Microscopic organisms that penetrate calcareous structures by actively dissolving the carbonate matrix, namely microendoliths, have an important influence on the breakdown of marine carbonates. The study of these microorganisms and the bioerosion traces they produce is crucial for understanding the impact of their bioeroding activity on the carbonate recycling in environments under global climate change. Traditionally, either the extracted microendoliths were studied by conventional microscopy or their traces were investigated using scanning electron microscopy (SEM) of epoxy resin casts. A visualisation of the microendoliths in situ, that is within their complex microbioerosion structures, was previously limited to the laborious and time-consuming double-inclusion cast-embedding technique. Here, we assess the applicability of various fluorescence staining methods in combination with confocal laser scanning microscopy (CLSM) for the study of fungal microendoliths in situ in partly translucent mollusc shells. Among the tested methods, specific staining with dyes against the DNA (nuclei) of the trace making organisms turned out to be a useful and reproducible approach. Bright and clearly delineated fluorescence signals of microendolithic nuclei allow, for instance, a differentiation between abandoned and still populated microborings. Furthermore, infiltrating the microborings with fluorescently stained resin seems to be of great capability for the visualisation and quantification of microbioerosion structures in their original spatial orientation. Potential fields of application are rapid assessments of endolithic bio- and ichnodiversity and the quantification of the impact of microendoliths on the overall calcium carbonate turnover. The method can be applied after CLSM of the stained microendoliths and retains the opportunity for a subsequent investigation of epoxy casts with SEM. This allows a three-fold approach in studying microendoliths in the context of their microborings, thereby fostering the integration of biological and ichnological aspects of microbial bioerosion.

中文翻译：

---

探索共聚焦激光扫描显微镜 (CLSM) 和荧光染色作为成像和量化海洋微生物侵蚀痕迹及其示踪微内石的工具

通过主动溶解碳酸盐基质（即微内石）来穿透钙质结构的微观生物对海洋碳酸盐的分解具有重要影响。研究这些微生物及其产生的生物侵蚀痕迹对于了解其生物侵蚀活动对全球气候变化环境中碳酸盐循环的影响至关重要。传统上，要么通过常规显微镜研究提取的微内石，要么使用环氧树脂铸件的扫描电子显微镜 (SEM) 研究它们的痕迹。原位微内石的可视化，即在其复杂的微生物侵蚀结构内，以前仅限于费力且耗时的双夹杂物铸造嵌入技术。这里，我们评估了各种荧光染色方法与共聚焦激光扫描显微镜 (CLSM) 相结合的适用性，用于研究部分半透明软体动物壳中的原位真菌微内石。在经过测试的方法中，用染料对示踪生物的 DNA（细胞核）进行特异性染色被证明是一种有用且可重复的方法。例如，微内石核的明亮而清晰的荧光信号允许区分废弃的和仍然存在的微孔。此外，用荧光染色树脂渗透微孔似乎对微生物侵蚀结构在其原始空间方向的可视化和量化具有很大的能力。潜在的应用领域是对内石生物和生物多样性的快速评估以及微内石对整体碳酸钙周转的影响的量化。该方法可以在染色的微内石 CLSM 之后应用, 并保留了随后用 SEM 研究环氧树脂铸件的机会。这允许在微孔的背景下研究微内石的三重方法，从而促进微生物生物侵蚀的生物学和工艺学方面的整合。