Details
| Original language | English |
|---|---|
| Pages (from-to) | 112-118 |
| Number of pages | 7 |
| Journal | CHEMOSPHERE |
| Volume | 161 |
| Early online date | 13 Jul 2016 |
| Publication status | Published - Oct 2016 |
Abstract
We combine confocal Raman microscopy (CRM) of wet samples with subsequent Fluorescent in situ hybridization (FISH) without significant limitations to either technique for analyzing the same sample of a microbial community on a cell-to-cell basis. This combination of techniques allows a much deeper, more complete understanding of complex environmental samples than provided by either technique alone. The minimalistic approach is based on laboratory glassware with micro-engravings for reproducible localization of the sample at cell scale combined with a fixation and de- and rehydration protocol for the respective techniques. As proof of concept, we analyzed a floc of nitrifying activated sludge, demonstrating that the sample can be tracked with cell-scale precision over different measurements and instruments. The collected information includes the microbial content, spatial shape, variant chemical compositions of the floc matrix and the mineral microparticles embedded within. In addition, the direct comparison of CRM and FISH revealed a difference in reported cell size due to the different cell components targeted by the respective technique. To the best of our knowledge, this is the first report of a direct cell-to-cell comparison of confocal Raman microscopy and Fluorescent in situ hybridization analysis performed on the same sample. An adaptation of the method to include native samples as a starting point is planned for the near future. The micro-engraving approach itself also opens up the possibility of combining other, functionally incompatible techniques as required for further in-depth investigations of low-volume samples.
Keywords
- Biofilms, In Situ Hybridization, Fluorescence, Microscopy, Confocal, Sewage, Spectrum Analysis, Raman, Activated sludge, Confocal Raman microscopy, Ammonium oxidizing bacteria, Imaging, Biofilm, FISH
ASJC Scopus subject areas
- Environmental Science(all)
- Pollution
- Chemistry(all)
- Environmental Science(all)
- Health, Toxicology and Mutagenesis
- Environmental Science(all)
- Environmental Engineering
- Environmental Science(all)
- Environmental Chemistry
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In: CHEMOSPHERE, Vol. 161, 10.2016, p. 112-118.
Research output: Contribution to journal › Article › Transfer › peer review
}
TY - JOUR
T1 - Confocal Raman microscopy and fluorescent in situ hybridization
T2 - A complementary approach for biofilm analysis
AU - Kniggendorf, Ann-Kathrin
AU - Nogueira, Regina
AU - Kelb, Christian
AU - Schadzek, Patrik
AU - Meinhardt-Wollweber, Merve
AU - Ngezahayo, Anaclet
AU - Roth, Bernhard
PY - 2016/10
Y1 - 2016/10
N2 - We combine confocal Raman microscopy (CRM) of wet samples with subsequent Fluorescent in situ hybridization (FISH) without significant limitations to either technique for analyzing the same sample of a microbial community on a cell-to-cell basis. This combination of techniques allows a much deeper, more complete understanding of complex environmental samples than provided by either technique alone. The minimalistic approach is based on laboratory glassware with micro-engravings for reproducible localization of the sample at cell scale combined with a fixation and de- and rehydration protocol for the respective techniques. As proof of concept, we analyzed a floc of nitrifying activated sludge, demonstrating that the sample can be tracked with cell-scale precision over different measurements and instruments. The collected information includes the microbial content, spatial shape, variant chemical compositions of the floc matrix and the mineral microparticles embedded within. In addition, the direct comparison of CRM and FISH revealed a difference in reported cell size due to the different cell components targeted by the respective technique. To the best of our knowledge, this is the first report of a direct cell-to-cell comparison of confocal Raman microscopy and Fluorescent in situ hybridization analysis performed on the same sample. An adaptation of the method to include native samples as a starting point is planned for the near future. The micro-engraving approach itself also opens up the possibility of combining other, functionally incompatible techniques as required for further in-depth investigations of low-volume samples.
AB - We combine confocal Raman microscopy (CRM) of wet samples with subsequent Fluorescent in situ hybridization (FISH) without significant limitations to either technique for analyzing the same sample of a microbial community on a cell-to-cell basis. This combination of techniques allows a much deeper, more complete understanding of complex environmental samples than provided by either technique alone. The minimalistic approach is based on laboratory glassware with micro-engravings for reproducible localization of the sample at cell scale combined with a fixation and de- and rehydration protocol for the respective techniques. As proof of concept, we analyzed a floc of nitrifying activated sludge, demonstrating that the sample can be tracked with cell-scale precision over different measurements and instruments. The collected information includes the microbial content, spatial shape, variant chemical compositions of the floc matrix and the mineral microparticles embedded within. In addition, the direct comparison of CRM and FISH revealed a difference in reported cell size due to the different cell components targeted by the respective technique. To the best of our knowledge, this is the first report of a direct cell-to-cell comparison of confocal Raman microscopy and Fluorescent in situ hybridization analysis performed on the same sample. An adaptation of the method to include native samples as a starting point is planned for the near future. The micro-engraving approach itself also opens up the possibility of combining other, functionally incompatible techniques as required for further in-depth investigations of low-volume samples.
KW - Biofilms
KW - In Situ Hybridization, Fluorescence
KW - Microscopy, Confocal
KW - Sewage
KW - Spectrum Analysis, Raman
KW - Activated sludge
KW - Confocal Raman microscopy
KW - Ammonium oxidizing bacteria
KW - Imaging
KW - Biofilm
KW - FISH
UR - http://www.scopus.com/inward/record.url?scp=84978197366&partnerID=8YFLogxK
U2 - 10.1016/j.chemosphere.2016.06.096
DO - 10.1016/j.chemosphere.2016.06.096
M3 - Article
C2 - 27423128
VL - 161
SP - 112
EP - 118
JO - CHEMOSPHERE
JF - CHEMOSPHERE
SN - 0045-6535
ER -