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.

Visualizing the nanoscale intracellular structures formed by nucleic acids, such as chromatin, in nonperturbed, structurally and dynamically complex cellular systems, will help expand our understanding of biological processes and open the next frontier for biological discovery. Traditional superresolution techniques to visualize subdiffractional macromolecular structures formed by nucleic acids require exogenous labels that may perturb cell function and change the very molecular processes they intend to study, especially at the extremely high label densities required for superresolution. However, despite tremendous interest and demonstrated need, label-free optical superresolution imaging of nucleotide topology under native nonperturbing conditions has never been possible. Here we investigate a photoswitching process of native nucleotides and present the demonstration of subdiffraction-resolution imaging of cellular structures using intrinsic contrast from unmodified DNA based on the principle of single-molecule photon localization microscopy (PLM). Using DNA-PLM, we achieved nanoscopic imaging of interphase nuclei and mitotic chromosomes, allowing a quantitative analysis of the DNA occupancy level and a subdiffractional analysis of the chromosomal organization. This study may pave a new way for label-free superresolution nanoscopic imaging of macromolecular structures with nucleotide topologies and could contribute to the development of new DNA-based contrast agents for superresolution imaging.superresolution fluorescence microscopy | label-free imaging | nucleic acids | chromatin topology | chromosome A dvances in genomics and molecular biology over the past decades revolutionized our knowledge of biological systems. Despite our expanded understanding of biological interactions, there continues to be a limited understanding of these complex molecular processes in nonperturbed, structurally and dynamically complex cellular systems (1). As such, it is of critical importance to develop methods that allow direct visualization of nanoscale structures where these processes take place in their native states. Recently, superresolution fluorescence microscopy techniques, including stimulated emission depletion microscopy, structured illumination microscopy, and photon localization microscopy (PLM), such as photoactivated localization microscopy and stochastic optical reconstruction microscopy (STORM), have extended the ultimate resolving power of optical microscopy far beyond the diffraction limit (2-6), facilitating access to the organization of cells at the nanoscale by optical means. Although superresolution imaging of biological structures using labeled proteins has been well documented due to a wide range of methodologies that provide desirable labeling properties (7,8), and despite tremendous interest and demonstrated need, there are few nanoscopic methods to image macromolecular structures formed by nucleic acids due to constraints in labeling (9-14). Likewise, the limited techniques that currently exist cannot ...

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.

We developed two-photon scanning patterned illumination microscopy (2P-SPIM) for super-resolution two-photon imaging. Our approach used a traditional two-photon microscopy setup with temporally modulated excitation to create patterned illumination fields. Combing nine different illuminations and structured illumination reconstruction, super-resolution imaging was achieved in two-photon microscopy. Using 2P-SPIM we achieved a lateral resolution of 141 nm, which represents an improvement by a factor of 1.9 over the corresponding diffraction limit. We further demonstrated super-resolution cellular imaging by 2P-SPIM to image actin cytoskeleton in mammalian cells and three-dimensional imaging in highly scattering retinal tissue.

The defect and impurity states in ZnO nanocrystals synthesized using the plasma arc technique can be modified to optimize the nonlinear optical properties for optoelectronic and biophotonic applications. Highly efficient second harmonic signals over a wide range of near-infrared wavelengths, spanning from 735 nm-980 nm, has been observed and can be used in biological imaging. The use of further high energy excitation ranging from 700 nm-755 nm leads to two-photon absorption and yields broadband two photon emission extending from the 370 nm-450 nm wavelength regime which can be useful for therapeutic applications.

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Anthracene dispersed in Polyphenylsiloxane (PPS) glass was synthesized on epitaxially grown zinc oxide (ZnO) to realize organic/inorganic hybrid semiconductors for efficient energy transfer. The photoluminescence (PL) from ZnO was modified by the presence of anthracene molecules due to resonant energy transfer. The UV-visible emission from anthracene molecule was also influenced due to resonant coupling with the excitonic and defect bound excitonic states in ZnO. Temperature dependence of PL of the hybrid system showed quenching of the defect bound emission of the ZnO to be due to energy transfer from anthracene. The PL lifetime in ZnO-anthracene/PPS hybrid structure at 4 K is relatively shorter and becomes comparable to the PL lifetimes in ZnO at 77 K. However, at room temperatures the PL lifetime of the hybrid structure is significantly longer than in ZnO and is comparable to the recombination lifetime in anthracene.

We investigated two-photon absorption-based photoacoustic generation and compared it with corresponding photoluminescence emission. Experimental results revealed expected quadratic dependences on the incident optical fluence in both photoacoustic and photoluminescence processes. We also investigated the influence of optical scattering on the generation of two-photon photoacoustic and photoluminescence signals and found that photoacoustic signals attenuated more slowly than photoluminescence signals when the optical scattering coefficient was increased, which was attributed to a weaker ultrasonic attenuation than that the optical attenuation in the scattering medium. Finally, we showed three-dimensional two-photon photoacoustic imaging.