dSTORM Imaging and Analysis of Desmosome Architecture
Desmosomes are cell-cell junctions responsible for mechanically integrating adjacent cells. Due to the small size of the junctions, their protein architecture cannot be elucidated using conventional fluorescence microscopy. Super-resolution microscopy tec
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dSTORM Imaging and Analysis of Desmosome Architecture Reena R. Beggs, William F. Dean, and Alexa L. Mattheyses Abstract Desmosomes are cell-cell junctions responsible for mechanically integrating adjacent cells. Due to the small size of the junctions, their protein architecture cannot be elucidated using conventional fluorescence microscopy. Super-resolution microscopy techniques, including dSTORM, deliver higher-resolution images which can reveal the localization or arrangement of proteins within individual desmosomes. Herein we describe an imaging and analysis method to determine the nanoscale architecture of desmosomes using super-resolution dSTORM. Key words Cell junctions, Microscopy, STORM, Super-resolution
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Introduction Fluorescence microscopy is an essential tool in cell and molecular biology because of its ability to reveal cellular organization. Proteins can be labeled with incredible specificity using genetically encoded fluorescent tags or fluorescently labeled antibodies. However, the spatial localization of these proteins is limited to several hundred nanometers, making it nearly impossible to resolve the architecture of small subcellular structures such as cell junctions. The resolution of an optical microscope refers to the smallest distance that can be distinguished between two objects. Resolution (r) is a result of diffraction and depends on the numerical aperture (NA) of the objective and the wavelength (λ) of the fluorescence being imaged. The Abbe resolution limit is defined by the equations rxy ¼ λ/2NA and rz ¼ 2λ/NA2 [1]. Based on the resolution limit, the smallest distance that can be measured by conventional widefield fluorescence microscopy is ~250 nm. Practically, this means that if fluorophores are separated by a distance less than ~250 nm, they cannot be resolved as individual points and will appear as one object. Given that the average size of single desmosome is approximately 500 nm, architectural details of these junctions are not accessible by conventional fluorescence microscopy. A number of super-resolution microscopy techniques have been developed to overcome this barrier and improve the spatial resolution of fluorescence images [2–5]. One family of super-
Reena R. Beggs et al.
resolution microscopy techniques is single-molecule localization microscopy (SMLM). SMLM techniques, including direct stochastic optical reconstruction microscopy (dSTORM), provide sub-diffraction resolution by compiling the precise localizations of individual fluorophores collected over many images. In dSTORM, organic fluorophores are driven into a dark state using high-power excitation and are stochastically switched between light and dark states, preventing the overlap of their emission signal. During these “blinking” events, many individual frames are captured of only a small population of individual fluorophores, which can be optically resolved. Single-molecule localizations are accumulated over many frames, compiled, and finally reconstructed into a super-resolved image (Fig. 1). The final dSTO
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