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Functionalized Buckyballs for Visualizing Microbial Species in Different States and Environments.

Cheng Q, Aravind A, Buckley M, Gifford A, Parvin B - Sci Rep (2015)

Bottom Line: To date, in situ visualization of microbial density has remained an open problem.Here, functionalized buckyballs (e.g., C60-pyrrolidine tris acid) are shown to be a versatile platform that allows internalization within a microorganism without either adhering to the cell wall and cell membrane or binding to a matrix substrate such as soil.We also demonstrate that cysteine-functionalized C60-pyrrolidine tris acid can differentiate live and dead microorganisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Electrical and Biomedical Engineering, University of Nevada, Reno, 1664 N Virginia Street, Reno NV, 89503, USA.

ABSTRACT
To date, in situ visualization of microbial density has remained an open problem. Here, functionalized buckyballs (e.g., C60-pyrrolidine tris acid) are shown to be a versatile platform that allows internalization within a microorganism without either adhering to the cell wall and cell membrane or binding to a matrix substrate such as soil. These molecular probes are validated via multi-scale imaging, to show association with microorganisms via fluorescence microscopy, positive cellular uptake via electron microscopy, and non-specific binding to the substrates through a combination of fluorescence and autoradiography imaging. We also demonstrate that cysteine-functionalized C60-pyrrolidine tris acid can differentiate live and dead microorganisms.

No MeSH data available.


Cellular uptake of fBSA labelled C60-pyrrolidine tris acid by E. coli and B. subtilis monitored by Confocal Laser Scanning Microscopy.(a) and (b) fBSA-C60 pyrrolidine tris acid uptake by E. coli (a) and B. subtilis (b) visualized by exciting fBSA fluorescence using the 488 nm laser. (c) and (d) Bright field (BF) images of the microorganisms. (e) and (f) Merged fluorescence and BF images indicate that the fluorescent signals co-localize with the presence of the microorganisms.
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f2: Cellular uptake of fBSA labelled C60-pyrrolidine tris acid by E. coli and B. subtilis monitored by Confocal Laser Scanning Microscopy.(a) and (b) fBSA-C60 pyrrolidine tris acid uptake by E. coli (a) and B. subtilis (b) visualized by exciting fBSA fluorescence using the 488 nm laser. (c) and (d) Bright field (BF) images of the microorganisms. (e) and (f) Merged fluorescence and BF images indicate that the fluorescent signals co-localize with the presence of the microorganisms.

Mentions: E. coli (Gram-negative) and B. subtilis (Gram-positive) are incubated with functionalized C60-pyrrolidine tris acid with fBSA for 30 min. Samples are then washed with DI H2O to remove excess probes, and samples are then imaged by confocal microscopy. Figure 2 indicates a positive association of C60-pyrrolidine tris acid with microorganisms, where in Fig. 2a,b, both E. coli and B. subtilis have fluorescent signal emission following excitation by a 488 nm laser. We confirmed that these fluorescent signals are solely from C60-pyrrolidine tris acid-fBSA, because neither E. coli nor B. subtilis has an auto-fluorescence signal under the same conditions in the absence of fluorescent C60 (Supplementary Figure 1). In addition, these fluorescent signals co-localize with E. coli and B. subtilis cells by combining bright field and fluorescent imaging, which suggests either internalization within the cell or binding to the cell wall. (Recall that CNTs were able to internalize inside the mammalian cells37, and similar behavior can be suggested for microbial species).


Functionalized Buckyballs for Visualizing Microbial Species in Different States and Environments.

Cheng Q, Aravind A, Buckley M, Gifford A, Parvin B - Sci Rep (2015)

Cellular uptake of fBSA labelled C60-pyrrolidine tris acid by E. coli and B. subtilis monitored by Confocal Laser Scanning Microscopy.(a) and (b) fBSA-C60 pyrrolidine tris acid uptake by E. coli (a) and B. subtilis (b) visualized by exciting fBSA fluorescence using the 488 nm laser. (c) and (d) Bright field (BF) images of the microorganisms. (e) and (f) Merged fluorescence and BF images indicate that the fluorescent signals co-localize with the presence of the microorganisms.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4561912&req=5

f2: Cellular uptake of fBSA labelled C60-pyrrolidine tris acid by E. coli and B. subtilis monitored by Confocal Laser Scanning Microscopy.(a) and (b) fBSA-C60 pyrrolidine tris acid uptake by E. coli (a) and B. subtilis (b) visualized by exciting fBSA fluorescence using the 488 nm laser. (c) and (d) Bright field (BF) images of the microorganisms. (e) and (f) Merged fluorescence and BF images indicate that the fluorescent signals co-localize with the presence of the microorganisms.
Mentions: E. coli (Gram-negative) and B. subtilis (Gram-positive) are incubated with functionalized C60-pyrrolidine tris acid with fBSA for 30 min. Samples are then washed with DI H2O to remove excess probes, and samples are then imaged by confocal microscopy. Figure 2 indicates a positive association of C60-pyrrolidine tris acid with microorganisms, where in Fig. 2a,b, both E. coli and B. subtilis have fluorescent signal emission following excitation by a 488 nm laser. We confirmed that these fluorescent signals are solely from C60-pyrrolidine tris acid-fBSA, because neither E. coli nor B. subtilis has an auto-fluorescence signal under the same conditions in the absence of fluorescent C60 (Supplementary Figure 1). In addition, these fluorescent signals co-localize with E. coli and B. subtilis cells by combining bright field and fluorescent imaging, which suggests either internalization within the cell or binding to the cell wall. (Recall that CNTs were able to internalize inside the mammalian cells37, and similar behavior can be suggested for microbial species).

Bottom Line: To date, in situ visualization of microbial density has remained an open problem.Here, functionalized buckyballs (e.g., C60-pyrrolidine tris acid) are shown to be a versatile platform that allows internalization within a microorganism without either adhering to the cell wall and cell membrane or binding to a matrix substrate such as soil.We also demonstrate that cysteine-functionalized C60-pyrrolidine tris acid can differentiate live and dead microorganisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Electrical and Biomedical Engineering, University of Nevada, Reno, 1664 N Virginia Street, Reno NV, 89503, USA.

ABSTRACT
To date, in situ visualization of microbial density has remained an open problem. Here, functionalized buckyballs (e.g., C60-pyrrolidine tris acid) are shown to be a versatile platform that allows internalization within a microorganism without either adhering to the cell wall and cell membrane or binding to a matrix substrate such as soil. These molecular probes are validated via multi-scale imaging, to show association with microorganisms via fluorescence microscopy, positive cellular uptake via electron microscopy, and non-specific binding to the substrates through a combination of fluorescence and autoradiography imaging. We also demonstrate that cysteine-functionalized C60-pyrrolidine tris acid can differentiate live and dead microorganisms.

No MeSH data available.