Exploring confocal laser scanning microscopy (CLSM) and fluorescence staining as a tool for imaging and quantifying traces of marine microbioerosion and their trace‐making microendoliths

Abstract

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 subsequentThis is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

54 years of microboring community history explored by machine learning in a massive coral from Mayotte (Indian Ocean)

“…The estimation of microborer filaments (Lukas, 1973;Priess et al, 2000) or traces (Chazottes et al, 1995;Carreiro-Silva et al, 2005) depends also on the observer, thus limiting comparisons of results. In the light of the new techniques developed recently by Salamon et al (2019) and Schätzle et al (2021) which combine specific fluorescent dyes and SEM or Confocal Laser Scanning Microscopy (CLSM) we believe that our method could be improved to quantify the abundance of a larger variety of microboring traces as here only two types were studied with the machine learning approach. Our method was limited by the grayscale SEM images reducing the ability of the CNN model to distinguish properly the various types of traces.…”

Microborings reveal alternating agitation, resting and sleeping stages of modern marine ooids

“…Furthermore, the analysis of microborings is a key tool in deciphering the microbiome of biogenic carbonates, such as fossil corals or mollusc shells (e.g. Salamon et al ., 2019; Schätzle et al ., 2021; Salamon & Kołodziej, 2022).…”