Subsurface Super-resolution Imaging of Unstained Polymer Nanostructures

Abstract

Optical imaging has offered unique advantages in material researches, such as spectroscopy and lifetime measurements of deeply embedded materials, which cannot be matched using electron or scanning-probe microscopy. Unfortunately, conventional optical imaging cannot provide the spatial resolutions necessary for many nanoscopic studies. Despite recent rapid progress, super-resolution optical imaging has yet to be widely applied to non-biological materials. Herein we describe a method for nanoscopic optical imaging of buried polymer nanostructures without the need for extrinsic staining. We observed intrinsic stochastic fluorescence emission or blinking from unstained polymers and performed spatial-temporal spectral analysis to investigate its origin. We further applied photon localization super-resolution imaging reconstruction to the detected stochastic blinking, and achieved a spatial resolution of at least 100 nm, which corresponds to a six-fold increase over the optical diffraction limit. This work demonstrates the potential for studying the static heterogeneities of intrinsic polymer molecular-specific properties at sub-diffraction-limited optical resolutions.

Super-resolution microscopy as a powerful tool to study complex synthetic materials

“…Tian, Li and Hu proposed 48 the use of spiropyran derivates to perform single molecule localization microscopy (PULSAR) and reconstructed images with a resolution down to 10-40 nm 52 Although polymers are usually fluorescently labeled in order to be studies with SRM, some polymers may exhibit intrinsic fluorescence in the visible range. 53 PMMA, polystyrene and Su-8 films were imaged using STORM without any external labelling showing intensive blinking events that enabled the reconstruction of a well-defined image. 53 The intrinsic fluorescent properties of several materials represent at the same time an advantage and a drawback as the spontaneous fluorescence generates a strong background that prevents high resolution visualization of the structure through specific labeling.…”

“…53 PMMA, polystyrene and Su-8 films were imaged using STORM without any external labelling showing intensive blinking events that enabled the reconstruction of a well-defined image. 53 The intrinsic fluorescent properties of several materials represent at the same time an advantage and a drawback as the spontaneous fluorescence generates a strong background that prevents high resolution visualization of the structure through specific labeling. Therefore, the development of novel blinking dyes that are emissive in the polymeric environment and the development of self-blinking materials suitable for label-free SMLM represent two interesting perspectives in SRM.…”

Superresolution intrinsic fluorescence imaging of chromatin utilizing native, unmodified nucleic acids for contrast

“…Consequently, the visible-light-induced autofluorescence of nucleic acids has likely been overlooked due to the broad availability of the nuclear counter stains Hoechst 33342 and DAPI, the ability to quickly eliminate the intrinsic autofluorescence within samples, and the common misperception that nucleic acids do not fluoresce within the visible range. A critical implication is that macromolecules, which are widely perceived as "dark," may be excited for superresolution imaging under different illumination conditions (27,28,53). The critical element in such an approach would depend on the capacity to ascertain molecular information from the intrinsic emissions of endogenous biological molecules (54).…”

Super‐resolution Fluorescence Imaging for Materials Science

“…However, there is interest in developing label‐free approaches. A recent example toward this direction showed that nanofabricated patterns on an “unstained” polymer (with no bulk absorption in the visible range) show blinking that can be used for SMLM, although the origin of this blinking has not been confirmed. Indeed, autofluorescence in polymers and other materials is not uncommon, and its origins are sometimes difficult to identify.…”