Super-resolution Microscopy Techniques Research Articles

Spectrally Resolved and Functional Super-resolution Microscopy via Ultrahigh-Throughput Single-Molecule Spectroscopy.

As an elegant integration of the spatial and temporal dimensions of single-molecule fluorescence, single-molecule localization microscopy (SMLM) overcomes the diffraction-limited resolution barrier of optical microscopy by localizing single molecules that stochastically switch between fluorescent and dark states over time. While this type of super-resolution microscopy (SRM) technique readily achieves remarkable spatial resolutions of ∼10 nm, it typically provides no spectral information. Meanwhile, current scanning-based single-location approaches for mapping the positions and spectra of single molecules are limited by low throughput and are difficult to apply to densely labeled (bio)samples. In this Account, we summarize the rationale, design, and results of our recent efforts toward the integration of the spectral dimension of single-molecule fluorescence with SMLM to achieve spectrally resolved SMLM (SR-SMLM) and functional SRM ( f-SRM). By developing a wide-field scheme for spectral measurement and implementing single-molecule fluorescence on-off switching typical of SMLM, we first showed that in densely labeled (bio)samples it is possible to record the fluorescence spectra and positions of millions of single molecules synchronously within minutes, giving rise to ultrahigh-throughput single-molecule spectroscopy and SR-SMLM. This allowed us to first show statistically that for many dyes, single molecules of the same species exhibit near identical emission in fixed cells. This narrow distribution of emission wavelengths, which contrasts markedly with previous results at solid surfaces, allowed us to unambiguously identify single molecules of spectrally similar dyes. Crosstalk-free, multiplexed SRM was thus achieved for four dyes that were merely 10 nm apart in emission spectrum, with the three-dimensional SRM images of all four dyes being automatically aligned within one image channel. The ability to incorporate single-molecule fluorescence measurement with SMLM was next utilized to achieve f-SRM. By encoding functional information into the spectral responses of environment-sensing fluorescent probes, f-SRM transcends the structural information provided by typical SRM techniques and reveals the spatiotemporal distribution of physicochemical parameters with single-molecule sensitivity and nanoscale spatial resolution. As one example, by employing the solvatochromic dye Nile Red to sense local chemical polarity, we revealed nanoscale heterogeneity in the membranes of live mammalian cells. This enabled us to unveil substantial polarity differences between the plasma membrane and the membranes of nanoscale intracellular organelles, a result we determined to be due to differences in local cholesterol levels. With the addition of cholesterol or cholera toxin, we further observed the formation of low-polarity, raftlike nanodomains in the plasma membrane. In another study, we generalized SR-SMLM to fluorogenic single-molecule reactions. As a wide-field technique, SR-SMLM readily captures the emission spectra of individual product fluorescent molecules that are stochastically produced from nonfluorescent reactants at random locations over large sample areas, and therefore, it provides the unique possibility to spectrally identify and characterize single product molecules in a high-throughput fashion. Using the ring-opening reaction of a photochromic spiropyran as an example, we demonstrated that the capability to resolve the emission spectra of single product molecules could unveil rich, multipath reaction pathways. In summary, by integrating the spatial, temporal, and spectral dimensions of single-molecule fluorescence, SR-SMLM and f-SRM add rich spectral and functional dimensions to SRM and thus open up new ways of probing biological and chemical systems at the single-molecule and nanoscale levels.

Highly biocompatible super-resolution fluorescence imaging using the fast photoswitching fluorescent protein Kohinoor and SPoD-ExPAN with Lp-regularized image reconstruction.

Far-field super-resolution fluorescence microscopy has enabled us to visualize live cells in great detail and with an unprecedented resolution. However, the techniques developed thus far have required high-power illumination (102-106 W/cm2), which leads to considerable phototoxicity to live cells and hampers time-lapse observation of the cells. In this study we show a highly biocompatible super-resolution microscopy technique that requires a very low-power illumination. The present technique combines a fast photoswitchable fluorescent protein, Kohinoor, with SPoD-ExPAN (super-resolution by polarization demodulation/excitation polarization angle narrowing). With this technique, we successfully observed Kohinoor-fusion proteins involving vimentin, paxillin, histone and clathrin expressed in HeLa cells at a spatial resolution of 70-80 nm with illumination power densities as low as ~1 W/cm2 for both excitation and photoswitching. Furthermore, although the previous SPoD-ExPAN technique used L1-regularized maximum-likelihood calculations to reconstruct super-resolved images, we devised an extension to the Lp-regularization to obtain super-resolved images that more accurately describe objects at the specimen plane. Thus, the present technique would significantly extend the applicability of super-resolution fluorescence microscopy for live-cell imaging.

Pinpointing Unlabeled RNA Sequences Within a Protein-RNA Complex with Atomic Force Microscopy

Structural information of biomolecules is crucial to understand their working mechanisms and to develop drugs. However, determining biological structures at sub-nanometer resolution often demands tedious sample preparation and vast instrumentational resources. In addition, although super-resolution microscopy techniques have enabled tracking fluorescent molecules at the nanometer scale, the requisite of labeling fluorophores remains a hurdle in studying the innate structure of biological complexes. We have developed a multicolor microscopy that shows chemical identities on top of topography at sub-nanometer resolution based on atomic force microscopy. We record torsional deflection of T-shaped cantilevers to observe single-molecule interaction force on the microsecond timescale, and by modifying the AFM tip with a probe molecule that generates a distinct force-distance profile for each interaction with multiple targets, multicolor imaging is made possible. Here we present label-free imaging of a histone mRNA-protein complex using a specifically-designed probe DNA tip. Histone mRNAs have a conserved stem-loop (SL) structure which forms a ternary complex with stem-loop binding protein and 3’-5’ exoribonuclease. In the crystal structure, SL is sandwiched in between the two protein domains and both of the 5’- and 3’-flanking sequences of the SL are exposed. We have designed a probe DNA that partially hybridizes to each flank sequence for the specific recognition, and the rupture of the transiently formed duplex marks the position of the flank sequence. Superimposing consecutive images of the same complex has shown that the detection signals for each sequence were separately clustered on the complex. This imaging platform can be used to study structures of biological complexes with minimal sample preparation in the physiologically relevant conditions.

Probing the Elongated Structure of a Streptococcal Surface Protein using Structural and Single-Molecule Biophysical Approaches

The protein Sgo0707 from Streptococcus gordonii is expressed on the bacterial cell surface and is proposed to be involved in binding to oral keratinocytes. Similar to several other surface proteins of Gram-positive bacteria, Sgo0707 contains a repetitive C-terminal region; in this case, repeats contain 84-88 amino acids. The aim of this work is to determine if the repetitive region forms a rod-like structure that projects the N-terminal functional domain of the protein from the bacterial cell surface. We have used SHRImP-TIRFM, a super-resolution fluorescence microscopy technique, to measure the end-to-end distance of a 7 repeat protein construct, and SEC-MALS-QELS to measure its hydrodynamic radius. Combined with crystal structures of both single and double repeat structures, we will assess the elongation of the repetitive region.

<em>In Vivo</em> Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal.

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

Superresolution imaging of the synaptonemal complex.

This chapter describes how two different superresolution microscopy techniques, namely, structured illumination microscopy and direct stochastic optical reconstruction microscopy, can be used to analyze the molecular architecture of the synaptonemal complex. The experimental protocols provided allow the construction of precise localization maps for different synaptonemal complex proteins.

Tracking HOCl concentrations across cellular organelles in real time using a super resolution microscopy probe.

BODIPY derivative, SF-1, exclusively shows a fluorescence ON response to HOCl and images endogenously generated HOCl in RAW 264.7 macrophages. Widefield and super resolution structured illumination microscopy images confirm localization in the Golgi complex and lysosomes, and hence specifically detects HOCl generated in these organelles. SF-1 is compatible with 3D-SIM imaging of individual cells.

Resolution improvement in STED super-resolution microscopy at low power using a phasor plot approach.

Stimulated emission depletion (STED) microscopy is a powerful super-resolution microscopy technique that has achieved significant results in breaking the resolution limit and relevant applications. In principle, STED super resolution is obtained by stimulated emission partially inhibiting the spontaneous emission in the periphery of a diffraction-limited area. However, very high depletion laser power is generally necessary for the enhancement of imaging resolution, which is harmful to live biological specimens due to its high phototoxicity and photo-bleaching effects. Therefore, further improving the STED resolution at a lower depletion power level has recently attracted increasing interest from researchers in various fields. In this work, a phasor plot approach combined with fluorescence lifetime imaging microscopy (FLIM) is used to resolve the abovementioned problem based on a long- and short-lifetime criterion. Firstly, the time-resolved data obtained by STED-FLIM is converted to the frequency domain via a phasor approach. Next, partial data is extracted according to the information on the phase and amplitude for resolution improvement. Then, fluorescent microspheres (100 nm in diameter) are observed under different depletion powers, resulting in a series of improved resolution through phasor plots. Finally, this method is applied to image human Nup153 in fixed HeLa cells, providing a 86 nm higher resolution than that in traditional STED imaging at a depletion power of 20 mW.

Hybrid Structured Illumination Expansion Microscopy Reveals Microbial Cytoskeleton Organization.

Recently developed tissue-hydrogel methods for specimen expansion now enable researchers to perform super-resolution microscopy with ∼65 nm lateral resolution using ordinary microscopes, standard fluorescent probes, and inexpensive reagents. Here we use the combination of specimen expansion and the optical super-resolution microscopy technique structured illumination microscopy (SIM) to extend the spatial resolution to ∼30 nm. We apply this hybrid method, which we call ExSIM, to study the cytoskeleton of the important human pathogen Giardia lamblia including the adhesive disc and flagellar axonemes. We determined the localization of two recently identified disc-associated proteins, including DAP86676 , which localizes to disc microribbons, and the functionally unknown DAP16263 , which primarily localizes to dorsal microtubules of the disc overlap zone and the paraflagellar rod of ventral axonemes. Based on its strong performance in revealing known and unknown details of the ultrastructure of Giardia, we find that ExSIM is a simple, rapid, and powerful super-resolution method for the study of fixed specimens, and it should be broadly applicable to other biological systems of interest.

Trade-offs between structural integrity and acquisition time in stochastic super-resolution microscopy techniques.

The applicability of widefield stochastic microscopy, such as PALM or STORM, is limited by their long acquisition times. Images are produced from the accumulation of a large number of frames that each contain a scarce number of super-resolved localizations. We show that the random and uneven distribution of localizations leads to a specific type of trade-off between the spatial and temporal resolutions. We derive analytical predictions for the minimal time required to obtain a reliable image at a given spatial resolution. We find that the image completion time scales logarithmically with the ratio of the image size to the spatial resolution volume, with second order corrections due to spurious localization within the background noise. We validate our predictions against experimental localization sequences of labeled microtubule filaments obtained by STORM. Our theoretical framework makes it possible to compare the efficiency of emitters, define optimal labeling strategies, and allow implementation of a stopping criterion for data acquisitions that can be performed using real-time monitoring algorithms.

Supramolecular Assembly of Peptide Amphiphiles.

ConspectusPeptide amphiphiles (PAs) are small molecules that contain hydrophobic components covalently conjugated to peptides. In this Account, we describe recent advances involving PAs that consist of a short peptide sequence linked to an aliphatic tail. The peptide sequence can be designed to form β-sheets among the amino acids near the alkyl tail, while the residues farthest from the tail are charged to promote solubility and in some cases contain a bioactive sequence. In water, β-sheet formation and hydrophobic collapse of the aliphatic tails induce assembly of the molecules into supramolecular one-dimensional nanostructures, commonly high-aspect-ratio cylindrical or ribbonlike nanofibers. These nanostructures hold significant promise for biomedical functions due to their ability to display a high density of biological signals on their surface for targeting or to activate pathways, as well as for biocompatibility and biodegradable nature.Recent studies have shown that supramolecular systems, such as PAs, often become kinetically trapped in local minima along their self-assembly reaction coordinate, not unlike the pathways associated with protein folding. Furthermore, the assembly pathway can influence the shape, internal structure, and dimension of nanostructures and thereby affect their bioactivity. We discuss methods to map the energy landscape of a PA structure as a function of thermal energy and ionic strength and vary these parameters to convert between kinetically trapped and thermodynamically favorable states. We also demonstrate that the pathway-dependent morphology of the PA assembly can determine biological cell adhesion and survival rates.The dynamics associated with the nanostructures are also critical to their function, and techniques are now available to probe the internal dynamics of these nanostructures. For example, by conjugating radical electron spin labels to PAs, electron paramagnetic resonance spectroscopy can be used to study the rotational diffusion rates within the fiber, showing a liquidlike to solidlike transition through the cross section of the nanofiber. PAs can also be labeled with fluorescent dyes, allowing the use of super-resolution microscopy techniques to study the molecular exchange dynamics between PA fibers. For a weak hydrogen-bonding PA, individual PA molecules or clusters exchange between fibers in time scales as short as minutes. The amount of hydrogen bonding within PAs that dictates the dynamics also plays an important role in biological function. In one case, weak hydrogen bonding within a PA resulted in cell death through disruption of lipid membranes, while in another example reduced hydrogen bonding enhanced growth factor signaling by increasing lipid raft mobility.PAs are a promising platform for designing advanced hybrid materials. We discuss a covalent polymer with a rigid aromatic imine backbone and alkylated peptide side chains that simultaneously polymerizes and interacts with a supramolecular PA structure with identical chemistry to that of the side chains. The covalent polymerization can be “catalyzed” by noncovalent polymerization of supramolecular monomers, taking advantage of the dynamic nature of supramolecular assemblies. These novel hybrid structures have potential in self-repairing materials and as reusable scaffolds for delivery of drugs or other chemicals. Finally, we highlight recent biomedical applications of PAs and related structures, ranging from bone regeneration to decreasing blood loss during internal bleeding.

Development of the fluorescent imaging method of single-molecule sRNA SgrS <italic>in situ</italic> in <italic>Escherichia coli</italic>

Single-cell gene expression studies could not only provide the real-time information on intracellular amount and subcellular localization of RNA, but also could reveal the regulatory mechanism of bacterial variation, which cannot be obtained based on traditional view of bacterial populations. Owing to the application of green fluorescent protein (GFP) reporters, most of studies have mainly focused on proteins. Recently, the quantitative and localization of bacterial RNAs have been also implemented at single transcript level. All of the non-coding RNAs who function as crucial regulators of gene expression are called regulatory small RNAs (sRNA) in bacteria. Single molecular imaging techniques for labeling bacterial sRNA can be used to detect the activity and abundance of sRNA, which will help us to understand its regulatory mechanism and biological function. SgrS, a conservative sRNA in γ-proteobacteria, is highly expressed under glucose-phosphate stress and involve in the sugar metabolism in bacteria. We chose SgrS as a model to establish the single-molecule fluorescence in situ hybridization (smFISH) method of RNA detection at the single-molecule level in Escherichia coli . It will be helpful for further studies on sRNA subcellular localization and gene regulation. We used kanamycin resistance box replace sgrS to successful construct sgrS deletion mutant Δ sgrS and subsequently cured the helper plasmid pKD46. The SgrS overexpression strain Δ sgrS -pBAD-SgrS was generated by introducing SgrS-overexpressed plasmid into the Δ sgrS strains. E. coli MG1655, Δ sgrS and Δ sgrS -pBAD-SgrS, all E. coli K-12 derivatives, were used in this study. The smFISH method on SgrS in E. coli was established. The optimal conditions are briefly stated as follows. The cells were fixed in 3.7% formaldehyde at room temperature for 30 minutes. Cells were permeabilized at 4°C for overnight. Washed cells with 40% wash solution was resuspended in 40% hybridization buffer. The cells were centrifuged and resuspended in the hybridization buffer with 1 μM sRNAs probes that denatured at 98°C for 10 minutes. Hybridization reactions were incubated in the hybridization oven at 40°C for 3 hours. The cells were washed five times with 40% wash solution. During wash, cells were resuspended and incubated at 40°C for 30 minutes. Cells were pelleted by 600× g centrifugation for 3 minutes, and the supernatant was removed. Cells were resuspended with 100 μL of freshly prepared 5 μg mL− 1 Alexa-555 WGA in 1×PBS, and incubated at room temperature for 10 minutes. Cells were pelleted by 600× g centrifugation for 3 minutes, and the supernatant was removed. The cells were resuspended in 5 μL of 2×SSC. Then 1 μL of sample and 1 μL of SlowFade Diamond Antifade Mountant were pipetted to poly-L-lysine treated borosilicate chambered coverslip. A coverslip was placed on the sample. Super-resolution imaging was performed on Nikon inverted microscope with a 100× oil immersion objective. SgrS was indicated as green fluorescence granular with diffuse distribution in cytoplasm, while cellular wall images exhibited red fluorescence upon being stained with Alexa-555 WGA. The combination of smFISH method and super-resolution microscopy techniques provides the potential clues and technical reference for further studies on revealing sRNA localization and sRNA-mediated regulatory mechanism.

Getting a bigger picture

Superresolution microscopy techniques allow researchers to observe objects tens of nanometers in size on or inside cells. But the methods can keep an eye on patches only 100 μm or less on a side at...

Coalescence of Nanoscale Cytoplasmic Signalosomes Contributes to T Cell Receptor Signaling to NF-κB

Abstract T cell receptor (TCR) activation of the transcription factor NF-κB is a crucial determinant of effector T lymphocyte function. The complex regulatory network surrounding this pathway remains poorly understood, particularly at time periods further removed from initial TCR triggering. We have previously demonstrated that following activation of the TCR, the proteins p62, Bcl10, and Malt1 rapidly combine to form a cytoplasmic filamentous signalosome called POLKADOTS, which recruits further signaling proteins and initiates the terminal steps in activation of NF-κB. Here, we examine the fate of POLKADOTS signalosomes following initial activation of NF-κB. We demonstrate via super-resolution and confocal microscopy techniques that POLKADOTS filaments converge on the microtubule-organizing center via microtubule transport, aggregating in a peri-nuclear aggresomal structure. Aggresomes are poorly understood structures thought to be depots of misfolded protein destined for degradation; however, our data suggest that the aggresomal accumulations of POLKADOTS continue to promote T cell activation. We show that the formation and maintenance of this aggresome corresponds with a secondary increase in NF-κB translocation. Additionally, the aggresomal structure prolongs phosphorylation of IKK and promotes the proteolytic activity of Malt1. Together, these results demonstrate that TCR signaling to NF-κB directs the orchestrated assembly, transport, and stable coalescence of nanoscale cytoplasmic signaling complexes. More broadly, our findings provide evidence that aggresomes can serve as stable platforms of signal transduction, thereby proposing a new role for these little understood structures.

Application of super-resolution microscopy to the study of B and T lymphocyte mitochondria morphology and metabolism

Abstract High-resolution microscopy techniques have advanced our understanding of lymphocyte biology. An emerging focus in the field of immunometabolism on activation-induced metabolic reprogramming and its effects on immune cell function will require in-depth analyses of mitochondria. However, even with newer imaging technologies, obtaining super-resolution images of lymphocyte mitochondria remains challenging due to their small cytoplasmic sizes. Here, we compared standard confocal laser microscopy with three commonly used super-resolution methods to analyze activation-induced mitochondrial changes in B and T cells. Naïve B cells were stimulated in vitro via Toll-like receptor 9 and/or the B cell receptor, and naïve T cells were differentiated in vitro into activated or T regulatory phenotypes. The cells were imaged using standard laser confocal microscopy, AiryScan, stimulated emission depletion (STED) microscopy, and computerized tomography electron microscopy (TEM). Mitochondrial morphology was best resolved by staining both the mitochondrial membrane and matrix simultaneously. In preliminary analyses, we observed activation-induced changes in the distribution of lymphocyte mitochondria. Of the imaging methods used, only TEM allowed for 3D reconstructions of the mitochondria’s internal structure, which revealed swelling of the inner matrix and significant loss of cristae in B cells, but not in T cells. In conclusion, the super-resolution light microscopy techniques we developed appear to be powerful tools for immunometabolism studies of lymphocyte mitochondria, having demonstrated clear visualization even at the molecular level, while TEM answered key questions about intra-mitochondrial morphology.

Review of combined isotopic and optical nanoscopy.

Investigating the detailed substructure of the cell is beyond the ability of conventional optical microscopy. Electron microscopy, therefore, has been the only option for such studies for several decades. The recent implementation of several super-resolution optical microscopy techniques has rendered the investigation of cellular substructure easier and more efficient. Nevertheless, optical microscopy only provides an image of the present structure of the cell, without any information on its long-temporal changes. These can be investigated by combining super-resolution optics with a nonoptical imaging technique, nanoscale secondary ion mass spectrometry, which investigates the isotopic composition of the samples. The resulting technique, combined isotopic and optical nanoscopy, enables the investigation of both the structure and the "history" of the cellular elements. The age and the turnover of cellular organelles can be read by isotopic imaging, while the structure can be analyzed by optical (fluorescence) approaches. We present these technologies, and we discuss their implementation for the study of biological samples. We conclude that, albeit complex, this type of technology is reliable enough for mass application to cell biology.

Super-resolution Microscopy - Applications in Plant Cell Research.

Most of the present knowledge about cell organization and function is based on molecular and genetic methods as well as cytological investigations. While electron microscopy allows identifying cell substructures until a resolution of ∼1 nm, the resolution of fluorescence microscopy is restricted to ∼200 nm due to the diffraction limit of light. However, the advantage of this technique is the possibility to identify and co-localize specifically labeled structures and molecules. The recently developed super-resolution microscopy techniques, such as Structured Illumination Microscopy, Photoactivated Localization Microscopy, Stochastic Optical Reconstruction Microscopy, and Stimulated Emission Depletion microscopy allow analyzing structures and molecules beyond the diffraction limit of light. Recently, there is an increasing application of these techniques in cell biology. This review evaluates and summarizes especially the data achieved until now in analyzing the organization and function of plant cells, chromosomes and interphase nuclei using super-resolution techniques.

Fluorescence Microscopy: A Concise Guide to Current Imaging Methods

The field of fluorescence microscopy is rapidly growing and offers ever more imaging capabilities for biologists. Over the past decade, many new technologies and techniques have been developed that allow for combinations of deeper, faster, and higher resolution imaging. These have included the commercialization of many super-resolution and light sheet fluorescence microscopy techniques. For the non-expert, it can be difficult to match the best imaging techniques to biological questions. Picking the most appropriate imaging modality requires a basic understanding of the underlying physics governing each of them, as well as information comparing potentially competing imaging properties in the context of the sample to be imaged. To address these issues, we provide here concise descriptions of a wide range of commercially available imaging techniques from wide-field to super-resolution microscopy, and provide a tabular guide to aid in comparisons among them. In this manner we provide a concise guide to understanding and matching the correct imaging modality to meet research needs. © 2017 by John Wiley & Sons, Inc.

High-numerical-aperture cryogenic light microscopy for increased precision of superresolution reconstructions.

Superresolution microscopy has fundamentally altered our ability to resolve subcellular proteins, but improving on these techniques to study dense structures composed of single-molecule-sized elements has been a challenge. One possible approach to enhance superresolution precision is to use cryogenic fluorescent imaging, reported to reduce fluorescent protein bleaching rates, thereby increasing the precision of superresolution imaging. Here, we describe an approach to cryogenic photoactivated localization microscopy (cPALM) that permits the use of a room-temperature high-numerical-aperture objective lens to image frozen samples in their native state. We find that cPALM increases photon yields and show that this approach can be used to enhance the effective resolution of two photoactivatable/switchable fluorophore-labeled structures in the same frozen sample. This higher resolution, two-color extension of the cPALM technique will expand the accessibility of this approach to a range of laboratories interested in more precise reconstructions of complex subcellular targets.