| Summary: | 碩士 === 國立陽明大學 === 醫學工程研究所 === 100 === Background: There are four major types of the autofluorescent proteins, Collagen, Nicotineamide Adenine Dinucleotide, Flavin Adenine Dinucleotide Sodium, and Elastin. The collagen and elastin is almost found from connective tissues, and then the Nictineamid Adenine Dinucleotide, and Flavin Adenine Dinucleotide Sodium are always found from cells’ Tricarboxyic Acid Cycle.Each tissue is composed of different autofluorescent proteins. It could be a feasible to find the spectral differences for the purpose of tissue discrimination. In this study, autoflurorescences will be observed at the tissue and cell level.
Method: In tissue level, the technique of heart perfusion was used in the rats for tissue fixation. Fluorescence microscopy (Leica DM IRB) with excitation of 330~385nm, 470~495nm, 530~550nm band-pass lights were appied to skeletal muscle and ligamentum falvum. The wmission band filters were 400~440nm, 500~540nm, and 555~595nm correspindingly. Meanwhile, laser confocal microscopy with excitation wavelengths of 405nm, 488nm and 543nm also applied to these tissues. The emission band filter were 420~470nm, 500~550nm and 600~650nm, correspondingly. The sample size for each examination was n=3.
In cell level, primary cultured skeletal muscle cells and ligamentum falvum cells were excited by band-passed lights of 405±20, 488±20 ,and 543±20nm, and the emission were foltered by 430~680nm, 510~680nm, and 565~680nm correspondingly and the spectra were measured by Fluorescence Spectrophotometer (Hitachi F-4500). In addition to these, collagen (type I) and elastin were also level examined by flurorescence microscopy. The sample size was n=3 for each tissue.
Result: In the tissues level examined by flurorescence microscopy, skeletal muscle tissue can be excited in the bands of 470~495nm, and555~595nm. However, the ligamentum flavum tissues can be excited in the bands of 400~440nm and 555~595nm. In laser confocal microscopy, muscle tissue can generate weak flurorescence by 405nm and 488nm, but none for 543nm, However, for ligamentum flavum tissue, all of the wxcitation wavelenght can generate fluorescent light.
At the cell level, both ligamentum flavum cells, and skeletal cells nearly have no response in flurorescent light emission to these excitation wavelenghts ( 488±20nm). However , for collagen type I and elastin, have both strong flourescenct respomses to 405±20nm excitation excitation waceelenght at shorter emission band of 430~680nm. Where the flourescence intensity decrease rapidly for elastin but it decays gradually collagen type I. For the rest excitation bands, 488±20nm, and 543±20nm, there is nearly no flourescent response for elastin, but collagen type I has similar profiles of flourescent response at 510~680nm and 565~680nm as it has in 430~680nm but with relatively weaker intensity.
Conclusion: It is clear that both laser confocal microscopu examined at tissue level and cell level in flourescent response to the corresoinding excitation wavelenghts consistent. These results, indicate that the tissue of ligamentum flavum has much more collagen type I than that of muscle tissue.
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