A Stimulated Emission Depletion (STED) Microscope of All Trades

Finzel, Lukas; Reuß, Matthias

doi:10.1017/s155192952200089x

Cited by 4 publications

(2 citation statements)

References 24 publications

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“…Scanning takes place as the sample is moved with a three-axis piezo positioner. Figure 8 also shows the point spread functions (PSFs) in multiple directions when using phase plates for ultrahigh lateral resolution (HR) and acquiring 3D images [ 64 ]. Details of the narrowing of the effective radiation area for 3D-STED are shown in Fig.…”

Section: Ultrastructural 3d Microscopy Methods Based On Super-resolut...mentioning

confidence: 99%

“…На рис. 8 также изображены функции рассеяния точки в нескольких направлениях, при использовании фазовых пластин для сверхвысокого латерального разрешения (HR) и получения 3D-изображений [64]. Сужение эффективной области излучения для 3D-STED подробнее показано на рис.…”

Section: методы ультраструктурной 3d-микроскопии на основе сверхразре...unclassified

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Ultrastructural 3D Microscopy for Biomedicine: Principles, Applications, and Perspectives

Mochalov,

Korzhov,

Altunina

et al. 2024

Acta Naturae

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Modern biomedical research often requires a three-dimensional microscopic analysis of the ultrastructure of biological objects and materials. Conceptual technical and methodological solutions for three-dimensional structure reconstruction are needed to improve the conventional optical, electron, and probe microscopy methods, which to begin with allow one to obtain two-dimensional images and data. This review discusses the principles and potential applications of such techniques as serial section transmission electron microscopy; techniques based on scanning electron microscopy (SEM) (array tomography, focused ion beam SEM, and serial block-face SEM). 3D analysis techniques based on modern super-resolution optical microscopy methods are described (stochastic optical reconstruction microscopy and stimulated emission depletion microscopy), as well as ultrastructural 3D microscopy methods based on scanning probe microscopy and the feasibility of combining them with optical techniques. A comparative analysis of the advantages and shortcomings of the discussed approaches is performed.

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Section: Ultrastructural 3d Microscopy Methods Based On Super-resolut...mentioning

confidence: 99%

Section: методы ультраструктурной 3d-микроскопии на основе сверхразре...unclassified

Ultrastructural 3D Microscopy for Biomedicine: Principles, Applications, and Perspectives

Mochalov,

Korzhov,

Altunina

et al. 2024

Acta Naturae

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A comparison of super-resolution microscopy techniques for imaging tightly packed microcolonies of an obligate intracellular bacterium

North,

Sharma,

Pyrgaki

et al. 2024

Preprint

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Conventional optical microscopy imaging of obligate intracellular bacteria is hampered by the small size of bacterial cells, tight clustering exhibited by some bacterial species and challenges relating to labelling such as background from host cells, a lack of validated reagents, and a lack of tools for genetic manipulation. In this study we imaged intracellular bacteria from the speciesOrientia tsutsugamushi(Ot) using five different fluorescence microscopy techniques: standard confocal, Airyscan confocal, instant Structured Illumination Microscopy (iSIM), three-dimensional Structured Illumination Microscopy (3D-SIM) and Stimulated Emission Depletion Microscopy (STED). We compared the ability of each to resolve bacterial cells in intracellular clumps in the lateral (xy) axis, using full width half maximum (FWHM) measurements of a labelled outer membrane protein (ScaA) and the ability to detect small, outer membrane vesicles external to the cells. We next compared the ability of each technique to sufficiently resolve bacteria in the axial (z) direction and found 3D-STED to be the most successful method for this. We then combined this approach with a custom 3D cell segmentation and analysis pipeline using the open-source, deep learning software, Cellpose to segment the cells and subsequently the commercial software Imaris to analyze their 3D shape and size. Using this combination, we demonstrated differences in bacterial shape, but not their size, when grown in different mammalian cell lines. Overall, we compare the advantages and disadvantages of different super-resolution microscopy techniques for imaging this cytoplasmic obligate intracellular bacterium based on the specific research question being addressed.

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A comparison of super‐resolution microscopy techniques for imaging tightly packed microcolonies of an obligate intracellular bacterium

North,

Sharma,

Pyrgaki

et al. 2024

Journal of Microscopy

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Conventional optical microscopy imaging of obligate intracellular bacteria is hampered by the small size of bacterial cells, tight clustering exhibited by some bacterial species and challenges relating to labelling such as background from host cells, a lack of validated reagents, and a lack of tools for genetic manipulation. In this study, we imaged intracellular bacteria from the species Orientia tsutsugamushi (Ot) using five different fluorescence microscopy techniques: standard confocal, Airyscan confocal, instant Structured Illumination Microscopy (iSIM), three‐dimensional Structured Illumination Microscopy (3D‐SIM) and Stimulated Emission Depletion Microscopy (STED). We compared the ability of each to resolve bacterial cells in intracellular clumps in the lateral (xy) axis, using full width half‐maximum (FWHM) measurements of a labelled outer membrane protein (ScaA) and the ability to detect small, outer membrane vesicles external to the cells. Comparing the techniques readily available to us (above), 3D‐SIM microscopy, in combination with the shortest‐wavelength dyes, was found overall to give the best lateral resolution. We next compared the ability of each technique to sufficiently resolve bacteria in the axial (z) direction and found 3D‐STED to be the most successful method for this. We then combined this 3D‐STED approach with a custom 3D cell segmentation and analysis pipeline using the open‐source, deep learning software, Cellpose to segment the cells and subsequently the commercial software Imaris to analyse their 3D shape and size. Using this combination, we demonstrated differences in bacterial shape, but not their size, when grown in different mammalian cell lines. Overall, we compare the advantages and disadvantages of different super‐resolution microscopy techniques for imaging this cytoplasmic obligate intracellular bacterium based on the specific research question being addressed.

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A Stimulated Emission Depletion (STED) Microscope of All Trades

Cited by 4 publications

References 24 publications

Ultrastructural 3D Microscopy for Biomedicine: Principles, Applications, and Perspectives

Ultrastructural 3D Microscopy for Biomedicine: Principles, Applications, and Perspectives

A comparison of super-resolution microscopy techniques for imaging tightly packed microcolonies of an obligate intracellular bacterium

A comparison of super‐resolution microscopy techniques for imaging tightly packed microcolonies of an obligate intracellular bacterium

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