組織學標本對於研究生物組織的微觀解剖和辨別組織中的疾病（組織病理學）至關重要。組織病理學常用的組織學製備方法有冷凍切片、固定和石蠟包埋三種。組織製備方法將不可避免地改變組織標本的內容。因此，探討製備方法對組織內容的影響勢在必行。

本論文旨在評估不同的標本製備方法將如何影響標本的組織形態、生物力學特性和化學成分。此研究使用椎間盤 (IVD) 組織，研究結果對其他軟組織也有重要參考價值。此研究採用奈米壓痕結合拉曼光譜來研究生物力學特性和化學成分。這些特性通過以下組織學標本製備方法進行探討：新鮮冷凍切片（對照組）；固定冷凍切片，石蠟包埋。

結果表明，用光學顯微鏡可以清晰分辨椎間盤組織中的髓核（NP）和纖維環（AF）。石蠟包埋的樣品顯示了最清晰的細節，尤其可以識別纖維環的薄片結構。組織形態的差異顯示組織製備影響樣品的微觀結構，同時還顯示組織製備影響組織的化學成分。樣品製備過程主要改變蛋白質並減少組織中的一些有機物含量，其中石蠟包埋的樣品比其他的樣品具有更顯著的變化。在生物力學性能方面，新鮮和固定樣品的冷凍切片之間沒有顯著差異（p>0.05）;相比之下，新鮮冷凍切片和石蠟包埋樣品顯示出明顯差異 (p<0.05)。總之，與石蠟包埋樣品相比，新鮮冷凍切片和固定冷凍切片樣品更適合用於組織學樣品中的生物力學評估。這項研究也提供研究組織樣品的機械性能和化學成分的重要參考，特別是對於椎間盤組織。

A histological specimen is essential to study the microscopic anatomy of biological tissues and identify disease in the tissues (histopathology). There are three types of histological preparation commonly used for histopathology including cryosection, fixation, and paraffin-embedded. A tissue preparation method will inevitably alter the tissue content. Therefore, it is imperative to investigate the impact of the preparation method on the tissue content.

This thesis aims to evaluate how different sample preparation methods will affect the tissue morphology, biomechanical properties, and chemical composition of samples. The study focused on the intervertebral disc (IVD) tissue and can be the further implication for other soft tissues. The study employed nanoindentation in conjunction with Raman spectroscopy to investigate the chemical composition and biomechanical properties. These properties were examined through the following histological specimen types: fresh cryosection (control); fixed cryosection, and paraffin-embedded.

The result showed that The IVD tissue could be localized using the optical microscope and the nucleus pulposus (NP) and annulus fibrosus (AF) can be seen. Paraffin-embedded samples showed the clearest details where the lamellae structure of AF could be identified. The difference in tissue morphology indicated the tissue preparations affect the microstructure of the samples. It was also found that the tissue preparations affected the chemical content of the tissues. Sample preparation was proved to alter mostly the protein and reduce some organic content of the tissues. Paraffin-embedded was found to have more significant changes among the other preparation. In terms of biomechanical properties, there was no significant difference between the cryosection of the fresh and fixed sample (p>0.05). In contrast, the fresh cryosection and paraffin-embedded samples showed a significant difference (p<0.05). In conclusion, fresh cryosection and fixed cryosection samples are more promising to work with for biomechanical assessment in histological tissue due to less manipulation compared to the paraffin-embedded sample. Moreover, the findings provided in this work address an essential gap in the literature by providing valuable insight into the mechanical characteristics of IVD at the nanoscale. This study also can become a reference for a better approach to check the mechanical properties and chemical content of tissues, especially for IVD tissues.

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