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...
Measurements of ultrafast fluorescence anisotropy decay in model branched dendritic molecules of different symmetry are reported. These molecules contain the fundamental branching center units of larger dendrimer macromolecules with either three (C(3))- or four (T(d), tetrahedral)-fold symmetry. The anisotropy for a tetrahedral system is found to decay on a subpicosecond time scale (880 fs). This decay can be qualitatively explained by Förster-type incoherent energy migration between chromophores. Alternatively, for a nitrogen-centered trimer system, the fluorescence anisotropy decay time (35 fs) is found to be much shorter than that of the tetramers, and the decay cannot be attributed to an incoherent hopping mechanism. In this case, a coherent interchromophore energy transport mechanism should be considered. The mechanism of the ultrafast energy migration process in the branched systems is interpreted by use of a phenomenological quantum mechanical model, which examines the two extreme cases of incoherent and coherent interactions.
Novel alkene and alkyne branched structures have been synthesized, and their two-photon absorption (2PA) properties are reported. This series of alkene and alkyne trimer systems tests the mechanistic approach for enhancing the 2PA process which is usually dictated by the pi-bridging, delocalization length, and corresponding charge transfer on the 2PA cross sections. The results suggest that alkene branched systems have higher 2PA cross sections. While steady-state absorption and emission measurements were not successful in predicting the observed trend of 2PA cross sections, time-resolved measurements have explained the trends observed. It was found that, upon photoexcitation, there is an ultrafast charge localization to an intramolecular charge-transfer (ICT) state, followed by the presence of a solvent and conformationally relaxed ICT state in these branched systems.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.