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Fluorophores are powerful tools for interrogating biological systems. Carbon nanotubes (CNTs) have long been attractive materials for biological imaging due to their near-infrared excitation and bright, tunable optical properties. The difficulty in synthesizing and functionalizing these materials with precision, however, has hampered progress in this area. Carbon nanohoops, which are macrocyclic CNT substructures, are carbon nanostructures that possess ideal photophysical characteristics of nanomaterials, while maintaining the precise synthesis of small molecules. However, much work remains to advance the nanohoop class of fluorophores as biological imaging agents. Herein, we report an intracellular targeted nanohoop. This fluorescent nanostructure is noncytotoxic at concentrations up to 50 μM, and cellular uptake investigations indicate internalization through endocytic pathways. Additionally, we employ this nanohoop for two-photon fluorescence imaging, demonstrating a high two-photon absorption cross-section (65 GM) and photostability comparable to a commercial probe. This work further motivates continued investigations into carbon nanohoop photophysics and their biological imaging applications.

Correlative light and electron microscopy (CLEM) is a powerful tool for defining the ultrastructural context of molecularly-labeled biological specimens, particularly when superresolution fluorescence microscopy (SRM) is used for CLEM. Current CLEM, however, is limited by the stark differences in sample preparation requirements between the two modalities. For CLEM using SRM, the small region of interest (ROI) of either or both modalities also leads to low success rate and imaging throughput. To overcome these limitations, here we present a CLEM workflow based on a novel focused ion beam/scanning electron microscope (FIB/SEM) compatible with common SRM for imaging biological specimen with ultrahigh 3D resolution and improved imaging throughput. By using a reactive oxygen source in a plasma FIB (PFIB) and a rotating sample stage, the novel FIB/SEM was able to achieve several hundreds of micrometer large area 3D analysis of resin embedded cells through a process named oxygen serial spin mill (OSSM). Compared with current FIB mechanisms, OSSM offers gentle erosion, highly consistent slice thickness, reduced charging during SEM imaging, and improved SEM contrast without increasing the dose of post-staining and fixation. These characteristics of OSSM-SEM allowed us to pair it with interferometric photoactivated localization microscopy (iPALM), a recent SRM technique that affords 10–20 nm isotropic spatial resolution on hydrated samples, for 3D CLEM imaging. We demonstrate a CLEM workflow generalizable to using other SRM strategies using mitochondria in human osteosarcoma (U2OS) cells as a model system, where immunostained TOM20, a marker for the mitochondrial outer membrane, was used for iPALM. Owing to the large scan area of OSSM-SEM, it is now possible to select as many FOVs as needed for iPALM and conveniently re-locate them in EM, this improving the imaging throughput. The significantly reduced dose of post-fixation also helped to better preserve the sample ultrastructures as evidenced by the excellent 3D registration between OSSM-SEM and iPALM images and by the accurate localization of TOM20 (by iPALM) to the peripheries of mitochondria (by OSSM-SEM). These advantages make OSSM-SEM an ideal modality for CLEM applications. As OSSM-SEM is still in development, we also discuss some of the remaining issues and the implications to biological imaging with SEM alone or with CLEM.

DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) facilitates multiplexing in superresolution microscopy but is practically limited by slow imaging speed. We have developed DNA-PAINT-ERS, where E=ethylene carbonate, R=repeating sequence, and S=spacer, for fast and multiplexed superresolution imaging with DNA-PAINT. Here we describe detailed procedures for DNA-PAINT-ERS including reagent preparation, sample labeling, as well as image acqusition and analysis.

DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) facilitates multiplexing in superresolution microscopy but is practically limited by slow imaging speed. To address this issue, we propose the additions of ethylene carbonate (EC) to the imaging buffer, sequence repeats to the docking strand, and a spacer between the docking strand and the affinity agent. Collectively termed DNA-PAINT-ERS (E = EC, R = Repeating sequence, and S = Spacer), these strategies can be easily integrated into current DNA-PAINT workflows for both accelerated imaging speed and improved image quality through optimized DNA hybridization kinetics and efficiency. We demonstrate the general applicability of DNA-PAINT-ERS for fast, multiplexed superresolution imaging using previously validated oligonucleotide constructs with slight modifications., DNA-PAINT is a powerful super-resolution imaging method but is limited in speed due to slow exchange kinetics of the imaging strand. Here the authors present a method involving the addition of ethylene carbonate to the imaging buffer and modifications to the docking strand to improve the quality and speed of DNA-PAINT.

Multicolor single-molecule localization microscopy (SMLM) expands our understanding of subcellular details and enables the study of biomolecular interactions through precise visualization of multiple molecules in a single sample with resolution of ~10-20 nm. Probe selection is vital to multicolor SMLM, as the fluorophores must not only exhibit minimal spectral crosstalk, but also be compatible with the same photochemical conditions that promote fluorophore photoswitching. While there are numerous commercially available photoswitchable fluorophores that are optimally excited in the standard Cy3 channel, they are restricted to short Stokes shifts (

Sample preparation is critical to biological electron microscopy (EM), and there have been continuous efforts on optimizing the procedures to best preserve structures of interest in the sample. However, a quantitative characterization of the morphological changes associated with each step in EM sample preparation is currently lacking. Using correlative EM and superresolution microscopy (SRM), we have examined the effects of different drying methods as well as osmium tetroxide (OsO4) post-fixation on cell morphology during scanning electron microscopy (SEM) sample preparation. Here, SRM images of the sample acquired under hydrated conditions were used as a baseline for evaluating morphological changes as the sample went through SEM sample processing. We found that both chemical drying and critical point drying lead to a mild cellular boundary retraction of ~60 nm. Post-fixation by OsO4 causes at least 40 nm additional boundary retraction. We also found that coating coverslips with adhesion molecules such as fibronectin prior to cell plating helps reduce cell distortion from OsO4 post-fixation. These quantitative measurements offer useful information for identifying causes of cell distortions in SEM sample preparation and improving current procedures.

Single-molecule localization microscopy (SMLM) image quality and resolution strongly depend on the photoswitching properties of fluorophores used for sample labeling. Development of fluorophores with optimized photoswitching will considerably improve SMLM spatial and spectral resolution. Currently, evaluating fluorophore photoswitching requires protein-conjugation before assessment mandating specific fluorophore functionality, which is a major hurdle for systematic characterization. Herein, we validated polyvinyl alcohol (PVA) as a single-molecule environment to efficiently quantify the photoswitching properties of fluorophores and identified photoswitching properties predictive of quality SMLM images. We demonstrated that the same fluorophore photoswitching properties measured in PVA films and using antibody adsorption, a protein-conjugation environment analogous to labeled cells, were significantly correlated to microtubule width and continuity, surrogate measures of SMLM image quality. Defining PVA as a fluorophore photoswitching screening platform will facilitate SMLM fluorophore development and optimal image buffer assessment through facile and accurate photoswitching property characterization, which translates to SMLM fluorophore imaging performance.

Millions of archived formalin-fixed, paraffin-embedded (FFPE) specimens contain valuable molecular insight into healthy and diseased states persevered in their native ultrastructure. To diagnose and treat diseases in tissue on the nanoscopic scale, pathology traditionally employs electron microscopy (EM), but this platform has significant limitations including cost and painstaking sample preparation. The invention of single molecule localization microscopy (SMLM) optically overcame the diffraction limit of light to resolve fluorescently labeled molecules on the nanoscale, leading to many exciting biological discoveries. However, applications of SMLM in preserved tissues has been limited. Through adaptation of the immunofluorescence workflow on FFPE sections milled at histological thickness, cellular architecture can now be visualized on the nanoscale using SMLM including individual mitochondria, undulations in the nuclear lamina, and the HER2 receptor on membrane protrusions in human breast cancer specimens. Using astigmatism imaging, these structures can also be resolved in three dimensions to a depth of ~800 nm. These results demonstrate the utility of SMLM in efficiently uncovering ultrastructural information of archived clinical samples, which may offer molecular insights into the physiopathology of tissues to assist in disease diagnosis and treatment using conventional sample preparation methods.

Single-molecule localization microscopy (SMLM) utilizes photoswitchable fluorophores to detect biological entities with 10-20 nm resolution. Multispectral superresolution microscopy (MSSRM) extends SMLM functionality by improving its spectral resolution up to 5 fold facilitating imaging of multicomponent cellular structures or signaling pathways. Current commercial fluorophores are not ideal for MSSRM as they are not designed to photoswitch and do not adequately cover the visible and far-red spectral regions required for MSSRM imaging. To obtain optimal MSSRM spatial and spectral resolution, fluorophores with narrow emission spectra and controllable photoswitching properties are necessary. Herein, a library of BODIPY-based fluorophores was synthesized and characterized to create optimal photoswitchable fluorophores for MSSRM. BODIPY was chosen as the core structure as it is photostable, has high quantum yield, and controllable photoswitching. The BODIPY core was modified through the addition of various aromatic moieties, resulting in a spectrally diverse library. Photoswitching properties were characterized using a novel polyvinyl alcohol (PVA) based film methodology to isolate single molecules. The PVA film methodology enabled photoswitching assessment without the need for protein conjugation, greatly improving screening efficiency of the BODIPY library. Additionally, image buffer conditions were optimized for the BODIPY-based fluorophores through systematic testing of oxygen scavenger systems, redox components, and additives. Through screening the photoswitching properties of BODIPY-based compounds in PVA films with optimized imaging buffer we identified novel fluorophores well suited for SMLM and MSSRM.

We propose a surface condensation method for assembling single-walled carbon nanotubes (SWNTs) on gold. The as-prepared long and randomly tangled SWNTs were cut into short pipes by chemical oxidation, allowing the nanotubes to be terminated by carboxyl functionalities. A surface condensation reaction was then performed by immersing an amino self-assembled monolayer (SAM)-modified gold substrate into the dimethylformamide suspension of carboxylic nanotubes with the aid of dicyclohexylcarbodiimide condensation agent. Raman spectroscopy and atomic force microscopy (AFM) results show that a highly aligned assembly of SWNTs has been formed on gold, with the nanotubes standing on the surface stable enough for a long ultrasonication. In combination with the microcontact printing (muCP) technique, we have fabricated patterned nanotube assemblies using this surface condensation method. Moreover, we found that the "giant" carbon nanotubes tend to form bundles on an amino-terminating surface, likely following a nucleation-growth model.

Super resolution microscopy (SRM) has overcome the historic spatial resolution limit of light microscopy, enabling fluorescence visualization of cellular structures and multi-protein complexes at the nanometer scale. Using single-molecule localization microscopy, the precise location of a stochastically activated population of photoswitchable fluorophores is determined during the collection of many images to form a single image with resolution of ~10–20 nm, an order of magnitude improvement over conventional microscopy. However, the spectral resolution of current SRM techniques are limited by existing fluorophores with only up to four colors imaged simultaneously, limiting the number of intracellular components that can be studied in a single sample. In the current work, a library of novel BODIPY-based fluorophores was synthesized using a solid phase synthetic platform with the goal of creating a set of photoswitchable fluorophores that can be excited by 5 distinct laser lines but emit throughout the spectral range (450–850 nm) enabling multispectral super resolution microscopy (MSSRM). The photoswitching properties of all new fluorophores were quantified for the following key photoswitching characteristics: (1) the number of photons per on cycle (2) the number of on cycles (switching events), (3) the percentage of time the fluorophore spends in the fluorescent on and off states, and (4) the susceptibility of the fluorophore to photobleaching (time of last event). To ensure the accuracy of our photoswitching measurements, our methodology to detect and quantitate the photoswitching properties of individual fluorophore molecules was validated by comparing measured photoswitching properties of three commercial dyes to published results.(1) We also identified two efficient methods to positionally isolate fluorophores on coverglass for screening of the BODIPY-based library.

Rapid advances of CARS microscopy occurred in the past few years, including significant improvements in sensitivity and imaging speed, understanding of contrast mechanisms, more reliable and less expensive laser sources. Many biomedical applications begin to emerge. Our latest work of CARS microscopy will be reported. Full-text article is not available.

Recent advances have improved the capability of CARS microscopy for imaging live cells with high sensitivity. In this contribution an overview of the developments in CARS microscopy is given along with applications to cell biology.