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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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Schematic of in situ fluid cell STEM and correlative fluorescence microscopy (not to scale).(a) Fluid cell microfluidic chamber consisting of two silicon microchips supporting two electron transparent SiN membranes. Cells of M. magneticum are attached to the top SiN window and imaged with STEM in the thin liquid layer. (b) The tip of the liquid cell is mounted on a glass slide and subsequently imaged in a fluorescence microscope with a 40X objective lens.
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f1: Schematic of in situ fluid cell STEM and correlative fluorescence microscopy (not to scale).(a) Fluid cell microfluidic chamber consisting of two silicon microchips supporting two electron transparent SiN membranes. Cells of M. magneticum are attached to the top SiN window and imaged with STEM in the thin liquid layer. (b) The tip of the liquid cell is mounted on a glass slide and subsequently imaged in a fluorescence microscope with a 40X objective lens.

Mentions: Fig. 1 shows the experimental setup used for the correlative STEM and fluorescence imaging of magnetotactic bacteria. Cells of Magnetospirillum magneticum, strain AMB-1, are attached to the top functionalized 100 nm SiN spacer chip (cf. Methods) and sandwiched with another 100 nm spacer chip, as schematically shown in Fig. 1a. The tip of the liquid cell is removed from the STEM holder platform, mounted on a glass slide and accepted by the fluorescence microscope for imaging with a 40X objective lens, as schematically shown in Fig. 1b.


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)

Schematic of in situ fluid cell STEM and correlative fluorescence microscopy (not to scale).(a) Fluid cell microfluidic chamber consisting of two silicon microchips supporting two electron transparent SiN membranes. Cells of M. magneticum are attached to the top SiN window and imaged with STEM in the thin liquid layer. (b) The tip of the liquid cell is mounted on a glass slide and subsequently imaged in a fluorescence microscope with a 40X objective lens.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Schematic of in situ fluid cell STEM and correlative fluorescence microscopy (not to scale).(a) Fluid cell microfluidic chamber consisting of two silicon microchips supporting two electron transparent SiN membranes. Cells of M. magneticum are attached to the top SiN window and imaged with STEM in the thin liquid layer. (b) The tip of the liquid cell is mounted on a glass slide and subsequently imaged in a fluorescence microscope with a 40X objective lens.
Mentions: Fig. 1 shows the experimental setup used for the correlative STEM and fluorescence imaging of magnetotactic bacteria. Cells of Magnetospirillum magneticum, strain AMB-1, are attached to the top functionalized 100 nm SiN spacer chip (cf. Methods) and sandwiched with another 100 nm spacer chip, as schematically shown in Fig. 1a. The tip of the liquid cell is removed from the STEM holder platform, mounted on a glass slide and accepted by the fluorescence microscope for imaging with a 40X objective lens, as schematically shown in Fig. 1b.

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