Real-time monitoring the variation of chlorophyll distributions and cellular structures in leaves during plant growth provides important information for understanding the physiological statuses of plants. Two-photon-excited autofluorescence imaging and second harmonic generation imaging of leaves can be used for monitoring the nature intrinsic fluorophores distribution and cellular structures of leaves by the use of the near-infrared region of light which has minimal light absorption by endogenous molecules and thus increases tissue penetration. However, the two-photon absorption peak of intrinsic fluorophores of a ficus benjamina leaf is 50 nm away from the second harmonic generation excitation wavelength, which cannot be effectively excited by a femtosecond laser beam with one central wavelength. This paper shows that a highly polarized supercontinuum light generated from a birefringent nonlinear photonic crystal fiber with two zero-dispersion wavelengths can effectively excite two-photon autofluorescence as well as second harmonic generation signals for simultaneously monitoring intrinsic fluorophore distributions and non-centrosymmetric structures of leaves.
Near-infrared laser-based microsurgery is promising for noninvasive cancer treatment. To make it a safe technique, a therapeutic process should be controllable and energy efficient, which requires the cancer cells to be identifiable and observable. In this work, for the first time we use a miniaturized nonlinear optical endomicroscope to achieve microtreatment of cancer cells labeled with gold nanorods. Due to the high two-photon-excited photoluminescence of gold nanorods, HeLa cells inside a tissue phantom up to 250 µm deep can be imaged by the nonlinear optical endomicroscope. This facilitates microsurgery of selected cancer cells by inducing instant damage through the necrosis process, or by stopping cell proliferation through the apoptosis process. The results indicate that a combination of nonlinear endomicroscopy with gold nanoparticles is potentially viable for minimally invasive cancer treatment.
Goblet cells are a requirement for the diagnosis of intestinal metaplasia of the stomach. The gastric mucosa is lined by a monolayer of columnar epithelium with some specialization at the crypts, but there are no goblet cells in normal gastric epithelium. The appearance of goblet cells in gastric epithelium is an indicator of potential malignant progression toward adenocarcinoma. Therefore, in vivo three-dimensional imaging of goblet cells is essential for diagnoses of a premalignant stage of gastric cancers called intestinal metaplasia. We used mouse intestine, which has goblet cells, as a model of intestinal metaplasia. One-photon confocal fluorescence endomicroscopy and two-photon fluorescence endomicroscopy are employed for 3-D imaging of goblet cells. The penetration depth, the sectioning ability, and the photobleaching information of imaging are demonstrated. Both endomicroscopy techniques can three-dimensionally observe goblet cells in mouse large intestine and achieve an imaging depth of 176 µm. The two-photon fluorescence endomicroscopy shows higher resolution and contrast of the imaging sections at each depth. In addition, two-photon fluorescence endomicroscopy also has advantages of sectioning ability and less photobleaching. These results prove that two-photon fluorescence endomicroscopy is advantageous in diagnoses of a premalignant stage of gastric cancers.
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