Photoactivated Localization Microscopy Imaging Research Articles

Characterizing locus specific chromatin structure and dynamics with correlative conventional and super-resolution imaging in living cells.

The dynamic rearrangement of chromatin is critical for gene regulation, but mapping both the spatial organization of chromatin and its dynamics remains a challenge. Many structural conformations are too small to be resolved via conventional fluorescence microscopy and the long acquisition time of super-resolution photoactivated localization microscopy (PALM) precludes the structural characterization of chromatin below the optical diffraction limit in living cells due to chromatin motion. Here we develop a correlative conventional fluorescence and PALM imaging approach to quantitatively map time-averaged chromatin structure and dynamics below the optical diffraction limit in living cells. By assigning localizations to a locus as it moves, we reliably discriminate between bound and unbound dCas9 molecules, whose mobilities overlap. Our approach accounts for changes in DNA mobility and relates local chromatin motion to larger scale domain movement. In our experimental system, we show that compacted telomeres move faster and have a higher density of bound dCas9 molecules, but the relative motion of those molecules is more restricted than in less compacted telomeres. Correlative conventional and PALM imaging therefore improves the ability to analyze the mobility and time-averaged nanoscopic structural features of locus specific chromatin with single molecule sensitivity and yields unprecedented insights across length and time scales.

CRISPR/Cas12a-mediated labeling of MET receptor enables quantitative single-molecule imaging of endogenous protein organization and dynamics.

SummarySingle-molecule localization microscopy (SMLM) reports on protein organization in cells with near-molecular resolution and in combination with stoichiometric labeling enables protein counting. Fluorescent proteins allow stoichiometric labeling of cellular proteins; however, most methods either lead to overexpression or are complex and time demanding. We introduce CRISPR/Cas12a for simple and efficient tagging of endogenous proteins with a photoactivatable protein for quantitative SMLM and single-particle tracking. We constructed a HEK293T cell line with the receptor tyrosine kinase MET tagged with mEos4b and demonstrate full functionality. We determine the oligomeric state of MET with quantitative SMLM and find a reorganization from monomeric to dimeric MET upon ligand stimulation. In addition, we measured the mobility of single MET receptors in vivo in resting and ligand-treated cells. The combination of CRISPR/Cas12a-assisted endogenous protein labeling and super-resolution microscopy represents a powerful tool for cell biological research with molecular resolution.

Characterization of Photophysical Properties of Photoactivatable Fluorescent Proteins for Super-Resolution Microscopy.

Photoactivatable fluorescent proteins (PA-FPs) are widely used in photoactivated localization microscopy (PALM), and information about their blinking properties is important for precise assembling of PALM images and for quantitative molecular counting with PALM. As the photophysical properties of fluorescent proteins depend sensitively on the local physiochemical environment, we developed a series of procedures to characterize the properties of the PA-FPs in fixed cells. As an example, we characterized the properties of Dendra2 in Escherichia coli and found that they are appreciably different from those in vitro. This highlights the importance of characterizing the photophysical properties of PA-FPs under the same experimental conditions as subsequent PALM imaging or molecular counting experiments. Other PA-FPs could be characterized using this strategy.

Strategy to Lengthen the On-Time of Photochromic Rhodamine Spirolactam for Super-resolution Photoactivated Localization Microscopy.

Rhodamine derivatives and analogues have been widely used for different super-resolution imaging techniques, including photoactivated localization microscopy (PALM). Among them, rhodamine spirolactams exhibit great superiority for PALM imaging due to a desirable bright-dark contrast during the photochromic switching process. Although considerable attention has been paid to the chemical modifications on rhodamine spirolactams, the on-time of photochromic switching, one of the key characteristics for PALM imaging, has never been optimized in previous developments. In this study, we proposed that simply installing a carboxyl group close to the lactam site could impose an intramolecular acidic environment, stabilize the photoactivated zwitterionic structure, and thus effectively increase the on-time. On the basis of this idea, we have synthesized a new rhodamine spirolactam, Rh-Gly, that demonstrated considerably longer on-time than the other tested analogues, as well as an enhancement of single-molecule brightness, an improvement on signal-to-noise ratio and an enlargement of total collected photons of a single molecule before photobleaching. Finally, super-resolution images of live cell mitochondria stained with Rh-Gly have been obtained with a good temporal resolution of 10 s, as well as a satisfactory localization precision of ∼25 nm. Through self-labeling protein tags, Rh-Gly modified with a HaloTag ligand enabled super-resolution imaging of histone H2B proteins in live HeLa cells; through immunostaining antibodies labeled with an isothiocyanate-substituted Rh-Gly, super-resolution imaging of microtubules was achieved in fixed cells. Therefore, our simple and effective strategy provides novel insight for developing further enhanced rhodamine spirolactams recommendable for PALM imaging.

Superresolution Imaging Reveals the Internal Organization of Syntaxin Clusters in Hippocampal Processes

N‐ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins are the universal molecular machinery used to mediate membrane fusion events. Syntaxins are SNARE proteins that reside in the target membrane and determine the allowable sites for membrane fusion. Clusters of syntaxin molecules release reactive syntaxin molecules for interactions with their partners in SNARE formation. In hippocampal neurons, syntaxin‐1a (Stx1a) mediates neurotransmitter release from synaptic vesicles whereas syntaxin‐3 (Stx3) facilitates activity‐dependent α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid(AMPA) receptor exocytosis. To explore the single‐molecule distribution of these syntaxin variants in hippocampal neurons, we implemented photoactivated localization microscopy (PALM), a superresolution fluorescence microscopy technique. PALM revealed single Stx1a molecules as well as clusters of Stx1a distributed throughout the hippocampal processes. The clusters of Stx1a varied markedly in size and the density of Stx1a molecules within the clusters was nonuniform with the greatest density of Stx1a molecules observed in the core of the clusters. The largest Stx1a clusters contained multiple density gradients of Stx1a within the cluster. PALM images revealed that Stx3 also forms clusters in hippocampal processes, and these Stx3 clusters likewise contained a density gradient of syntaxin molecules with Stx3 density most pronounced in the cluster core. This work is supported by National Institutes of Health grant 2 R15 NS40425‐03.

Mechanisms of localized activation of the T cell antigen receptor inside clusters

The T cell antigen receptor (TCR) has been shown to cluster both before and upon engagement with cognate antigens. However, the effect of TCR clustering on its activation remains poorly understood. Here, we used two-color photo-activated localization microscopy (PALM) to visualize individual molecules of TCR and ZAP-70, as a marker of TCR activation and phosphorylation, at the plasma membrane of uniformly activated T cells. Imaging and second-order statistics revealed that ZAP-70 recruitment and TCR activation localized inside TCR clusters. Live cell PALM imaging showed that the extent of localized TCR activation decreased, yet remained significant, with cell spreading. Using dynamic modeling and Monte-Carlo simulations we evaluated possible mechanisms of localized TCR activation. Our simulations indicate that localized TCR activation is the result of long-range cooperative interactions between activated TCRs, or localized activation by Lck and Fyn. Our results demonstrate the role of molecular clustering in cell signaling and activation, and are relevant to studying a wide range of multi-molecular complexes. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.

Development of a Slow-Switching Dronpa Variant for 2-Color Super Resolution Imaging of Drp1 During Mitochondrial Fission

We studied the single-molecule photo-switching properties of Dronpa, a green photo-switchable fluorescent protein (PS-FP) and a popular marker for photo-activated localization microscopy (PALM). We found the excitation light photo-activates as well as deactivates Dronpa single molecules, hindering temporal separation and limiting super resolution. To resolve this limitation, we have developed a novel Dronpa variant, rsKame. The increased steric hindrance generated by the mutation reduced the excitation light-induced photo-conversion from the dark to fluorescent state. To demonstrate the applicability of rsKame, we paired it with PAmCherry1 in a 2-color PALM imaging method to observe the inner and outer mitochondrial membrane structures and selectively labeled dynamin related protein 1 (Drp1), responsible for membrane scission during mitochondrial fission. We quantified Drp1 helical ring diameters and lengths showing that the rings undergo changes in diameter between different stages of mitochondrial fission without significant modification of their length. These results support the twistase model of Drp1 constriction with some loss of subunits at the helical ends.

Single-Molecule Fluorescence Imaging Reveals Mismatch Repair Dynamics in Live Bacillus Subtilis

The error-free progression of DNA replication is essential for all organisms, and deficiencies in DNA repair mechanisms can have severe consequences, from increased antibiotic resistance in bacteria to human cancers. The mismatch repair (MMR) pathway corrects base-pairing errors that are incorporated during genome replication. This pathway is critical for maintaining genomic fidelity: loss of MMR increases the mutation rate several hundred-fold in bacteria and humans alike. From prokaryotes to human cells, the highly conserved MMR protein MutS and its homologs recognize mismatched nucleotides and recruits downstream MMR proteins for repair. Despite the crucial role of MutS in MMR, the mechanism by which MutS first locates base-pairing errors remains unclear.We have developed new methods of super-resolution imaging to reveal the in vivo single-molecule distributions and dynamics of MutS in live Bacillus subtilis. This Gram-positive bacterium has served as the model for studying DNA replication and repair due to its genetic competence and high homology with corresponding pathways in humans. Based on Photoactivated Localization Microscopy (PALM) and single-particle tracking (SPT), we localized and tracked MutS-PAmCherry and the replisome subunit DnaX-mCitrine with 20-nm precision to reveal the spatial relationship and diffusive behaviors of these two proteins. We observed transient, mismatch-dependent colocalization between MutS-PAmCherry and DnaX-mCitrine with two-color PALM imaging, and SPT revealed dramatic changes in MutS-PAmCherry diffusion rates and confinement behavior upon mutagen treatment. To develop a mechanistic understanding, we have studied wt MutS as well as mutant strains defective for mismatch recognition and mismatch unbinding, respectively, and studied how the interactions between MutS-PAmCherry and the replisome are changed by such mutations. Together, our results provide strong evidence supporting that MutS recruitment to the replisome is required for MMR and precedes mismatch binding events in vivo.

Correlative Photoactivated Localization and Scanning Electron Microscopy

The ability to localize proteins precisely within subcellular space is crucial to understanding the functioning of biological systems. Recently, we described a protocol that correlates a precise map of fluorescent fusion proteins localized using three-dimensional super-resolution optical microscopy with the fine ultrastructural context of three-dimensional electron micrographs. While it achieved the difficult simultaneous objectives of high photoactivated fluorophore preservation and ultrastructure preservation, it required a super-resolution optical and specialized electron microscope that is not available to many researchers. We present here a faster and more practical protocol with the advantage of a simpler two-dimensional optical (Photoactivated Localization Microscopy (PALM)) and scanning electron microscope (SEM) system that retains the often mutually exclusive attributes of fluorophore preservation and ultrastructure preservation. As before, cryosections were prepared using the Tokuyasu protocol, but the staining protocol was modified to be amenable for use in a standard SEM without the need for focused ion beam ablation. We show the versatility of this technique by labeling different cellular compartments and structures including mitochondrial nucleoids, peroxisomes, and the nuclear lamina. We also demonstrate simultaneous two-color PALM imaging with correlated electron micrographs. Lastly, this technique can be used with small-molecule dyes as demonstrated with actin labeling using phalloidin conjugated to a caged dye. By retaining the dense protein labeling expected for super-resolution microscopy combined with ultrastructural preservation, simplifying the tools required for correlative microscopy, and expanding the number of useful labels we expect this method to be accessible and valuable to a wide variety of researchers.

Photoactivated Localization Microscopy (PALM) of adhesion complexes.

Key to understanding a protein's biological function is the accurate determination of its spatial distribution inside a cell. Although fluorescent protein markers allow the targeting of specific proteins with molecular precision, much of this information is lost when the resultant fusion proteins are imaged with conventional, diffraction-limited optics. In response, several imaging modalities that are capable of resolution below the diffraction limit (∼200 nm) have emerged. Here, both single- and dual-color superresolution imaging of biological structures using photoactivated localization microscopy (PALM) are described. The examples discussed focus on adhesion complexes: dense, protein-filled assemblies that form at the interface between cells and their substrata. A particular emphasis is placed on the instrumentation and photoactivatable fluorescent protein (PA-FP) tags necessary to achieve PALM images at ∼20 nm resolution in 5 to 30 min in fixed cells.

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring. PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with <20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules. Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring and can be applied to other proteins of interest.

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells7, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring8. PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with <20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules. Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10 and can be applied to other proteins of interest.

Time-Lapse Super-Resolution Imaging of Apical Membrane Protein Domains in Live Filamentous Fungi

The core mechanism of the asymmetric and the polarized cell growth is a conserved process among many organisms. This process involves complex concerted interplay of dynamic rearrangements of cytoskeletons, the mobilization of proteins from intracellular pools, active transport of vesicles to fusion sites and the accumulation of “cell end marker” proteins. While many of the key components for the polarized growth are known, the detailed mechanism of these complex processes is still unclear. TeaR is one of the cell end marker protein that plays a crucial role in the initiation and the maintenance of straight-growth in a filamentous fungi, Aspergillus nidulans. While many end marker proteins show a single accumulation of the domain near the end of the cell, widefield fluorescence microscopy data shows a distribution of multiple TeaR domains. Our current colocalization studies strongly suggest that majority of TeaR domains consist of accumulated secretory vesicles that are docked near the plasma membrane. This suggests that these domains mark the exocytosis sites of the hypha. In order to elucidate the detailed architecture of TeaR domains near the hypha tip, we have performed a super-resolution microscopy imaging, photoactivated localization microscopy (PALM) in live filamentous fungi. PALM imaging of TeaR fused with a photoconvertible fluorescent protein, mEosFPthermo, shows a cluster size distribution centered around 140 nm. Time-lapse PALM imaging further reveals that these clusters in growing cells are highly dynamic. Processes resemble vesicle trafficking along the cytoskeleton, docking, accumulation of vesicles, dispersion of proteins along the membrane and the growth of the membrane has been observed. We present the affect of key deletion mutants that influence the directionality of the hypha growth on these dynamics.

A Super-Resolution Study of the Structural Organization of the E. Coli FTSZ-Ring by ZapA and ZapB

The first step in E. coli cell division is the localization of the FtsZ protein at midcell, where it polymerizes into a ring-like structure called the Z-ring. FtsZ is widely conserved throughout bacteria and is a structural homolog of eukaryotic tubulin. Understanding the organization of the Z-ring will provide insight into the force generation mechanism of bacterial cell division.In our previous work we used a super-resolution imaging method, photoactivated localization microscopy (PALM), to characterize the arrangement of FtsZ protofilaments inside the Z-ring1. We showed that the Z-ring is likely composed of a loose association of protofilaments overlapping in both the circumferential and radial direction of the Z-ring. Given that FtsZ protofilaments exhibit a low intrinsic self-interaction, how they are associated in vivo remains unknown.Recently a family of Z-ring associated proteins (Zap) have been shown to promote FtsZ polymerization in vitro and Z-ring formation in vivo. In this study, we investigated how ZapA and ZapB affect the organization of the Z-ring.We used live-cell PALM imaging to characterize the structure of the Z-ring in the absence of ZapA and ZapB. We found that in the absence of ZapA or ZapB, FtsZ adopts a variety of non-native structures characterized by a fractured appearance containing dispersed clusters of FtsZ. Similar FtsZ clusters have been observed to precede wild-type Z-ring assembly and are likely composed of multiple FtsZ protofilaments. Comparison of the FtsZ clusters observed in ΔzapA and ΔzapB to those observed in wild-type suggest that ZapA and ZapB may function to promote Z-ring assembly by corralling and consolidating higher-ordered FtsZ groupings.[1] G. Fu, T. Huang, J. Buss, C. Coltharp, Z. Hensel, J. Xiao, PLoS One 2010, 5, e12680.

Resolving multi-molecular protein interactions by photoactivated localization microscopy

Multi-molecular protein complexes are critical to many cellular functions, including signaling, DNA transcription and enzymatic reactions. In spite of their importance, current research techniques such as biochemistry and diffraction-limited microscopy cannot resolve the heterogeneity and nanoscale organization of protein complexes in intact cells. Here we describe a technique that enables the study of multi-molecular protein complexes at the single molecule level in intact cells. The technique uses photoactivated localization microscopy (PALM) to resolve individual proteins with a resolution down to 20nm in intact cells, and second-order statistics to study the spatial interactions of the proteins. We demonstrate the feasibility of this technique by studying signaling complexes that form in activated T cells. We first use single color PALM imaging and univariate second-order statistics to resolve the clustering of Linker for Activation of T cells (LAT) at the plasma membrane (PM) of the cells. We then use two color PALM and bivariate second-order statistics to resolve the interaction of LAT with key interacting proteins. We discuss potential caveats in studying molecular clustering and the robustness of the technique to study bimolecular interactions. Our proposed technique, combined with older techniques, could help shed new light on the nature of multimolecular protein complexes and their significance to cell function.

Plasticity of the asialoglycoprotein receptor deciphered by ensemble FRET imaging and single-molecule counting PALM imaging.

The stoichiometry and composition of membrane protein receptors are critical to their function. However, the inability to assess receptor subunit stoichiometry in situ has hampered efforts to relate receptor structures to functional states. Here, we address this problem for the asialoglycoprotein receptor using ensemble FRET imaging, analytical modeling, and single-molecule counting with photoactivated localization microscopy (PALM). We show that the two subunits of asialoglycoprotein receptor [rat hepatic lectin 1 (RHL1) and RHL2] can assemble into both homo- and hetero-oligomeric complexes, displaying three forms with distinct ligand specificities that coexist on the plasma membrane: higher-order homo-oligomers of RHL1, higher-order hetero-oligomers of RHL1 and RHL2 with two-to-one stoichiometry, and the homo-dimer RHL2 with little tendency to further homo-oligomerize. Levels of these complexes can be modulated in the plasma membrane by exogenous ligands. Thus, even a simple two-subunit receptor can exhibit remarkable plasticity in structure, and consequently function, underscoring the importance of deciphering oligomerization in single cells at the single-molecule level.

Combining Super-Resolution Imaging and Single Particle Tracking in Living Cells to Probe Interactions Between Actin and Plasma Membrane Proteins

Cortical actin is a complex meshwork essential for the dynamic organization and localization of plasma membrane proteins. In order to characterize dynamics of the cytoskeleton of living mammalian cells with resolution beyond the diffraction limit, we used photoactivated localization microscopy (PALM). We transfected ND7/23 and HEK 293 cells to express Dendra2 labeled actin. The cells were imaged in a custom-built total internal reflection microscope maintaining the cells under physiological conditions for prolonged periods of time. Dynamic PALM images of the actin cytoskeleton were reconstructed in a 5-second sliding time window. A resolution of 50 nm was achieved.By combining dynamic super-resolution actin images and single particle tracking in the plasma membrane, we studied the actin cytoskeleton's role in the organization of Kv2.1 channels into segregated microdomains. Kv2.1 forms stable clusters in hippocampal neurons and transfected HEK cells, but the mechanism by which the clusters are formed is not well understood. By labeling Kv2.1 channels with quantum dots which are spectrally well separated from Dendra2, two-color images were obtained which revealed complex interactions between the actin cytoskeleton and Kv2.1 channels. This work demonstrates that PALM combined with single particle tracking is an effective technique to probe the dynamic interactions between cortical actin and membrane proteins.

Assessing the localization of centrosomal proteins by PALM/STORM nanoscopy

The structure of the centrosome was resolved by EM many years ago to reveal a pair of centrioles embedded in a dense network of proteins. More recently, the molecular composition of the centrosome was catalogued by mass spectroscopy and many novel components were identified. Determining precisely where a novel component localizes to within the centrosome remains a challenge, and until now it has required the use of immuno-EM. This technique is both time-consuming and unreliable, as it often fails due to problems with antigen accessibility. We have investigated the use of two nanoscopic techniques, photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), as alternative techniques for localizing centrosomal proteins. The localization of a known centrosomal component, the distal appendage protein Cep164 was investigated by direct STORM (dSTORM) and resolved with a high spatial resolution. We further validated the use of nanoscopic PALM imaging by showing that the previously uncharacterized centrosomal protein CCDC123 (Cep123) localizes to the distal appendages, forming ring-like structures with a diameter of 500 nm. Our results demonstrate that both PALM and STORM imaging have great potential as alternatives to immuno-EM.

Quantitative Photo Activated Localization Microscopy: Unraveling the Effects of Photoblinking

In this work we discuss how to use photophysical information for improved quantitative measurements using Photo Activated Localization Microscopy (PALM) imaging. We introduce a method that reliably estimates the number of photoblinking molecules present in a biological sample and gives a robust way to quantify proteins at the single-cell level from PALM images. We apply this method to determine the amount of β2 adrenergic receptor, a prototypical G Protein Coupled Receptor, expressed on the plasma membrane of HeLa cells.

Confined activation and subdiffractive localization enables whole-cell PALM with genetically expressed probes

We demonstrate 3D superresolution microscopy in whole fixed cells using photoactivated localization microscopy (PALM). The use of the bright, genetically expressed fluorescent marker photoactivatable mCherry (PA-mCherry1) in combination with near diffraction-limited confinement of photoactivation using two-photon illumination and 3D localization methods allowed us to investigate a variety of cellular structures at <50 nm lateral and <100 nm axial resolution. Compared to existing methods, we substantially reduce excitation and bleaching of unlocalized markers, enabling 3D PALM imaging with high localization density in thick structures. Our 3D localization algorithms based on cross-correlation do not rely on idealized noise models or specific optical configurations, allowing flexible instrument design. Generation of appropriate fusion constructs and expression in Cos7 cells allowed us to image invaginations of the nuclear membrane, vimentin fibrils, the mitochondrial network, and the endoplasmic reticulum at depths greater than 8 μm.