Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function

Abstract

We demonstrate single-molecule fluorescence imaging beyond the optical diffraction limit in 3 dimensions with a wide-field microscope that exhibits a double-helix point spread function (DH-PSF). The DH-PSF design features high and uniform Fisher information and has 2 dominant lobes in the image plane whose angular orientation rotates with the axial (z) position of the emitter. Single fluorescent molecules in a thick polymer sample are localized in single 500-ms acquisitions with 10-to 20-nm precision over a large depth of field (2 m) by finding the center of the 2 DH-PSF lobes. By using a photoactivatable fluorophore, repeated imaging of sparse subsets with a DH-PSF microscope provides superresolution imaging of high concentrations of molecules in all 3 dimensions. The combination of optical PSF design and digital postprocessing with photoactivatable fluorophores opens up avenues for improving 3D imaging resolution beyond the Rayleigh diffraction limit.microscopy ͉ photoactivation ͉ superresolution ͉ computational imaging ͉ PSF engineering F luorescence microscopy is ubiquitous in biological studies because light can noninvasively probe the interior of a cell with high signal-to-background and remarkable label specificity. Unfortunately, optical diffraction limits the transverse (x-y) resolution of a conventional fluorescence microscope to approximately /(2NA), where is the optical wavelength and NA is the numerical aperture of the objective lens (1). This limitation requires that point sources need to be Ͼ Ϸ200 nm apart in the visible wavelength region to be distinguished with modern high-quality fluorescence microscopes. Diffraction causes the image of a single-point emitter to appear as a blob (i.e., the point-spread function or PSF) with a width given by the diffraction limit. However, if the shape of the PSF is measured, then the center position of the blob can be determined with a far greater precision (termed superlocalization) that scales approximately as the diffraction limit divided by the square root of the number of photons collected, a fact noted as early as Heisenberg in the context of electron localization with photons (2) and later extended to point objects (3, 4) and single-molecule emitters (5-8). Because single-molecule emitters are only a few nanometers in size, they represent particularly useful point sources for imaging, and superlocalization of single molecules at room temperature has been pushed to the 1-nm regime (9) in transverse (2-dimensional) imaging. In the third (z) dimension, diffraction also limits resolution to Ϸ2n /NA 2 with n the index of refraction, corresponding to a depth of field of Ϸ500 nm in the visible wavelength region with modern microscopes. Improvements in 3D localization beyond this limit are also possible by using astigmatism (10, 11), defocusing (12), or simultaneous multiplane viewing (13).Until recently, superlocalization of individual molecules was unable to provide true resolution beyond the diffraction limit (superresolution) because the concentration of emi...

Quantitative evaluation of software packages for single-molecule localization microscopy

“…In the future, our reference data will include more features, such as drift, 3D localization (PSF engineering 57,58 and multiple planes 59,60 , additional levels of molecule density, multiple fluorescent channels, asymmetrical PSF due to dipole effect 61 , scattering effects, and a richer variety of noise models associated with various types of cameras, EMCCD, sCMOS 62,63 . It will also be interesting to generate benchmarking data to test the impact of clustering (spatial aggregation) and diffusion for single-particle tracking.…”

Segmentation of 3D Trajectories Acquired by TSUNAMI Microscope: An Application to EGFR Trafficking

“…Whereas SPT has made important discoveries that change our view of plasma membrane organization (17,19) and molecular motor dynamics (20), the use of SPT in monitoring ''intracellular'' processes is rather limited because of the lack of three-dimensional (3D) tracking capacity that can follow a single particle inside a live cell for a long period of time. In the past decade, new SPT techniques have been developed to visualize molecular motion in the 3D space (termed 3D-SPT), including multiple imaging planes (21,22), orbital tracking (23)(24)(25), point spread function engineering (26,27), and confocal tracking (28,29). Although allowing for direct observation of transport processes from membrane to cytoplasm, current 3D-SPT methods often suffer from shallow imaging depth (because of the use of one-photon excitation) and limited z-tracking range (e.g., astigmatismbased, nonfeedback tracking systems (27)), which prevent these methods from tracking single molecules inside multicellular models such as spheroids (see our review in (8)).…”

From microscopy to nanoscopy via visible light

“…In this way, the fluorescent spot was spread along perpendicular directions before and after the ideal focal plane, so that the axial position can be determined with nanometer accuracy. A similar idea that uses double-helix PSF to create the difference was proposed by Pavani et al 97 in 2009. There were also some other techniques developed to achieve 3D imaging during the same period, such as biplane, 98 and dual-objective PALM/STORM, 99,100 and the highest resolution achieved until now is beyond 20 nm 99 in both lateral and axial directions.…”