ABSTRACT
Volumetric subcellular imaging has long been essential for studying structures and dynamics in cells and tissues. However, due to limited imaging speed and depth of field, it has been challenging to perform live-cell imaging and single-particle tracking. Here we report a 2.5D fluorescence microscopy combined with highly inclined illumination beams, which significantly reduce not only the image acquisition time but also the out-of-focus background by â¼2-fold compared to epi-illumination. Instead of sequential z-scanning, our method projects a certain depth of volumetric information onto a 2D plane in a single shot using multi-layered glass for incoherent wavefront splitting, enabling high photon detection efficiency. We apply our method to multi-color immunofluorescence imaging and volumetric super-resolution imaging, covering â¼3-4⯵m thickness of samples without z-scanning. Additionally, we demonstrate that our approach can substantially extend the observation time of single-particle tracking in living cells.
ABSTRACT
Membrane fission involves a crucial step of lipid remodeling, in which the dynamin collar constricts and severs the tubulated lipid membrane at the neck of budding vesicles. Nevertheless, the difficulty in accurately determining the rotational dynamics of live endocytotic vesicles poses a limit on the elucidation of dynamin-induced membrane remodeling for endocytotic vesicle scission. Herein, we designed a DNA-modified gold homodimer (AuHD)-based anisotropic plasmonic probe with uniform surface chemistry, minimizing orientational fluctuation within vesicle encapsulation. Using AuHDs as cargos to image the dynamics of cargo-containing vesicles during endocytosis, we showed that, prior to detachment from plasma membrane, the cargo-containing vesicles underwent multiple intermittent twists of ~4° angular orientation relative to plasma membrane with a ~0.2 s dwell time. These findings suggest that the membrane torques resulting from dynamin actions in vivo constitute the pathway to membrane fission, potentially shedding light on how dynamin-mediated lipid remodeling orchestrates membrane fission.
ABSTRACT
The efficiency of virus internalization into target cells is a major determinant of infectivity. SARS-CoV-2 internalization occurs via S-protein-mediated cell binding followed either by direct fusion with the plasma membrane or endocytosis and subsequent fusion with the endosomal membrane. Despite the crucial role of virus internalization, the precise kinetics of the processes involved remains elusive. We developed a pipeline, which combines live-cell microscopy and advanced image analysis, for measuring the rates of multiple internalization-associated molecular events of single SARS-CoV-2-virus-like particles (VLPs), including endosome ingression and pH change. Our live-cell imaging experiments demonstrate that only a few minutes after binding to the plasma membrane, VLPs ingress into RAP5-negative endosomes via dynamin-dependent scission. Less than two minutes later, VLP speed increases in parallel with a pH drop below 5, yet these two events are not interrelated. By co-imaging fluorescently labeled nucleocapsid proteins, we show that nucleocapsid release occurs with similar kinetics to VLP acidification. Neither Omicron mutations nor abrogation of the S protein polybasic cleavage site affected the rate of VLP internalization, indicating that they do not confer any significant advantages or disadvantages during this process. Finally, we observe that VLP internalization occurs two to three times faster in VeroE6 than in A549 cells, which may contribute to the greater susceptibility of the former cell line to SARS-CoV-2 infection. Taken together, our precise measurements of the kinetics of VLP internalization-associated processes shed light on their contribution to the effectiveness of SARS-CoV-2 propagation in cells.
Subject(s)
COVID-19 , Endosomes , SARS-CoV-2 , Virus Internalization , SARS-CoV-2/physiology , SARS-CoV-2/metabolism , Humans , Kinetics , COVID-19/virology , COVID-19/metabolism , Endosomes/metabolism , Endosomes/virology , Endocytosis , Animals , Hydrogen-Ion Concentration , Chlorocebus aethiops , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Cell Membrane/metabolism , Cell Membrane/virology , Virion/metabolismABSTRACT
The spatial organization characteristics and redox status of the extracellular space (ECS) are crucial in the development of brain diseases. However, it remains a challenge to simultaneously capture dynamic changes in microstructural features and redox states at the submicron level within the ECS. Here, we developed a reversible glutathione (GSH)-responsive nanoprobe (RGN) for mapping the spatial organization features and redox status of the ECS in brain tissues with nanoscale resolution. The RGN is composed of polymer nanoparticles modified with GSH-responsive molecules and amino-functionalized methoxypoly(ethylene glycol), which exhibit exceptional single-particle brightness and excellent free diffusion capability in the ECS of brain tissues. Tracking single RGNs in acute brain slices allowed us to dynamically map spatial organizational features and redox levels within the ECS of brain tissues in disease models. This provides a powerful super-resolution imaging method that offers a potential opportunity to study the dynamic changes in the ECS microenvironment and to understand the physiological and pathological roles of the ECS in vivo.
Subject(s)
Brain , Extracellular Space , Glutathione , Nanoparticles , Oxidation-Reduction , Brain/metabolism , Brain/diagnostic imaging , Animals , Extracellular Space/metabolism , Extracellular Space/chemistry , Glutathione/chemistry , Glutathione/metabolism , Nanoparticles/chemistry , Mice , Polyethylene Glycols/chemistryABSTRACT
AMPA-type receptors (AMPARs) are rapidly inserted into synapses undergoing plasticity to increase synaptic transmission, but it is not fully understood if and how AMPAR-containing vesicles are selectively trafficked to these synapses. Here, we developed a strategy to label AMPAR GluA1 subunits expressed from their endogenous loci in cultured rat hippocampal neurons and characterized the motion of GluA1-containing vesicles using single-particle tracking and mathematical modeling. We find that GluA1-containing vesicles are confined and concentrated near sites of stimulation-induced structural plasticity. We show that confinement is mediated by actin polymerization, which hinders the active transport of GluA1-containing vesicles along the length of the dendritic shaft by modulating the rheological properties of the cytoplasm. Actin polymerization also facilitates myosin-mediated transport of GluA1-containing vesicles to exocytic sites. We conclude that neurons utilize F-actin to increase vesicular GluA1 reservoirs and promote exocytosis proximal to the sites of synaptic activity.
Subject(s)
Actins , Dendrites , Hippocampus , Neuronal Plasticity , Polymerization , Receptors, AMPA , Animals , Receptors, AMPA/metabolism , Actins/metabolism , Rats , Neuronal Plasticity/physiology , Dendrites/metabolism , Hippocampus/metabolism , Hippocampus/cytology , Protein Transport , Neurons/metabolism , Cells, Cultured , ExocytosisABSTRACT
All DNA-binding proteins in vivo exist as a population of freely diffusing molecules and of DNA-bound molecules. The molecules bound to DNA can be split into specifically/tightly and nonspecifically bound proteins. Single-molecule tracking (SMT) is a method allowing to visualize protein dynamics in living cells, revealing their behavior in terms of mode of motion, diffusion coefficient/speed, change of dwell times, and unveiling preferred subcellular sites of dwelling. Bleaching-type SMT or fluorescent protein-tagged SMT involves rapid laser-induced bleaching of most fluorophore-labeled molecules. The remaining single fluorescent proteins are then continuously tracked. The trajectories of several fluorescent molecules per cell for a population of cells are analyzed and combined to permit a robust analysis of average behavior of single molecules in live cells, including analyses of protein dynamics in mutant cells or cells exposed to changes in environmental conditions.In this chapter, we describe the preparation of Bacillus subtilis cells, the recording of movies of those cells expressing a monomeric variant of a yellow fluorescent protein (mNeonGreen) fused to a protein of choice, and the subsequent curation of the movie data including the statistical analysis of the protein dynamics. We present a short overview of the analysis program SMTracker 2.0, highlighting its ability to analyze SMT data by non-expert scientists.
Subject(s)
Bacillus subtilis , DNA-Binding Proteins , Single Molecule Imaging , Single Molecule Imaging/methods , Bacillus subtilis/metabolism , Bacillus subtilis/genetics , DNA-Binding Proteins/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Microscopy, Fluorescence/methods , Luminescent Proteins/metabolism , Luminescent Proteins/geneticsABSTRACT
Receptor for advanced glycation endproducts (RAGE) and toll-like receptor 4 (TLR4) are pattern-recognition receptors that bind to molecular patterns associated with pathogens, stress, and cellular damage. Diffusion plays an important role in receptor functionality in the cell membrane. However, there has been no prior investigation of the reciprocal effect of RAGE and TLR4 diffusion properties in the presence and absence of each receptor. This study reports how RAGE and TLR4 affect the mobility of each other in the human embryonic kidney (HEK) 293 cell membrane. Diffusion properties were measured using single-particle tracking (SPT) with quantum dots (QDs) that are selectively attached to RAGE or TLR4. The Brownian diffusion coefficients of RAGE and TLR4 are affected by the presence of the other receptor, leading to similar diffusion coefficients when both receptors coexist in the cell. When TLR4 is present, the average Brownian diffusion coefficient of RAGE increases by 40%, while the presence of RAGE decreases the average Brownian diffusion coefficient of TLR4 by 32%. Diffusion in confined membrane domains is not altered by the presence of the other receptor. The mobility of the cell membrane lipid remains constant whether one or both receptors are present. Overall, this work shows that the presence of each receptor can affect a subset of diffusion properties of the other receptor without affecting the mobility of the membrane.
Subject(s)
Cell Membrane , Receptor for Advanced Glycation End Products , Toll-Like Receptor 4 , Humans , Toll-Like Receptor 4/metabolism , Receptor for Advanced Glycation End Products/metabolism , HEK293 Cells , Cell Membrane/metabolism , DiffusionABSTRACT
The analysis of nucleocytoplasmic transport of proteins and messenger RNA has been the focus of advanced microscopic approaches. Recently, it has been possible to identify and visualize individual pre-ribosomal particles on their way through the nuclear pore complex using both electron and light microscopy. In this review, we focused on the transport of pre-ribosomal particles in the nucleus on their way to and through the pores.
Subject(s)
Active Transport, Cell Nucleus , Cell Nucleolus , Cytoplasm , Nuclear Pore , Cell Nucleolus/metabolism , Nuclear Pore/metabolism , Cytoplasm/metabolism , Humans , Animals , Ribosomes/metabolism , Cell Nucleus/metabolismABSTRACT
Individuals tend to move freely when there is enough room but would act collectively for their survival under external stress. In the case of living cells, for instance, when a drop of low-density flagellated bacterial solution is transferred onto the agar surface, the initially disordered movement of individual bacteria would be replaced with coordinated cell swarming after a lag phase of a few hours. Here, we study how such cooperation is established while overcoming the disorder at the onset of the lag phase with single nanoparticle tracking. Upon the spreading of the droplet, the bacteria in the solution cluster and align near the almost immobilized contact line confining the drop, forming a narrow ring of cells. As individual cells move in and out of the ring continuously, certain flow patterns emerge in the inter-bacterial fluid. We reveal high-speed long-distance unidirectional flows with definite chirality along the outside of the ring, along the inside of the ring and across the ring. We speculate that these flows enable the fast and efficient transport, facilitating the communication and unification of the bacterial community.
ABSTRACT
Nanoscale Metal-Organic Frameworks (nanoMOFs) are widely implemented in a host of assays involving drug delivery, biosensing catalysis, and bioimaging. However, the cell pathways and cell fate remain poorly understood. Here, a new fluorescent nanoMOF integrating ATTO 655 into surface defects of colloidal UiO-66 is synthesized, allowing to track the spatiotemporal localization of Single nanoMOF in live cells. Density functional theory reveals the stronger binding of ATTO 655 to the Zr6 cluster nodes compared with phosphate and Alendronate Sodium. Parallelized tracking of the spatiotemporal localization of thousands of nanoMOFs and analysis using machine learning platforms reveals whether nanoMOFs remain outside as well as their cellular internalization pathways. To quantitatively assess their colocalization with endo/lysosomal compartments, a colocalization proxy approach relying on the nanoMOF detection of particles in one channel to the signal in the corresponding endo/lysosomal compartments channel, considering signal versus local background intensity ratio and signal-to-noise ratio is developed. This strategy mitigates colocalization value inflation from high or low signal expression in endo/lysosomal compartments. The results accurately measure the nanoMOFs' colocalization from early to late endosomes and lysosomes and emphasize the importance of understanding their intracellular dynamics based on single-particle tracking for optimal and safe drug delivery.
Subject(s)
Drug Carriers , Lysosomes , Metal-Organic Frameworks , Metal-Organic Frameworks/chemistry , Humans , Drug Carriers/chemistry , Lysosomes/metabolism , Nanoparticles/chemistry , Endosomes/metabolism , Zirconium/chemistryABSTRACT
The dynamics of DNA in the cell nucleus plays a role in cellular processes and fates but the interplay of DNA mobility with the hierarchical levels of DNA organization is still underexplored. Here, we made use of DNA replication to directly label genomic DNA in an unbiased genome-wide manner. This was followed by live-cell time-lapse microscopy of the labeled DNA combining imaging at different resolutions levels simultaneously and allowing one to trace DNA motion across organization levels within the same cells. Quantification of the labeled DNA segments at different microscopic resolution levels revealed sizes comparable to the ones reported for DNA loops using 3D super-resolution microscopy, topologically associated domains (TAD) using 3D widefield microscopy, and also entire chromosomes. By employing advanced chromatin tracking and image registration, we discovered that DNA exhibited higher mobility at the individual loop level compared to the TAD level and even less at the chromosome level. Additionally, our findings indicate that chromatin movement, regardless of the resolution, slowed down during the S phase of the cell cycle compared to the G1/G2 phases. Furthermore, we found that a fraction of DNA loops and TADs exhibited directed movement with the majority depicting constrained movement. Our data also indicated spatial mobility differences with DNA loops and TADs at the nuclear periphery and the nuclear interior exhibiting lower velocity and radius of gyration than the intermediate locations. On the basis of these insights, we propose that there is a link between DNA mobility and its organizational structure including spatial distribution, which impacts cellular processes.
Subject(s)
DNA , DNA/chemistry , Humans , Chromosomes/metabolism , Chromosomes/chemistry , Chromatin/chemistry , Chromatin/metabolismABSTRACT
Mucus is a dynamic biological hydrogel, composed primarily of the glycoprotein mucin, exhibits unique biophysical properties and forms a barrier protecting cells against a broad-spectrum of viruses. Here, this work develops a polyglycerol sulfate-based dendronized mucin-inspired copolymer (MICP-1) with ≈10% repeating units of activated disulfide as cross-linking sites. Cryo-electron microscopy (Cryo-EM) analysis of MICP-1 reveals an elongated single-chain fiber morphology. MICP-1 shows potential inhibitory activity against many viruses such as herpes simplex virus 1 (HSV-1) and SARS-CoV-2 (including variants such as Delta and Omicron). MICP-1 produces hydrogels with viscoelastic properties similar to healthy human sputum and with tuneable microstructures using linear and branched polyethylene glycol-thiol (PEG-thiol) as cross-linkers. Single particle tracking microrheology, electron paramagnetic resonance (EPR) and cryo-scanning electron microscopy (Cryo-SEM) are used to characterize the network structures. The synthesized hydrogels exhibit self-healing properties, along with viscoelastic properties that are tuneable through reduction. A transwell assay is used to investigate the hydrogel's protective properties against viral infection against HSV-1. Live-cell microscopy confirms that these hydrogels can protect underlying cells from infection by trapping the virus, due to both network morphology and anionic multivalent effects. Overall, this novel mucin-inspired copolymer generates mucus-mimetic hydrogels on a multi-gram scale. These hydrogels can be used as models for disulfide-rich airway mucus research, and as biomaterials.
Subject(s)
Herpesvirus 1, Human , Hydrogels , Mucus , SARS-CoV-2 , Hydrogels/chemistry , Hydrogels/pharmacology , Herpesvirus 1, Human/drug effects , Humans , Mucus/metabolism , SARS-CoV-2/drug effects , Mucins/chemistry , Mucins/metabolism , Antiviral Agents/pharmacology , Antiviral Agents/chemistry , Polymers/chemistry , Polymers/pharmacology , Animals , Disulfides/chemistry , Polyethylene Glycols/chemistry , Cryoelectron Microscopy , COVID-19/virology , GlycerolABSTRACT
Super-resolution microscopy (SRM) approaches revolutionize cell biology by providing insights into the nanoscale organization and dynamics of macromolecular assemblies and single molecules in living cells. A major hurdle limiting SRM democratization is post-acquisition data analysis which is often complex and time-consuming. Here, we present OneFlowTraX, a user-friendly and open-source software dedicated to the analysis of single-molecule localization microscopy (SMLM) approaches such as single-particle tracking photoactivated localization microscopy (sptPALM). Through an intuitive graphical user interface, OneFlowTraX provides an automated all-in-one solution for single-molecule localization, tracking, as well as mobility and clustering analyses. OneFlowTraX allows the extraction of diffusion and clustering parameters of millions of molecules in a few minutes. Finally, OneFlowTraX greatly simplifies data management following the FAIR (Findable, Accessible, Interoperable, Reusable) principles. We provide a detailed step-by-step manual and guidelines to assess the quality of single-molecule analyses. Applying different fluorophores including mEos3.2, PA-GFP, and PATagRFP, we exemplarily used OneFlowTraX to analyze the dynamics of plant plasma membrane-localized proteins including an aquaporin, the brassinosteroid receptor Brassinosteroid Insensitive 1 (BRI1) and the Receptor-Like Protein 44 (RLP44).
ABSTRACT
This paper describes how branch lengths of anisotropic nanoparticles can affect interactions between grafted ligands and cell-membrane receptors. Using live-cell, single-particle tracking, we found that DNA aptamer-gold nanostar nanoconstructs with longer branches showed improved binding efficacy to human epidermal growth factor receptor 2 (HER2) on cancer cell membranes. Inhibiting nanoconstruct-HER2 binding promoted nonspecific interactions, which increased the rotational speed of long-branched nanoconstructs but did not affect that of short-branched constructs. Bivariate analysis of the rotational and translational dynamics showed that longer branch lengths increased the ratio of targeting to nontargeting interactions. We also found that longer branches increased the nanoconstruct-cell interaction times before internalization and decreased intracellular trafficking velocities. Differences in binding efficacy revealed by single-particle dynamics can be attributed to the distinct protein corona distributions on short- and long-branched nanoconstructs, as validated by transmission electron microscopy. Minimal protein adsorption at the high positive curvature tips of long-branched nanoconstructs facilitated binding of DNA aptamer ligands to HER2. Our study reveals the significance of nanoparticle branch length in regulating local chemical environment and interactions with live cells at the single-particle level.
Subject(s)
Aptamers, Nucleotide , Cell Membrane , Gold , Metal Nanoparticles , Receptor, ErbB-2 , Humans , Anisotropy , Gold/chemistry , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/metabolism , Cell Membrane/metabolism , Cell Membrane/chemistry , Receptor, ErbB-2/metabolism , Receptor, ErbB-2/chemistry , Metal Nanoparticles/chemistry , Cell Line, Tumor , LigandsABSTRACT
Single-molecule localization microscopy has proved promising to unravel the dynamics and molecular architecture of thin biological samples down to nanoscales. For applications in complex, thick biological tissues shifting single-particle emission wavelengths to the shortwave infrared (SWIR also called NIR II) region between 900 to 2100 nm, where biological tissues are more transparent is key. To date, mainly single-walled carbon nanotubes (SWCNTs) enable such applications, but they are inherently 1D objects. Here, 0D ultra-small luminescent gold nanoclusters (AuNCs, <3 nm) and ≈25 nm AuNC-loaded-polymeric particles that can be detected at the single-particle level in the SWIR are presented. Thanks to high brightness and excellent photostability, it is shown that the dynamics of the spherical polymeric particles can be followed at the single-particle level in solution at video rates for minutes. We compared single particle tracking of AuNC-loaded-polymeric particles with that of SWCNT diffusing in agarose gels demonstrating the specificity and complementarity of diffusion properties of these SWIR-emitting nano-objects when exploring a complex environment. This extends the library of photostable SWIR emitting nanomaterials to 0D nano-objects of variable size for single-molecule localization microscopy in the second biological window, opening unprecedented possibilities for mapping the structure and dynamics of complex biological systems.
ABSTRACT
Neural tracing proteins like horseradish peroxidase-conjugated wheat germ agglutinin (WGA-HRP) can target the central nervous system (CNS) through anatomic retrograde transport without crossing the blood-brain barrier (BBB). Conjugating WGA-HRP to nanoparticles may enable the creation of BBB-bypassing nanomedicine. Microfluidics and two-photon confocal microscopy is applied to screen nanocarriers for transport efficacy and gain mechanistic insights into their interactions with neurons. Protein modification of gold nanoparticles alters their cellular uptake at the axonal terminal and activates fast retrograde transport. Trajectory analysis of individual endosomes carrying the nanoparticles reveals a run-and-pause pattern along the axon with endosomes carrying WGA-HRP-conjugated gold nanoparticles exhibiting longer run duration and faster instantaneous velocity than those carrying nonconjugated nanoparticles. The results offer a mechanistic explanation of the different axonal transport dynamics as well as a cell-based functional assay of neuron-targeted nanoparticles with the goal of developing BBB-bypassing nanomedicine for the treatment of nervous system disorders.
ABSTRACT
During liver fibrosis, recurrent hepatic injuries lead to the accumulation of collagen and other extracellular matrix components in the interstitial space, ultimately disrupting liver functions. Early stages of liver fibrosis may be reversible, but opportunities for diagnosis at these stages are currently limited. Here, we show that the alterations of the interstitial space associated with fibrosis can be probed by tracking individual fluorescent single-walled carbon nanotubes (SWCNTs) diffusing in that space. In a mouse model of early liver fibrosis, we find that nanotubes generally explore elongated areas, whose lengths decrease as the disease progresses, even in regions where histopathological examination does not reveal fibrosis yet. Furthermore, this decrease in nanotube mobility is a purely geometrical effect as the instantaneous nanotube diffusivity stays unmodified. This work establishes the promise of SWCNTs both for diagnosing liver fibrosis at an early stage and for more in-depth studies of the biophysical effects of the disease.
Subject(s)
Liver Cirrhosis , Nanotubes, Carbon , Nanotubes, Carbon/chemistry , Animals , Liver Cirrhosis/pathology , Mice , Liver/pathology , Extracellular Matrix/metabolism , Fluorescent Dyes/chemistry , Disease Models, Animal , DiffusionABSTRACT
Emerging single-molecule protein sensing techniques are ushering in a transformative era in biomedical research. Nevertheless, challenges persist in realizing ultra-fast full-length protein sensing, including loss of molecular integrity due to protein fragmentation, biases introduced by antibodies affinity, identification of proteoforms, and low throughputs. Here, a single-molecule method for parallel protein separation and tracking is introduced, yielding multi-dimensional molecular properties used for their identification. Proteins are tagged by chemo-selective dual amino-acid specific labels and are electrophoretically separated by their mass/charge in custom-designed thin silicon channel with subwavelength height. This approach allows analysis of thousands of individual proteins within a few minutes by tracking their motion during the migration. The power of the method is demonstrated by quantifying a cytokine panel for host-response discrimination between viral and bacterial infections. Moreover, it is shown that two clinically-relevant splice isoforms of Vascular endothelial growth factor (VEGF) can be accurately quantified from human serum samples. Being non-destructive and compatible with full-length intact proteins, this method opens up ways for antibody-free single-protein molecule quantification.
Subject(s)
Silicon , Vascular Endothelial Growth Factor A , Silicon/chemistry , Humans , Vascular Endothelial Growth Factor A/metabolism , Proteins/chemistry , Proteins/metabolism , Single Molecule Imaging/methodsABSTRACT
Single-particle tracking (SPT) is a powerful technique to unveil molecular behaviors crucial to the understanding of many biological processes, but it is limited by factors such as probe photostability and spectral orthogonality. To overcome these limitations, we develop upconverting nanoparticles (UCNPs), which are photostable over several hours at the single-particle level, enabling long-term multicolor SPT. We investigate the brightness of core-shell UCNPs as a function of inert shell thickness to minimize particle size while maintaining sufficient signal for SPT. We explore different rare-earth dopants to optimize for the brightest probes and find that UCNPs doped with 2% Tm3+/30% Yb3+, 10% Er3+/90% Yb3+, and 15% Tm3+/85% Yb3+ represent the optimal probes for blue, green, and near-infrared emission, respectively. The multiplexed 10 nm probes enable three-color single-particle tracking on live HeLa cells for tens of minutes using a single, near-infrared excitation source. These photostable and multiplexed probes open new avenues for numerous biological applications.
ABSTRACT
Single-particle tracking (SPT) of biomolecules in the plant endoplasmic reticulum has the potential to inform on the formation of protein-protein complexes, metabolons, and the transport of molecules through both the ER membrane and lumen. Plant cells are particularly challenging for observing and tracking single molecules due to their unique structure, size, and considerable autofluorescence. However, by using variable-angle or highly inclined epifluorescence microscopy (VAEM) and transient expression in tobacco, it is possible to observe single-particle dynamics in the ER. Selecting the appropriate fluorophore, and ensuring the correct fluorophore density in the ER, is essential for successful SPT. By using tuneable fluorophores, which can be photoconverted and photoactivated, it is possible to vary the density of visible fluorophores in the ER dynamically. Here we describe methods to prepare plant samples for VAEM and two methods for determining and analyzing single-particle tracks from VAEM time series.