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Correlative electron and fluorescence microscopy of magnetotactic bacteria in liquid: toward in vivo imaging.

Woehl TJ, Kashyap S, Firlar E, Perez-Gonzalez T, Faivre D, Trubitsyn D, Bazylinski DA, Prozorov T - Sci Rep (2014)

Bottom Line: Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms.Fluorescently labeled cells were immobilized on microchip window surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity.Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions.

View Article: PubMed Central - PubMed

Affiliation: Emergent Atomic and Magnetic Structures, Division of Materials Sciences and Engineering, Ames Laboratory, Ames, IA 50011, USA.

ABSTRACT
Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip window surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria.

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Related in: MedlinePlus

Composite fluorescence images of stained cells of M. magneticum (a) 40 minutes and (b) 110 minutes after sealing the fluid cell tip.The scale bar in (b) is 10 μm. (c) Percentage of cells with intact membranes (green fluorescence only) as a function of time from fluid cell assembly. Percent viability is determined by dividing green fluorescent bacterial cells by the total number of bacteria cells in the fluid cell. Each data point is the mean of three trials, the error bars are two standard deviations of the mean.
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f6: Composite fluorescence images of stained cells of M. magneticum (a) 40 minutes and (b) 110 minutes after sealing the fluid cell tip.The scale bar in (b) is 10 μm. (c) Percentage of cells with intact membranes (green fluorescence only) as a function of time from fluid cell assembly. Percent viability is determined by dividing green fluorescent bacterial cells by the total number of bacteria cells in the fluid cell. Each data point is the mean of three trials, the error bars are two standard deviations of the mean.

Mentions: From Fig. 5c, it appears that simply loading the magnetotactic bacteria in the fluid cell drastically decreased their viability even before STEM imaging. We acquired fluorescence images of cells attached to a BioPlus chip every 10 minutes to systematically test bacteria viability as a function of time in the fluid cell (Fig. 6). Figs. 6a and 6b show example fluorescence images taken after 40 and 110 minutes in the fluid cell, respectively. There was a 40 minute lag time between fluid cell assembly and the first fluorescence image to allow for sample preparation and transportation to the fluorescence microscope. Fig. 6c shows a plot of the percent viable bacterial cells as a function of time. After 40 minutes in the fluid cell, approximately half of the bacteria were viable. Over the next 70 minutes, several cells lost their green fluorescence and began to exhibit red fluorescence, with the bacterial viability decreasing at a rate of ~0.5% min−1. After 110 minutes, the bacterial viability decreased to ~30%.


Correlative electron and fluorescence microscopy of magnetotactic bacteria in liquid: toward in vivo imaging.

Woehl TJ, Kashyap S, Firlar E, Perez-Gonzalez T, Faivre D, Trubitsyn D, Bazylinski DA, Prozorov T - Sci Rep (2014)

Composite fluorescence images of stained cells of M. magneticum (a) 40 minutes and (b) 110 minutes after sealing the fluid cell tip.The scale bar in (b) is 10 μm. (c) Percentage of cells with intact membranes (green fluorescence only) as a function of time from fluid cell assembly. Percent viability is determined by dividing green fluorescent bacterial cells by the total number of bacteria cells in the fluid cell. Each data point is the mean of three trials, the error bars are two standard deviations of the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215306&req=5

f6: Composite fluorescence images of stained cells of M. magneticum (a) 40 minutes and (b) 110 minutes after sealing the fluid cell tip.The scale bar in (b) is 10 μm. (c) Percentage of cells with intact membranes (green fluorescence only) as a function of time from fluid cell assembly. Percent viability is determined by dividing green fluorescent bacterial cells by the total number of bacteria cells in the fluid cell. Each data point is the mean of three trials, the error bars are two standard deviations of the mean.
Mentions: From Fig. 5c, it appears that simply loading the magnetotactic bacteria in the fluid cell drastically decreased their viability even before STEM imaging. We acquired fluorescence images of cells attached to a BioPlus chip every 10 minutes to systematically test bacteria viability as a function of time in the fluid cell (Fig. 6). Figs. 6a and 6b show example fluorescence images taken after 40 and 110 minutes in the fluid cell, respectively. There was a 40 minute lag time between fluid cell assembly and the first fluorescence image to allow for sample preparation and transportation to the fluorescence microscope. Fig. 6c shows a plot of the percent viable bacterial cells as a function of time. After 40 minutes in the fluid cell, approximately half of the bacteria were viable. Over the next 70 minutes, several cells lost their green fluorescence and began to exhibit red fluorescence, with the bacterial viability decreasing at a rate of ~0.5% min−1. After 110 minutes, the bacterial viability decreased to ~30%.

Bottom Line: Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms.Fluorescently labeled cells were immobilized on microchip window surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity.Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions.

View Article: PubMed Central - PubMed

Affiliation: Emergent Atomic and Magnetic Structures, Division of Materials Sciences and Engineering, Ames Laboratory, Ames, IA 50011, USA.

ABSTRACT
Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip window surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria.

Show MeSH
Related in: MedlinePlus