KEYWORDS: Super resolution, Multiplexing, Denoising, Signal to noise ratio, Molecules, Microscopy, Algorithm development, Spectroscopy, Spectral resolution, Single molecule spectroscopy
The spectroscopic Single-Molecule Localization Microscopy (sSMLM) is an emerging tool offering exciting new capabilities for single-molecule localization and tracking. By simultaneously capturing the spatial location and full spectra of fluorescent emission from single molecules, sSMLM can significantly extend the number of distinct target species for multiplexed super-resolution imaging and offer desirable resolving power for functional super-resolution imaging. However, extracting accurate spectral information in sSMLM remains challenging due to the poor signal-to-noise ratio (SNR) of spectral images set by limited photon budget from single-molecule fluorescent emission and inherent electronic noise during the image acquisition using digital cameras. Here, we developed an unsupervised learning-based method consisting of a convolutional autoencoder and an unsupervised clustering algorithm for high-fidelity spectral denoising and high-accurate molecular discrimination. Compared to existing learning-based spectral classification methods used in sSMLM, highly accurate molecular discrimination can be achieved even at low photon budget while high-SNR spectra can be simultaneously recovered without undermining the fidelity of the spectral details. Since the developed data processing flow can be directly integrated into any current sSMLM hardware, we hope the unsupervised spectral denoising and classification method can facilitate highly accurate molecular discrimination for multiplexed super-resolution imaging and single-molecule tracking.
The field of view (FOV) of single-molecule localization microscopy (SMLM) is practically restricted to an area of approximately 50×50 μm^2 as only relatively perfect point spread functions (PSFs) in the center of the objective FOV can be used for accurate super-resolution image reconstruction. Here we present a systematical study to show that optical aberrations, such as spherical aberration, coma, field curvature, and chromatic aberration, commonly presented in the FOV periphery can significantly compromise the localization precision and accuracy and produce unreliable imaging results when using currently available localization algorithm for SMLM image reconstruction.
The existence of fluorescent impurities has been a long-standing obstacle in single-molecule imaging, which results in sample misidentification and higher localization uncertainty. Spectroscopic single-molecule localization microscopy can record the full fluorescent spectrum of every stochastic single-molecule emission event. This capability allows us to quantify the spatial and spectral characteristics of fluorescent impurities introduced by sample preparation steps, based on which we developed a method to effectively separate fluorescent impurities from target molecules.
Fluorescence photo-switching of native, unmodified DNA using visible light enables label-free, nanoscale, single-molecule photon localization microscopy (PLM) of chromatin structure. Compared with conventional label-based super resolution imaging techniques, the label-free DNA-PLM has the advantage of faithfully resolving the native nucleotides under non-perturbing conditions, thus allowing a reliable analysis of the chromatin organization. Recently, we have developed an algorithm to quantify the chromatin spatial distribution based on label-free DNA-PLM images by calculating the fractal dimension from the chromatin cluster size and the number of photon emission events. For demonstration, we employed label-free DNA-PLM with TIRF illumination, and imaged the nuclei of ovarian cancer cells with three descending chromatin heterogeneities: the P53 mutation (M248), the wild type (A2780), and the wild type treated with a commonly-used chemotherapeutic drug celecoxib (Cele). Using the algorithm, we extracted the fractal dimensions for nuclear chromatin. We found that the fractal dimension is between 2 to 3 for all cells, which lies in the range of reported values from other techniques (e.g., TEM). We also observed that M248 has the highest fractal dimension while Cele has the lowest, a perfect match with the experimental expectations. We believe this study can provide a new approach to quantify label-free super-resolution imaging of macromolecular structures and could contribute to our knowledge of native in-vitro nuclear chromatin configurations.
High-resolution optical longitudinal cortical imaging usually uses cranial window, which involves removing a skull portion and sealing the exposed brain area with a transparent cover glass, allowing ballistic photons to reach the cortex with minimal disturbance of the brain function. It enables obtaining high-resolution brain images in extended periods of time for long-term neuronal activity studies using confocal and two-photon microscopies. Photoacoustic microscopy (PAM), as the only imaging method that directly measure absorption contrast, is a complementary functional imaging method to provide absorption related brain information, such as total concentration of hemoglobin and oxygen saturation of hemoglobin. However, the use of traditional piezoelectric transducers (PZT) to collect ultrasound signal greatly limits the versatility of PAM. Though highly sensitive, PZT transducers are usually bulky and optically opaque. It blocks the light and is hard to be inserted into the limited distance between the optical objective and imaging sample, which are normally less than one millimeter when using a high- numerical aperture (NA) objective to achieve submicron resolution.
Here, we developed a simple and cost-efficient soft nanoimprint lithography (NIL) process to fabricate fully embedded micro-ring resonator ultrasound detectors on optically transparent substrates, and integrated the detector onto a cranial window, making cranial window itself an ultrasonic detector. We implanted this functional cranial window on mouse head and achieved longitudinal monitoring of cortex vasculature using PAM. Our low-cost, disposable, and optically transparent detector may potentially reshape the longitudinal functional brain imaging using PAM in small animals.
The spectroscopic information and the corresponding polarization states of a single-molecule emission possess wealth molecule-specific signatures that can be used to reveal the unique molecular electronic state, conformation, and its interactions with the host media. However, existing spectroscopic methods and advanced image analysis techniques, which can potentially provide quantitative analytical tools for the study of cellular dynamics, are yet limited by the diffraction limit. Therefore, developing a nanoscopic imaging platform for simultaneous acquisition of multiple molecular specific properties is highly desirable. Here we report a three-dimensional (3D), polarization-sensitive, spectroscopic photon localization microscopy (3D-Polar-SPLM) that simultaneously captures nanoscopic location of individual fluorescent emitters and their corresponding optical spectra and polarization states. To evaluate the capability of the imaging system, we imaged model system consisting quantum rods (QRs). Using 3D-Polar-SPLM, we spatially localized individual QRs with a lateral localization precision of 8 nm and an axial localization precision of 35 nm. In addition, we achieved a spectral resolution of 2 nm and a polarization angle measuring precision of 8 degrees. The spectral profile of the fluorescence emission provided a particle-specific signature for identifying individual QRs among the heterogeneous population, which significantly improved the fidelity in parallel 3D tracking of multiple QRs at a temporal resolution of 10 ms. Except its versatility, 3D-Polar-SPLM further provides advantageous in practical applications since it only employs a single light-path and therefore, is compatible with existing PALM/STORM, potentially bringing immediate impact to the broader research community, across physics, chemistry, material science and biology.
Recently, 3D printing has gone beyond being an industrial prototyping process and has gradually evolved as the tool to manufacture production-quality parts that are otherwise challenging by using traditional methods. Especially, translating 3D printing technique into the optical realm would dramatically improve the time- and cost-efficiency of customized optical elements, while conventional methods such as multiaxial lathes polishing, magnetorheological finishing, molding techniques are relatively expensive and time consuming. However, 3D printing also suffers from the inherent drawback: the reduced surface quality associated with the stair-stepping effect as a direct result of the layered deposition of the material. In this paper, we have demonstrated a time- and cost-effective single photon micro-stereolithography based 3D printing method to eliminate the layer stair-stepping effect. This method supports not only sub-voxel accuracy (~ 2 μm) of the surface in the range of 2 mm diameter, but also features deep sub-wavelength roughness (< 10 nm) of the surfaces and extremely good reproducibility. Furthermore, we designed and rapidly prototyped the aspherical lenses which not only feature low distortion, but also show remarkable optical quality in a broadband wavelength range from 400 nm to 800 nm.
Imaging the nanoscale intracellular structures formed by nucleic acids, such as chromatin, in non-perturbed, structurally and dynamically complex cellular systems, will help improve our understanding of biological processes and open the next frontier for biological discovery. Current optical super-resolution fluorescence techniques require exogenous labels that may disrupt cell function and alter the subdiffractional macromolecular structures they are used to visualize. As a means for label-free optical super-resolution imaging, we examined the discovery of stochastic fluorescence switching of unmodified nucleic acids under visible light illumination. Utilizing this phenomenon and a single-molecule photon localization approach we generated subdiffraction-resolution images down to ~20nm using intrinsic fluorescence from nucleic acids. Specifically, the nanoscale organization of interphase nuclei and mitotic chromosomes were imaged. Using such a method for visualization, we performed a quantitative analysis of the DNA occupancy level and a subdiffractional analysis of the chromosomal organization. These experiments demonstrate a new method for visualizing the nanoscopic features of macromolecular structures composed of nucleic acids without the need for exogenous labels.
Distinguishing minute differences in spectroscopic signatures is crucial for revealing the fluorescence heterogeneity among fluorophores to achieve a high molecular specificity. Here we report spectroscopic photon localization microscopy (SPLM), a newly developed far-field spectroscopic imaging technique, to achieve nanoscopic resolution based on the principle of single-molecule localization microscopy while simultaneously uncovering the inherent molecular spectroscopic information associated with each stochastic event (Dong et al., Nature Communications 2016, in press). In SPLM, by using a slit-less monochromator, both the zero-order and the first-order diffractions from a grating were recorded simultaneously by an electron multiplying charge-coupled device to reveal the spatial distribution and the associated emission spectra of individual stochastic radiation events, respectively. As a result, the origins of photon emissions from different molecules can be identified according to their spectral differences with sub-nm spectral resolution, even when the molecules are within close proximity. With the newly developed algorithms including background subtraction and spectral overlap unmixing, we established and tested a method which can significantly extend the fundamental spatial resolution limit of single molecule localization microscopy by molecular discrimination through spectral regression. Taking advantage of this unique capability, we demonstrated improvement in spatial resolution of PALM/STORM up to ten fold with selected fluorophores. This technique can be readily adopted by other research groups to greatly enhance the optical resolution of single molecule localization microscopy without the need to modify their existing staining methods and protocols. This new resolving capability can potentially provide new insights into biological phenomena and enable significant research progress to be made in the life sciences.
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