TSB without using any carrier or surfactant.(2) DPA-TSB nanoparticle solution has two-photon absorption properties, two-photon fluorescence can be seen by 800nm laser excitation.(3) It has strong stability, there is no visible precipitation when placing 6 months, moreover, particle morphology and its optical properties have no significant changes.(4) Nanoparticles with human gastric cancer cell SGC-7901 or human cervical cancer (Hela) cells were co-cultured for 6h, a large number of rich nanoparticles are visible in the cytoplasm and show specific two-photon fluorescence by confocal fluorescence microscope.(5) Cells and nanoparticles were co-cultured for 24h, there are no obvious morphological changes between staining cells and normal cells. This initial proves the biocompatibility of Nanoparticles and cells.(6) To select the appropriate concentration range, using the methods of WST-1 and LDH activity to detection of cell proliferation and damage of cell membrane, after different concentrations of DPA-TSB nanoparticles and cells were cultured in different time.In summary, DPA-TSB aqueous dispersed nanoparticles can be easily uptaked by tumor cells. In vitro, confocal microscopy has demonstrated that these nanoparticles are avidly internalized into cells with bright fluorescence. The good cell membrane permeability of the resulting two-photon functional DPA-TSB nanoparticles make them promising candidates for an efficent and stable bioimaging staining agent for the deeper tissue.Meanwhile, we preliminary evaluate the biological safety of such material by different cell toxicity detections.In addition, these results will give experimental evidence available to the further study, such as two-photon photodynamic therapy.Background: The incidence and mortality of gastric cancer ranks first in gastrointetinal neoplasm. Endoscopy is still the main way to make early diagnosis of gastrointetinal neoplasm. The confocal laser endomicroscope is a brand new technique, which integrate confocal microscope to the dital end of convetional endoscope. With exogenous fluorecein, the new device can provide real-time,high magnification,continuous cross-sectional images of gastrointestinal epithelium during routine endoscopy. The greatest advantage of the endoscopy its approximately 1000-fold magnification, which readily provides images of cell or even subcell structure. The real-time highly magnified images of the gastrointestinal tract mucosa permits a histological diagnosis during endoscopy without the need of biopsy and thus the technique has been termed"optical biopsy"and"cell CT". Several investigators have reported that the technique is of value. Some CLE diagnosis standard of Barrett's esophagus has been made acorrding to cell type, whether there is goblet cell or certain vascular pattern. The diagnosis sensitivity, specility and accuracy is 95%, 90% and 85%. The different pattern of intrapapillary capillary loop between normal eaphageal and eaophageal squamous cancer under CLE is obvious, and some researchers have reported that the diagnosis of esophageal squamous cancer can be made on this ground. CLE allows not only the the observation of gastric epithlium crypt but also the gastric intestinal metaplasia and inflammation of lamina propria layer. CLE permits optical biopsy of gastric mucosa and guides the target biopsy of suspected tumor.Objective: This experiment is designed to evaluate CLE in imaging of gastric cancer cell and to learn the images of gastric cancer or precancerous lesions. The agreement of diagonosis of gastric cancer between CLE and histopathology was assessed.Methods: Patients who were diagnosed gastric cancer by conventional endoscope underwent CLE. Then, biopsy the very same spot where the CLE scaned. The histological diagnosis was taken as the"golden standard". The agreement of diagonosis of gastric cancer between CLE and histopathology was assessed, and evaluated the traits of these images.Results: 13 patients diagnosed gastric cancer by traditional endoscopy underwent CLE, 104 sites from these patients'stamoch were scaned and biopsied, every patient was biopsied 7-10 sites. The total number of CLE images is 3993, 1758 of them are of good quality, 1816 of them are of normal quality, and 419 are of bad quality. 89 sites were diagnosed malignant by CLE. The kappa value is 0.78. 10 patients were diagnosed gastric cancer histologically.Conclusion: The agreement between the CLE and hisological diagnosis for gastric cancer is regarded as good. With CLE ,the real-time histological examination can be performed and repaeated noninvasively. CLE is a usful and potentially important method for the diagnosis of gastric cancer.
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