Direct optical nanoscopy with axially localized detection

Abstract

Evanescent light excitation is widely used in super-resolution fluorescence microscopy to confine light and reduce background noise.Herein we propose a method of exploiting evanescent light in the context of emission. When a fluorophore is located in close proximity to a medium with a higher refractive index, its near-field component is converted into light that propagates beyond the critical angle. This so-called Supercritical-Angle Fluorescence (SAF) can be captured using a high-NA objective and used to determine the axial position of the fluorophore with nanometer precision. We introduce a new technique for 3D nanoscopy that combines direct STochastic Optical Reconstruction Microscopy (dSTORM) imaging with dedicated detection of SAF emission. We demonstrate that our approach of a Direct Optical Nanoscopy with Axially Localized Detection (DONALD) yields a typical isotropic 3D localization precision of 20-nm. 2Super-localization methods in optical microscopy such as PALM, (F)PALM and dSTORM, have shattered the spatial resolution barrier imposed by the diffraction limit. These techniques are based on the sequential detection of several thousands individual fluorescent molecules [1,2,3,4]. In their simplest implementations, these methods typically improve lateral resolution by one order of magnitude, however their axial resolution is still limited by diffraction. Specific techniques must be developped to tackle this strong anisotropy resolution, which compromises 3D imaging. Super-localization techniques must be combined with Point Spread Function (PSF) engineering methods, to measure the depth position of each detected fluorophore [5,6,7,8,9]. Single-[5] and double- [7] cylindrical-lens methods have achieved axial-localization precisions 60 and 20 nm, respectively. The former is very stable and straightforward to implement, whereas the higher precision of the latter comes at the cost of increased complexity. Similarly, alternative elaborate methods such as interferometric PALM (iPALM) [10] and the SelfBending PSF (SB-PSF) [11] can achieve axial localization precision of 10 nm and 15 nm, respectively [12]. In addition, all these strategies provide only the relative axial positions of the fluorophores with respect to an arbitrary focal plane. Hence, 3D optical nanoscopy is in need of a method that combines high nanometer axial precision, simplicity of implementation and absolute axial positioning.Herein we report a new approach termed "Direct Optical Nanoscopy with This dipole is also endowed with a non propagative near field component that depends on the surrounding refractive index n m . In the presence of an interface with a medium with a refractive index of n g > n m , the transmitted light follows Snell-Decartes law of refraction (Fig. 1a). The refracted light is emitted within a cone that is limited by the critical angle θ c = arcsin (n m /n g ). Plane (BFP). These aplanetic objectives satisfy Abbe sine relation: light emitted with an angle θ lies within a circle of radius ρ = nf sin(θ) in the BFP where n...

Three-Dimensional Localization of Single Molecules for Super-Resolution Imaging and Single-Particle Tracking

“…This method has been demonstrated for in vitro experiments with 3D origami nanostructures and for single-molecule imaging very near the interface in cells. (76, 77)…”

Combining 3D single molecule localization strategies for reproducible bioimaging

“…7). Afterwards, the axial positions were calculated: the values of z SAF were obtained using the theoretical curve provided in [15] whereas those of z astigmatic could be retrieved by fitting w U AF x − w U AF y to the calibration curve (see the Astigmatism calibration section) using a least squares calculation. Lateral drifts were then corrected using a temporal crosscorrelation algorithm.…”

“…Combined with SMLM, this technique, called Direct Optical Nanoscopy with Axially Localized Detection (DONALD) or Supercritical Angle Localization Microscopy (SALM), yields absolute axial positions (i.e. independent of the focus position) in the first 500 nm beyond the coverslip with a precision down to 15 nm [15,16]. The principle relies on the comparison between the SAF and the Undercritical Angle Fluorescence (UAF) components to extract the absolute axial position.…”

Breaking the Axial Diffraction Limit: A Guide to Axial Super‐Resolution Fluorescence Microscopy

“…By analyzing these two signals, super‐resolution localization precision could be obtained. Reproduced with permission . Copyright 2015, Springer Nature.…”

“…Similar to TIRFM, SAFM can also provide good optical sectioning capacity and high SBR, which has been applied to highly sensitive biosensing, cell adhesion and motility quantification, plasma membrane association and protein diffusion measurement, and near‐membrane refractometry implementation . In addition, SAFM can be combined with existing SMLM methods to perform 3D super‐resolution imaging . For example, coupling with STORM yields a 20‐nm isotropic 3D localization precision within ≈150‐nm axial range.…”