Resolving Single-Molecule Assembled Patterns with Superresolution Blink-Microscopy

Abstract

In this paper we experimentally combine a recently developed AFM-based molecule-by-molecule assembly (single-molecule cut-and-paste, SMCP) with subdiffraction resolution fluorescence imaging. Using "Blink-Microscopy", which exploits the fluctuating emission of single molecules for the reconstruction of superresolution images, we resolved SMCP assembled structures with features below the diffraction limit. Artificial line patterns then served as calibration structures to characterize parameters, such as the labeling density, that can influence resolution of Blink-Microscopy besides the localization precision of a single molecule. Finally, we experimentally utilized the adjustability of blink parameters to demonstrate the general connection of photophysical parameters with spatial resolution and acquisition time in superresolution microscopy.

Measuring localization performance of super-resolution algorithms on very active samples

“…Achieving high structural resolution with localization microscopy has three prerequisites: The fluorophore density must be sufficiently high according to the Nyquist-Shannon sampling theorem [15], the imaging speed must surpass the sample dynamics, and the density of generated spots must be small enough to be optically resolvable to avoid imaging artifacts and false localizations [9,16,17]. While these prerequisites are easily fulfilled as long as only extended filaments, e.g.…”

“…While these prerequisites are easily fulfilled as long as only extended filaments, e.g. microtubulin and actin, are imaged, super-resolution imaging of complex and densely labeled structures necessitates the use of photoswitchable fluorophores with highly stable off states and appropriately set photoswitching rates [16,17]. Three-dimensional and dynamic super-resolution imaging with high spatiotemporal resolution [5,8,9,18,19] or single turnover counting for spatially resolved observation of catalysis [20,21] are even more challenging.…”

Localization microscopy coming of age: from concepts to biological impact

“…centrosomes comprising two orthogonally arranged centrioles, each consisting of nine triplet microtubules (Sillibourne et al, 2011;Lau et al, 2012;Lukinavičius et al, 2013), or pre-and post-synaptic proteins separated by the synaptic cleft (Dani et al, 2010), can also be used as natural biological calibration samples (Box 2). Artificial calibration samples for 2D and 3D localization microscopy include DNA origami (Steinhauer et al, 2009), single-molecule assembled patterns generated by cut-and-paste technology (SMCP) (Kufer et al, 2008;Cordes et al, 2010) and DNA bricks (Box 2) (Ke et al, 2012).…”

Advanced-Microscopy Techniques for the Characterization of Cellulose Structure and Cellulose-Cellulase Interactions

“…These assemblies typically contain fluorescently labeled molecules that can be selectively excited to emit photons, and their emission profile can be localized within the field of view with high precision in a manner analogous to the SM localization and tracking approaches described above. SRFM includes methodologies that take advantage of nonlinear optical effects to condition or reduce the size of the excitation point spread function (Stimulated Emission-Depletion or STED, Saturated Structured Illumination microscopy, SSIM)(87-91), or that reconstruct super-resolution images from the emission profiles of individual fluorescent molecules that are selectively (Photo-Activated Localization Microscopy or PALM, and related techniques) (92,93) or randomly (Stochastic Optical Reconstruction Microscopy, STORM, blink microscopy or dSTORM) (94)(95)(96)(97)(98) switched to emit fluorescence. While all these approaches obtain similar resolution (< 50 nm), they have different attributes and equipment requirements that make them more or less amenable to implementation, and a number of reviews exist that compare them.…”

“…Recently, Tinnefeld and collaborators have reported the ability to perform SRFM with conventional fluorescent dyes, provided that suitable imaging wavelength intensities and aqueous medium can be utilized. (95)(96)(97) This imaging medium can be utilized with protein or nucleic acid systems, and could be well suited for imaging of cellulose-protein interactions. (101) Despite the difference in operational mechanisms, the principle behind SRFM techniques (Figure 4) is that in a sample that contains a large number of fluorescent molecules, under appropriate experimental conditions, only a few of them are conditioned to emit fluorescence at any given time.…”