Lipophilic Fluorophore Research Articles

Fluorogenic in-situ Labelling of Gelatin Polymer in Aqueous Solution and Hydrogel.

Gelatin polymers made from partially degraded collagen are important biomaterials, but their in-situ analysis suffers from uncontrollable covalent labelling and poor spatial-temporal imaging resolution. Herein, three tetrazolate-tagged tetraphenylethylene fluorophores (TPE-TAs) are introduced for practical fluorogenic labelling of gelatin in aqueous phase and hydrogels. These probes with aggregation-induced emission characteristics offer negligible background and elicit turn-on fluorescence by simply mixing with the gelatin in aqueous phase, giving a detection limit of 0.15 mg/L over a linear dynamic range up to 100 mg/L. This method does not work for collagens and causes minimal interference with gelatin properties. Mechanistic studies reveal a key role for multivalent electrostatic interactions between the abundant basic residues in gelatin (e. g., lysine, hydroxylysine, arginine) and anionic tetrazolate moieties of the lipophilic fluorophore synergistically in spatially rigid macromolecular encapsulation to achieve fluorogenic labelling. The AIE strategy by forming non-covalent fluorophore-gelatin complexes was developed for novel hydrogels that exhibited reversible fluorescence in response to dynamic microstructural changes in the hydrogel scaffold upon salting-in/out treatments, and enabled high spatial-temporal imaging of the fiber network in lyophilized samples. This work may open up avenues for in-situ imaging analysis and evaluation of gelatin-based biomaterials during processes such as in vivo degradation and mineralization.

Lipid Droplets Specific Fluorophore for Demarcation of Normal and Diseased Tissues.

Using high-fidelity, permeable, lipophilic, and bright fluorophores for imaging lipid droplets (LDs) in tissues holds immense potential in diagnosing conditions such as diabetic or alcoholic fatty liver disease. In this work, we utilized linear and Λ-shaped polarity-sensitive fluorescent probes for imaging LDs in both cellular and tissue environments, specifically in rats with diabetic and alcoholic fatty liver disease. The fluorescent probes possess several key characteristics, including high permeability, lipophilicity, and brightness, which make them well-suited for efficient LD imaging. Notably, the probes exhibit a substantial Stokes shift, with 143 nm for DCS and 201 nm for DCN with selective targeting of the lipid droplets. Our experimental investigations successfully differentiated morphological variations between diseased and normal tissues in three distinct tissue types: liver, adipose, and small intestine. They could help provide pointers for improved detection and understanding of LD-related pathologies.

Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment.

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing dysfunction caused by free fatty acids (FAs). The liver, given its critical role in the body's fat metabolism, is persistently threatened by the intracellular accumulation of LDs in the form of both microvesicular and macrovesicular hepatic steatosis. The histologic characterization of LDs is typically based on lipid-soluble diazo dyes, such as Oil Red O (ORO) staining, but a number of disadvantages consistently hamper the use of this analysis with liver specimens. More recently, lipophilic fluorophores 493/503 have become popular for visualizing and locating LDs due to their rapid uptake and accumulation into the neutral lipid droplet core. Even though most applications are well-described in cell cultures, there is less evidence demonstrating the reliable use of lipophilic fluorophore probes as an LD imaging tool in tissue samples. Herein, we propose an optimized boron dipyrromethene (BODIPY) 493/503-based protocol for the evaluation of LDs in liver specimens from an animal model of high-fat diet (HFD)-induced hepatic steatosis. This protocol covers liver sample preparation, tissue sectioning, BODIPY 493/503 staining, image acquisition, and data analysis. We demonstrate an increased number, intensity, area ratio, and diameter of hepatic LDs upon HFD feeding. Using orthogonal projections and 3D reconstructions, it was possible to observe the full content of neutral lipids in the LD core, which appeared as nearly spherical droplets. Moreover, with the fluorophore BODIPY 493/503, we were able to distinguish microvesicles (1 µm < d ≤ 3 µm), intermediate vesicles (3 µm < d ≤ 9 µm), and macrovesicles (d > 9 µm), allowing the successful discrimination of microvesicular and macrovesicular steatosis. Overall, this BODIPY 493/503 fluorescence-based protocol is a reliable and simple tool for hepatic LD characterization and may represent a complementary approach to the classical histological protocols.

Homotypic Membrane-Enhanced Blood-Brain Barrier Crossing and Glioblastoma Targeting for Precise Surgical Resection and Photothermal Therapy.

The crossing of blood-brain barrier (BBB) is essential for glioblastoma (GBM) therapy, and homotypic targeting is an effective strategy to achieve BBB crossing. In this work, GBM patient-derived tumor cell membrane (GBM-PDTCM) is prepared to cloak gold nanorods (AuNRs). Relying on the high homology of the GBM-PDTCM to the brain cell membrane, GBM-PDTCM@AuNRs realize efficient BBB crossing and selective GBM targeting. Meanwhile, owing to the functionalization of Raman reporter and lipophilic fluorophore, GBM-PDTCM@AuNRs are able to generate fluorescence and Raman signals at GBM lesion, and almost all tumor can be precisely resected in 15 min by the guidance of dual signals, ameliorating the surgical treatment for advanced GBM. In addition, photothermal therapy for orthotopic xenograft mice is accomplished by intravenous injection of GBM-PDTCM@AuNRs, doubling the median survival time of the mice, which improves the nonsurgical treatment for early GBM. Therefore, benefiting from homotypic membrane-enhanced BBB crossing and GBM targeting, all-stage GBM can be treated with GBM-PDTCM@AuNRs in distinct ways, providing an alternative idea for the therapy of tumor in the brain.

Spatially selective cell treatment and collection for integrative drug testing using hydrodynamic flow focusing and shifting.

Hydrodynamic focusing capable of readily producing and controlling laminar flow facilitates drug treatment of cells in existing microfluidic culture devices. However, to expand applications of such devices to multiparameter drug testing, critical limitations in current hydrodynamic focusing microfluidics must be addressed. Here we describe hydrodynamic focusing and shifting as an advanced microfluidics tool for spatially selective drug delivery and integrative cell-based drug testing. We designed and fabricated a co-flow focusing, three-channel microfluidic device with a wide cell culture chamber. By controlling inlet flow rates of sample and two side solutions, we could generate hydrodynamic focusing and shifting that mediated precise regulation of the path and width of reagent and drug stream in the microfluidic device. We successfully validated a hydrodynamic focusing and shifting approach for spatially selective delivery of DiI, a lipophilic fluorophore, and doxorubicin, a chemotherapeutic agent, to tumor cells in our device. Moreover, subsequent flowing of a trypsin EDTA solution over the cells that were exposed to doxorubicin flow allowed us to selectively collect the treated cells. Our approach enabled downstream high-resolution microscopy of the cell suspension to confirm the nuclear delivery of doxorubicin into the tumor cells. In the device, we could also evaluate in situ the cytotoxic effect of doxorubicin to the tumor cells that were selectively treated by hydrodynamic flow focusing and shifting. These results show that hydrodynamic focusing and shifting enable a fast and robust approach to spatially treat and then collect cells in an optimized microfluidic device, offering an integrative assay tool for efficient drug screening and discovery.

Improved lipophilic probe for visualizing lipid droplets in erastin-induced ferroptosis.

Studying the viscosity of lipid droplets (LDs) provides insights into various diseases associated with LD viscosity. Ferroptosis is one such process in which LD viscosity increases due to the abnormal accumulation of lipid ROS (reactive oxygen species) caused by peroxidation. For investigating the LD imaging and ferroptosis, we developed two molecules (NNS and DNS) that show significant Stokes shifts (182-232 nm) and utilized them for sub-cellular imaging. Excellent localization is noted with the lipid droplets. Subsequently, DNS was used to monitor the variations in the LD viscosity during erastin-induced ferroptosis followed by ferroptosis inhibition. Additionally, we explored variations in the LD quantity, size, and accumulation when subjected to oleic acid stimulation. Extensive DFT and TDDFT investigations have been employed to understand the effect of NO2 substitution on the linear and branched molecular derivatives. Our results with the improved lipophilic fluorophore, exhibiting excellent colocalization with LDs, offer valuable insights into sensing erastin-induced ferroptosis and have the potential for real-time diagnostic applications.

Highly lipophilic coumarin fluorophore with excimer-monomer transition property for lipid droplet imaging

Lipid droplet (LD) fluorescent imaging plays an important role in the detection of lipid-related diseases. Due to their poor photostability and low hydrophobicity of currently available LD imaging fluorophores, LD imaging is limited by its short imaging period and low imaging contrast. Herein, we reasonably designed a highly lipophilic compound Cou-Flu with excellent photostability and excimer-monomer transition property. It exhibited weak excimer emission in cytoplasm, but strong monomer emission in LDs, enabling high contrast LD imaging and LD movement tracing in cells. Zebrafish imaging study demonstrated that Cou-Flu was also suitable for in vivo LD detection with excellent sensitivity. We anticipate that Cou-Flu could be widely applied to understand LD-related intracellular activities and even LD-related diseases in the future.

Novel Lipophilic Fluorophores with Highly Acidity-Dependent Two-Photon Response.

Lipophilic fluorophores are widely implemented in nonlinear microscopy; however, few existing membrane-specific probes combine the high brightness of two-photon excited fluorescence (2PEF) with pH sensitivity. Herein we describe four novel two-photon excited fluorophores, based on a coumarin 151 core structure, where lipophilicity is induced by a covalently attached phosphazene moiety. Changing the environmental acidity using trifluoromethanesulfonic (triflic) acid leads to profound changes in the linear fluorescence and 2PEF characteristics, due to chromophores' switching between neutral- and protonated forms. We characterize this dependence by measuring the two-photon absorption (2PA) spectra over the region λ2PA =550-1000 nm, observing 2PA cross sections of σ2PA =10-20 GM, with an associated 2PEF brightness of 10-13 GM, in neutral solutions of both acetonitrile and n-octanol. Although quantum chemical modelling and NMR measurements show that, at high chromophore concentrations, protonation may be accompanied by a dimerization process, these dimers likely do not form at the lower concentrations used in optical spectroscopy.

Chemically Tuned, Reversible Fluorogenic Electrophile for Live Cell Nanoscopy.

We report a chemically tuned fluorogenic electrophile designed to conduct live-cell super-resolution imaging by exploiting its stochastic reversible alkylation reaction with cellular nucleophiles. Consisting of a lipophilic BODIPY fluorophore tethered to an electrophilic cyanoacrylate warhead, the new probe cyanoAcroB remains nonemissive due to internal conversion along the cyanoacrylate moiety. Intermittent fluorescence occurs following thiolate Michael addition to the probe, followed by retro-Michael reaction, tuned by the cyano moiety in the acrylate warhead and BODIPY decoration. This design enables long-term super-resolved imaging of live cells by preventing fluorescent product accumulation and background increase, while preserving the pool of the probe. We demonstrate the imaging capabilities of cyanoAcroB via two methods: (i) single-molecule localization microscopy imaging with nanometer accuracy by stochastic chemical activation and (ii) super-resolution radial fluctuation. The latter tolerates higher probe concentrations and low imaging powers, as it exploits the stochastic adduct dissociation. Super-resolved imaging with cyanoAcroB reveals that electrophile alkylation is prevalent in mitochondria and endoplasmic reticulum. The 2D dynamics of these organelles within a single cell are unraveled with tens of nanometers spatial and sub-second temporal resolution through continuous imaging of cyanoAcroB extending for tens of minutes. Our work underscores the opportunities that reversible fluorogenic probes with bioinspired warheads bring toward illuminating chemical reactions with super-resolved features in live cells.

The use of LipidGreen2 for visualization and quantification of intracellular Poly(3-hydroxybutyrate) in Cupriavidus necator

Numerous studies have been conducted to develop a rapid protocol for the quantification of poly(3-hydroxybutyrate) during bacterial fermentation as an alternative to time-consuming gravimetric or analytical methods. Fluorescence spectroscopy is one of the most promising approaches. In this study, it could be demonstrated that the novel fluorescent probe LipidGreen2 is able to stain selectively poly(3-hydroxybutyrate) in Cupriavidus necator. Optimal excitation and emission wavelengths were evaluated using 3D-Excitation-Emission-Matrix, displaying the best intensities between 440-460 nm and 490–520 nm for excitation and emission, respectively. The lipophilic fluorophore LipidGreen2 showed a high long-term stability even when incubated under ambient lighting. Due to a strong linear relationship between side scatter and biomass concentration, the influence of the inner filter effects could be incorporated, and adjusting the sample to a specific OD is thus superfluous. The developed method allows a very accurate quantification of poly(3-hydroxybutyrate) in just 15 min, following a comprehensible and simple protocol. It is also excellently suited for bioimaging of intracellular poly(3-hydroxybutyrate) granules.

The Composition of Reconstituted High-Density Lipoproteins (rHDL) Dictates the Degree of rHDL Cargo- and Size-Remodeling via Direct Interactions with Endogenous Lipoproteins.

The application of reconstituted high-density lipoproteins (rHDL) as a drug-carrier has during the past decade been established as a promising approach for effective receptor-mediated drug delivery, and its ability to target tumors has recently been confirmed in a clinical trial. The rHDL mimics the endogenous HDL, which is known to be highly dynamic and undergo extensive enzyme-mediated remodulations. Hence, to reveal the physiological rHDL stability, a thorough characterization of the dynamics of rHDL in biologically relevant environments is needed. We employ a size-exclusion chromatography (SEC) method to evaluate the dynamics of discoidal rHDL in fetal bovine serum (FBS), where we track both the rHDL lipids (by the fluorescence from lipid-conjugated fluorophores) and apoA-I (by human apoA-I ELISA). We show by using lipoprotein depleted FBS and isolated lipoproteins that rHDL lipids can be transferred to endogenous lipoproteins via direct interactions in a nonenzymatic process, resulting in rHDL compositional- and size-remodeling. This type of dynamics could lead to misinterpretations of fluorescence-based rHDL uptake studies due to desorption of labile lipophilic fluorophores or off-target side effects due to desorption of incorporated drugs. Importantly, we show how the degree of rHDL remodeling can be controlled by the compositional design of the rHDL. Understanding the correlation between the molecular properties of the rHDL constituents and their collective dynamics is essential for improving the rHDL-based drug delivery platform. Taken together, our work highlights the need to carefully consider the compositional design of rHDL and test its stability in a biological relevant environment, when developing rHDL for drug delivery purposes.

Assessment of lipid load in tumor-infiltrating Tregs by flow cytometry.

Regulatory T cells (Tregs), expressing the transcription factor Foxp3, are defined as immunosuppressive cells able to modulate a variety of immune cells in order to avoid unwanted and excessive immune responses; however, in the tumor context, Treg function contribute to inhibit immune surveillance, thus promoting cancer progression. In tumor microenvironment, where the availability of metabolic resources is strongly limited, Tregs are expanded and may exploit different metabolic routes to achieve a metabolic advantage, prevailing over effector cells. In this context an important role of lipid metabolism has been described thanks to the possibility to evaluate the intracellular lipid content selectively in tumor-infiltrating Tregs (TUM-Tregs). Taking into account the heterogeneous and complex build of tumor mass, we set-up a combined protocol that optimizes tumor-infiltrating lymphocytes (TIL) extraction from the tissue through a Percoll density gradient, and assesses ex vivo the lipid load in whole TUM-Treg population, evaluating by flow cytometry the incorporation of an intensely fluorescent lipophilic fluorophore able to specifically stain neutral lipids. This method provides an important advantage compared to the traditional technique based on microscopy, whose lipid level evaluation is limited to a tissue section, and hence may not be representative of the entire population.

Albumin tailoring fluorescence and photothermal conversion effect of near-infrared-II fluorophore with aggregation-induced emission characteristics

Fluorophores with donor-acceptor-donor groups with the emission spanning the second near-infrared window (NIR-II) have recently received great attention for biomedical application. Yet, the mechanism underlying the equilibrium between fluorescence (radiative decay) and photothermal effect (non-radiative decay) of these fluorophores remains elusive. Here, we demonstrate that a lipophilic NIR-II fluorophore, BPBBT, possesses both twisted intramolecular charge transfer (TICT) and aggregation-induced emission (AIE) characteristics. Human serum albumin (HSA) binds to BPBBT, which changes the planarity of the fluorophore and restricts its intramolecular rotation. The binding results in alteration to the equilibrium between AIE and TICT state of BPBBT, tailoring its fluorescence and photothermal efficiency. Under the guidance of intraoperative NIR-II fluorescence image, the prepared HSA-bound BPBBT nanoparticles delineate primary orthotopic mouse colon tumor and metastatic lesions with dimensions as small as 0.5 mm × 0.3 mm, and offer photothermal ablation therapy with optimized timing, dosing and area of the laser irradiation.

Visualization of intracellular lipid droplets using lipophilic benzothiazole-based push-pull fluorophores at ultralow concentration

In this study we describe the development of new lipophilic and biocompatible benzothiazole-based push-pull fluorophores as potent fluorogenic probes for the specific staining of intracellular lipid droplets (LDs) at ultralow concentration (≤100 nM). These new fluorophores, harboring a D-π-A framework featuring an extended π-conjugated spacer, a lipophilic tertiary amine (D) and hydrophobic aryl groups (A), were prepared by an efficient and practical synthetic route. The photophysical studies of these compounds underlined their high polarity sensitivity, characterized by a strong solvatochromic effect and the large Stokes shifts values (λem 465–630 nm, Δλ up to 6751 cm−1). Finally, the in cellulo investigations of D1 revealed its ability to selectivity accumulate in LDs and to allow their remarkable visualization using confocal fluorescence microscopy.

Pitfalls associated with lipophilic fluorophore staining of extracellular vesicles for uptake studies

ABSTRACTPost-staining of extracellular vesicles (EVs) with lipid-anchored fluorophores (LAFs) such as PKH67 is a widely used strategy for studying EVs but it is associated with several pitfalls. The pitfalls discussed in this commentary are related to LAF labelling of non-EV species due to (1) lipoprotein contamination in EV samples, (2) desorption of the LAF reporters from vesicles into proteins and lipoproteins in blood and serum, and (3) the capability of the amphiphilic LAF compounds to form EV-like particles. Awareness of these challenges and developing solutions to overcome these are important to ensure that we make relevant interpretations when using LAFs to track EVs.

Interface-Targeting Strategy Enables Two-Photon Fluorescent Lipid Droplet Probes for High-Fidelity Imaging of Turbid Tissues and Detecting Fatty Liver

Lipid droplets (LDs) with unique interfacial architecture not only play crucial roles in protecting a cell from lipotoxicity and lipoapoptosis but also closely relate with many diseases such as fatty liver and diabetes. Thus, as one of the important applied biomaterials, fluorescent probes with ultrahigh selectivity for in situ and high-fidelity imaging of LDs in living cells and tissues are critical to elucidate relevant physiological and pathological events as well as detect related diseases. However, available probes only utilizing LDs' waterless neutral cores but ignoring the unique phospholipid monolayer interfaces exhibit low selectivity. They cannot differentiate neutral cores of LDs from intracellular other lipophilic microenvironments, which results in extensively cloud-like background noise and severely limited their bioapplications. Herein, to design LD probes with ultrahigh selectivity, the exceptional interfacial architecture of LDs is considered adequately and thus an interface-targeting strategy is proposed for the first time. According to the novel strategy, we have developed two amphipathic fluorescent probes (N-Cy and N-Py) by introducing different cations into a lipophilic fluorophore (nitrobenzoxadiazole (NBD)). Consequently, their cationic moiety precisely locates the interfaces through electrostatic interaction and simultaneously NBD entirely embeds into the waterless core via hydrophobic interaction. Thus, high-fidelity and background-free fluorescence imaging of LDs are expectably realized in living cells in situ. Moreover, LDs in turbid tissues like skeletal muscle slices have been clearly imaged (up to 82 μm depth) by a two-photon microscope. Importantly, using N-Cy, we not only intuitively monitored the variations of LDs in number, size, and morphology but also clearly revealed their abnormity in hepatic tissues resulting from fatty liver. Therefore, these unique probes provide excellent imaging tools for elucidating LD-related physiological and pathological processes and the interface-targeting strategy possesses universal significance for designing probes with ultrahigh selectivity.

Normalized neutral lipid quantitation by flow cytometry

Interest in measuring tissue lipids has increased as the link between fat-laden tissues and metabolic disease has become obvious; however, linking disease to a specific cell type within a tissue has been hampered by methodological limitations. Flow cytometry (FC) has been used to assess relative lipid levels in cells. Unfortunately, its usefulness is limited because comparisons between samples generated over several hours is problematic. We show that: 1) in lipophilic fluorophore stained cells, fluorescence intensity measured by FC reflects lipid levels; 2) this technique can be used to assess lipid levels in a mixed cell population; 3) normalizing to a control condition can decrease experiment-to-experiment variation; and 4) fluorescence intensity increases linearly with lipid levels. This allows triacylglycerol (TG) mass to be estimated in mixed cell populations comparing cells with known fluorescence and TG levels. We exploited this strategy to estimate lipid levels in monocytes within a mixed population of cells isolated from human blood. Using this strategy, we also confirmed that perilipin (PLIN)1 increases TG accumulation by ectopically expressing fluorescently tagged PLIN1 in Huh7 cells. In both examples, biochemically assaying for TG in specific cell populations is problematic due to limited cell numbers and isolation challenges. Other advantages are discussed.

The Detection of Nanoscale Membrane Bending with Polarized Localization Microscopy

The curvature of biological membranes at the nanometer scale is critically important for vesicle trafficking, organelle morphology, and disease propagation. The initiation of membrane bending occurs at a length scale that is irresolvable by most superresolution optical microscopy methods. Here, we report the development of polarized localization microscopy (PLM), a pointillist optical imaging technique for the detection of nanoscale membrane curvature in correlation with single-molecule dynamics and molecular sorting. PLM combines polarized total internal reflection fluorescence microscopy and single-molecule localization microscopy to reveal membrane orientation with subdiffraction-limited resolution without reducing localization precision by point spread function manipulation. Membrane curvature detection with PLM requires fewer localization events to detect curvature than three-dimensional single-molecule localization microscopy (e.g., photoactivated localization microscopy or stochastic optical reconstruction microscopy), which enables curvature detection 10× faster via PLM. With rotationally confined lipophilic fluorophores and the polarized incident fluorescence excitation, membrane-bending events are revealed with superresolution. Engineered hemispherical membrane curvature with a radius ≥24 nm was detected with PLM, and individual fluorophore localization precision was 13 ± 5 nm. Further, deciphering molecular mobility as a function of membrane topology was enabled. The diffusion coefficient of individual DiI molecules was 25 ± 5× higher in planar supported lipid bilayers than within nanoscale membrane curvature. Through the theoretical foundation and experimental demonstration provided here, PLM is poised to become a powerful technique for revealing the underlying biophysical mechanisms of membrane bending at physiological length scales.

Bipolar and fixable probe targeting mitochondria to trace local depolarization via two-photon fluorescence lifetime imaging.

Polarization/depolarization levels of different single mitochondria in a cell are inhomogeneous, and always varying. Because depolarization is an indicator of mitochondrial dysfunction, tracing local depolarization is highly desirable. The existing fluorescent probes, however, are not well suited for this task, although they are applicable to assess the average polarization extents of whole cells. A multifunctional and bipolar probe MITFPS is thus developed, which includes a positively charged hydrophilic group and an environment sensitive fluorophore. In the probe design, the hydrophilic anchoring unit is chemically immobilized on a membrane protein, while the lipophilic fluorophore can be inserted deep into the phospholipid layer. The probe exhibits a sensitive response to the local variation in polarization by changing its fluorescence lifetime. MITFPS's applicability is confirmed by real-time in situ imaging of the complete process of an uncoupler-induced depolarization under a two-photon fluorescence lifetime microscope. The imaging result reveals that one mitochondrion could have quite different polarization than the other, even though they are in the same cell.

Imaging Sub-Diffraction Membrane Curvature Dynamics during Clathrin Mediated Endocytosis

Proteins can generate and recognize curved membranes that in turn, modulate protein clustering, diffusion and biochemical reactions. The biophysical mechanisms of this dynamic relationship influence numerous biologically important events including receptor signal transduction, endocytosis, exocytosis and viral assembly. New approaches are needed to measure the dynamics of membrane bending in conjunction with protein recruitment and assembly. Here, I will discuss our work using back focal plane (BFP) spinning total internal reflection fluorescence (360-TIRF) microscopy to create uniform evanescent fields for quantitative imaging of molecular recruitment and assembly. We have paired these methods with BFP positioning for polarized-TIRF microscopy of membrane-oriented lipophilic fluorophores to image the sub-resolution membrane curvature. Using these methods, we have imaged the membrane bending dynamics during assembly of clathrin and dynamin at single endocytic sites in living cells. Our data demonstrate that this method is capable of imaging sub-resolution endocytic events and within the context of their surrounding topography. Together, these methods are enabling new insights into the interplay between assembly, reaction dynamics and membrane topography in living cells.