Near-isotropic 3D optical nanoscopy with photon-limited chromophores

Abstract

Imaging approaches based on single molecule localization break the diffraction barrier of conventional fluorescence microscopy, allowing for bioimaging with nanometer resolution. It remains a challenge, however, to precisely localize photon-limited single molecules in 3D. We have developed a new localization-based imaging technique achieving almost isotropic subdiffraction resolution in 3D. A tilted mirror is used to generate a side view in addition to the front view of activated single emitters, allowing their 3D localization to be precisely determined for superresolution imaging. Because both front and side views are in focus, this method is able to efficiently collect emitted photons. The technique is simple to implement on a commercial fluorescence microscope, and especially suitable for biological samples with photon-limited chromophores such as endogenously expressed photoactivatable fluorescent proteins. Moreover, this method is relatively resistant to optical aberration, as it requires only centroid determination for localization analysis. Here we demonstrate the application of this method to 3D imaging of bacterial protein distribution and neuron dendritic morphology with subdiffraction resolution.3D bioimaging | nanoimaging | bacterial imaging | neuron imaging | biophotonics F luorescence microscopy allows the observation of biological samples in a highly sensitive, selective, and relatively noninvasive fashion. Moreover, fluorescence imaging tools can probe deeply into tissues to obtain volume images without physically isolating different sections. However, traditional fluorescence microscopy suffers from diffraction-limited resolution, far larger than actual molecular sizes. Recent development of two different superresolution (SR) fluorescence imaging methods has broken the diffraction limit and enables direct optical observation of biological features near molecular scales. One approach is to create a subdiffraction excitation pattern by stimulated emission depletion (1, 2) or structured illumination (3, 4), and the other approach is based on single molecule localization (5-9). An essential part of the single molecule localization methods involves centroid fitting of serial single molecule images at the detection plane in order to determine the lateral localization of each fluorescent molecule with precision limited not by diffraction, but mainly by its number of emitted photons. A superresolution 2D image of the sample can then be rendered by overlapping all single molecule localizations from all image frames. This principle of localization has also been applied to single particle tracking (10)(11)(12)(13)(14).Single molecule-based superresolution microscopy requires individual chromophores to be turned "on" and "off" sequentially, either by photoswitching or by other stochastic photophysical processes. Compared to synthetic photoswitchable probes, photoactivatable fluorescent proteins (PA-FPs) (15-20) are superior in many regards: PA-FPs can be genetically fused to target proteins and endogenously...

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

“…Unfortunately, current algorithms used for single-molecule localization microscopy mainly focus on spatial localization precision and computational speed and rely on well-separated input spots [24][25][26]. The lack of efficient algorithms results, in our opinion, primarily from the difficulties to check the performance of the algorithm on experimental data obtained from densely labeled samples.…”

Distinct, but not completely separate spatial transport routes in the nuclear pore complex

“…Unfortunately, the currently available 3D imaging methods generally cannot capture or distinguish such rapid diffusion in a sub-micrometer bio-cavity such as the NPC. [70][71][72][73][74][75] Finally, the complexity of natively unfolded FG Nups in the NPC makes mimicking real nuclear transport in artificial pores challenging. [76][77][78] Therefore, an imaging technique that can map real-time nuclear transport at an exceptionally high spatiotemporal resolution in 3D in the NPCs of living cells is urgently needed.…”

Super-resolution photon-efficient imaging by nanometric double-helix point spread function localization of emitters (SPINDLE)

“…Resolution below 50 nm in the transverse direction and 100 nm in depth are now possible. 3D microscopes with astigmatic response [9], bi-plane detection [10], interferometric detection (IPALM) [11], double-helix point spread function (DH-PSF) [12], tilted mirrors [13] and phase ramps [14] have been applied to super-resolution imaging.…”