Super-resolution spectroscopic microscopy via photon localization

Abstract

Traditional photon localization microscopy analyses only the spatial distributions of photons emitted by individual molecules to reconstruct super-resolution optical images. Unfortunately, however, the highly valuable spectroscopic information from these photons have been overlooked. Here we report a spectroscopic photon localization microscopy that is capable of capturing the inherent spectroscopic signatures of photons from individual stochastic radiation events. Spectroscopic photon localization microscopy achieved higher spatial resolution than traditional photon localization microscopy through spectral discrimination to identify the photons emitted from individual molecules. As a result, we resolved two fluorescent molecules, which were 15 nm apart, with the corresponding spatial resolution of 10 nm—a four-fold improvement over photon localization microscopy. Using spectroscopic photon localization microscopy, we further demonstrated simultaneous multi-colour super-resolution imaging of microtubules and mitochondria in COS-7 cells and showed that background autofluorescence can be identified through its distinct emission spectra.

Multiplexed RNAi therapy against brain tumor-initiating cells via lipopolymeric nanoparticle infusion delays glioblastoma progression

“…Super-Resolution SMLM Analysis. To enhance the optical resolution of subcellular organelles and nanoscale LPNP aggregates further after biological uptake into the cytosol of BTICs, we performed super-resolution imaging of BTICs after LPNP uptake at different time points, using a custom SMLM super-resolution nanoscopy (79,80). To perform SMLM imaging, 5-μL cell suspensions of each sample were mixed with the imaging buffer and deposited at the center of a freshly cleaned glass slide.…”

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

“…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).…”

Spatial and spectral imaging of point-spread functions using a spatial light modulator

“…The presented SLM-based system is flexible and adjustable for various experimental conditions and compatible with many existing PSF-engineered optical imaging methods [29]. In particular, the method provides an accessible and alternative system design for the reported spectrally-resolved techniques for single-molecule measurement and super-resolution fluorescence microscopy [22,23]. Furthermore, the use of the SLM allows the incorporation of phase patterns that encode axial information for 3D localization.…”

“…However, the spectral dimension of molecular emissions has long been overlooked in super-resolution microscopy. Addressing this demand, several emerging techniques have successfully extracted the spectral information from the PSFs, implementing new PSF engineering concepts in the spectral domain using various dispersive optical elements, such as a dual-objective system consisting a prism in one channel [22], spectral photon localization using a diffraction grating [23,24], mechanical slits [25], and spectrally-dependent multi-color PSF engineering [26]. These methods enable single-molecule spectroscopy without compromising the 3D localization, allowing the simultaneous identification of both spatial and spectral dimensions of molecular emissions.…”