A simple cleaning and fluorescent staining protocol for recent and fossil diatom frustules

Abstract

The analysis of diatom frustules by fluorescence microscopy and, in particular, confocal laser scanning microscopy requires adequate cleaning and staining methods prior to imaging. Here, a set of tools for an imaging procedure for fossil and living diatoms is presented. A simple and inexpensive cleaning protocol and four easily synthesized fluorescent dyes will give diatomists the possibility of analysing mostly organic-free diatom frustules at different wavelengths. The cleaning of cultured diatoms can easily be conducted at ambient temperature using commercially available bleaches based on hypochlorite solutions. This method quickly leads to clean and stainable frustules. Besides the known fluorescent dye, fluorescein isothiocyanate–(3-aminopropyl)trimethoxysilane (FITC–APS), three related new dyes, rhodamine B isothiocyanate–(3-aminopropyl)trimethoxysilane (RBITC–APS), eosin 5-isothiocyanate–(3-aminopropyl)trimethoxysilane (EITC–APS) and tetramethylrhodamine B isothiocyanate–(3-aminopropyl)trimethoxysilane (TRITC–APS) are introduced. This protocol is suitable for confocal laser scanning microscopy and the creation of three-dimensional models of diatom frustules for palaeontologists and biologists alike, allowing detailed morphological studies.

Keywords:

• CLSM
• fluorescence dye
• 3D imaging
• cleaning procedure

Acknowledgements

I wish to thank Rocky Torgrimson of Dicalite Minerals Corp. for the diatomite samples and Friedel Hinz for SEM support. This work was funded by the virtual institute ‘PlanktonTech’ of the Helmholtz Society.