Fluorescence in situ hybridisation coupled to ultra small immunogold detection to identify prokaryotic cells using transmission and scanning electron microscopy.
We describe a method based on fluorescence in situ hybridisation (FISH) that allows the identification of individual cells by electron microscopy. We hybridised universal and specific fluorescein-labelled oligonucleotide probes to the ribosomal RNA of prokaryotic microorganisms in heterogeneous cell mixtures. We then used antibodies against fluorescein coupled to sub-nanometer gold particles to label the hybridised probes in the ribosome. After increasing the diameter of the metal particles by silver enhancement, the specific gold-silver signal was visualised by optical microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). It is the first time that SEM is applied to the detection of gold nanoparticles hybridised to an intracellular target, such as the ribosome. The possibility to couple phylogenetic identification by FISH to cell surface and ultrastructure observation at electron microscopy resolution has promising potential applications in microbial ecology.
Gérard
Emmanuelle
E
Laboratoire de Géosciences Marines, CNRS UMR 7097, Institut de Physique du Globe de Paris, 4 place Jussieu, 75005 Paris, France. emmanuelle.gerard@ese.u-psud.fr
Guyot
François
F
Philippot
Pascal
P
López-García
Purificación
P
D034301:Deinococcus / Q000302:isolation & purification; D004926:Escherichia coli / Q000302:isolation & purification; D005453:Fluorescence; D007089:Image Enhancement / Q000379:methods; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008828:Microbiological Techniques / Q000379:methods*; D008853:Microscopy; D008854:Microscopy, Electron / Q000379:methods*; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D015345:Oligonucleotide Probes; D012335:RNA, Ribosomal / Q000378:metabolism; D013045:Species Specificity
D015345:Oligonucleotide Probes; D012335:RNA, Ribosomal