| dc.description.abstract | The development of biomedical engineering has gradually improved, and molecular imaging has been one of its most important techniques. To solve the limitation of a conventional microscope that merely detects the morphology of samples, we’ve combined hyperspectral imaging with two photon fluorescence microscopy and designed the setup of two photon fluorescence hyperspectral microscopy. This neoteric technique includes the advantages of both microscopies, providing deeper depth penetration, less photo damage to sample, higher axial resolution, and the addition of receiving spectral information of the targeted sample simultaneously. With these properties, it’s more suitable for biomedical research in vivo, it also provides more information of the samples to researchers, and reduces the misjudgment probabilities of researches that are dependent on the morphology.
Using the previously developed system, a non-de-scanned two-photon fluorescence hyperspectral microscope with parallel recording, experiments of cells and sectioning skin samples were carried out to test the efficiency of the system. Furthermore, the major aim is to try to actually apply this microscopy to biological researches and to overcome the limitation of multiple fluorescence labeling samples. Multispectral images might generate crosstalk and increase the difficulty of fluorescence discrimination. Therefore, we applied the applicably spectral analysis, linear unmixing, to improve the spectral discriminating ability.
In the biological research, there were two parts of experiments. In the first part, cultured cell lines were labeled with multiple fluorescence dyes and the hyperspectral images of these cells were analyzed to show the ability and correctness of linear unmixing. In the second part, mouse skin tissues with hair follicles being fluorescently labeled were used as samples. According to the experimental results and the spectral analyses, the fluorescence and second harmonic generation signals was easily separated. The unexpected signal sources, second harmonic generation, can be removed to avoid influencing doctor’s diagnoses. The two-photon hyperspectral imaging combined with linear unmixing has shown its ability to biomedical fields. | en_US |