PMID- 21515722
OWN - NLM
STAT- MEDLINE
DCOM- 20110912
LR  - 20211020
IS  - 1098-5336 (Electronic)
IS  - 0099-2240 (Print)
IS  - 0099-2240 (Linking)
VI  - 77
IP  - 12
DP  - 2011 Jun
TI  - Direct image-based correlative microscopy technique for coupling identification 
      and structural investigation of bacterial symbionts associated with metazoans.
PG  - 4172-9
LID - 10.1128/AEM.02461-10 [doi]
AB  - Coupling prokaryote identification with ultrastructural investigation of 
      bacterial communities has proven difficult in environmental samples. Prokaryotes 
      can be identified by using specific probes and fluorescence in situ hybridization 
      (FISH), but resolution achieved by light microscopes does not allow 
      ultrastructural investigation. In the case of symbioses involving bacteria 
      associated with metazoan tissues, FISH-based studies often indicate the 
      co-occurrence of several bacterial types within a single host species. The 
      ultrastructure is then relevant to address host and bacterial morphology and the 
      intra- or extracellular localization of symbionts. A simple protocol for 
      correlative light and electron microscopy (CLEM) is presented here which allows 
      FISH-based identification of specific 16S rRNA phylotypes and transmission 
      electron microscopy to be performed on a same sample. Image analysis tools are 
      provided to superimpose images obtained and generate overlays. This procedure has 
      been applied to two symbiont-bearing metazoans, namely, aphids and deep-sea 
      mussels. The FISH protocol was modified to take into account constraints 
      associated with the use of electron microscopy grids, and intense and specific 
      signals were obtained. FISH signals were successfully overlaid with bacterial 
      morphotypes in aphids. We thus used the method to address the question of 
      symbiont morphology and localization in a deep-sea mussel. Signals from a type I 
      methanotroph-related phylotype were associated with morphotypes displaying the 
      stacked internal membranes typical for this group and three-dimensional electron 
      tomography was performed, confirming for the first time the correspondence 
      between morphology and phylotype. CLEM is thus feasible and reliable and could 
      emerge as a potent tool for the study of prokaryotic communities.
FAU - Halary, Sebastien
AU  - Halary S
AD  - Universite Pierre et Marie Curie, 4 Place Jussieu, 75 005 Paris, France.
FAU - Duperron, Sebastien
AU  - Duperron S
FAU - Boudier, Thomas
AU  - Boudier T
LA  - eng
PT  - Evaluation Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20110422
PL  - United States
TA  - Appl Environ Microbiol
JT  - Applied and environmental microbiology
JID - 7605801
RN  - 0 (RNA, Bacterial)
RN  - 0 (RNA, Ribosomal, 16S)
SB  - IM
MH  - Animals
MH  - Aphids/microbiology
MH  - Bacteria/*cytology
MH  - *Bacterial Physiological Phenomena
MH  - Bivalvia/microbiology
MH  - Image Processing, Computer-Assisted/*methods
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Microscopy, Electron, Transmission/*methods
MH  - RNA, Bacterial/genetics
MH  - RNA, Ribosomal, 16S/genetics
MH  - *Symbiosis
PMC - PMC3131643
EDAT- 2011/04/26 06:00
MHDA- 2011/09/13 06:00
PMCR- 2011/12/01
CRDT- 2011/04/26 06:00
PHST- 2011/04/26 06:00 [entrez]
PHST- 2011/04/26 06:00 [pubmed]
PHST- 2011/09/13 06:00 [medline]
PHST- 2011/12/01 00:00 [pmc-release]
AID - AEM.02461-10 [pii]
AID - 2461-10 [pii]
AID - 10.1128/AEM.02461-10 [doi]
PST - ppublish
SO  - Appl Environ Microbiol. 2011 Jun;77(12):4172-9. doi: 10.1128/AEM.02461-10. Epub 
      2011 Apr 22.