Two-color Fluorescence Microscopy Research Articles

Purification Analysis, Intracellular Tracking, and Colocalization of Extracellular Vesicles Using Atomic Force and 3D Single-Molecule Localization Microscopy.

Extracellular vesicles (EVs) play a key role in cell-cell communication and thus have great potential to be utilized as therapeutic agents and diagnostic tools. In this study, we implemented single-molecule microscopy techniques as a toolbox for a comprehensive characterization as well as measurement of the cellular uptake of HEK293T cell-derived EVs (eGFP-labeled) in HeLa cells. A combination of fluorescence and atomic force microscopy revealed a fraction of 68% fluorescently labeled EVs with an average size of ∼45 nm. Two-color single-molecule fluorescence microscopy analysis elucidated the 3D dynamics of EVs entering HeLa cells. 3D colocalization analysis of two-color direct stochastic optical reconstruction microscopy (dSTORM) images revealed that 25% of EVs that experienced uptake colocalized with transferrin, which has been linked to early recycling of endosomes and clathrin-mediated endocytosis. The localization analysis was combined with stepwise photobleaching, providing a comparison of protein aggregation outside and inside the cells.

Diffusion Analysis of NAnoscopic Ensembles: A Tracking-Free Diffusivity Analysis for NAnoscopic Ensembles in Biological Samples and Nanotechnology.

The rapid development of microscopic techniques over the past decades enables the establishment of single molecule fluorescence imaging as a powerful tool in biological and biomedical sciences. Single molecule fluorescence imaging allows to study the chemical, physicochemical, and biological properties of target molecules or particles by tracking their molecular position in the biological environment and determining their dynamic behavior. However, the precise determination of particle distribution and diffusivities is often challenging due to high molecule/particle densities, fast diffusion, and photobleaching/blinking of the fluorophore. A novel, accurate, and fast statistical analysis tool, Diffusion Analysis of NAnoscopic Ensembles (DANAE), that solves all these obstacles is introduced. DANAE requires no approximations or any a priori input regarding unknown system-inherent parameters, such as background distributions; a requirement that is vitally important when studying the behavior of molecules/particles in living cells. The superiority of DANAE with various data from simulations is demonstrated. As experimental applications of DANAE, membrane receptor diffusion in its natural membrane environment, and cargo mobility/distribution within nanostructured lipid nanoparticles are presented. Finally, the method is extended to two-color channel fluorescence microscopy.

Encoding Multiple Virtual Signals in DNA Barcodes with Single-Molecule FRET.

DNA barcoding provides a way to label a myriad of different biological molecules using the extreme programmability in DNA sequence synthesis. Fluorescence imaging is presumably the most easy-to-access method for DNA barcoding, yet large spectral overlaps between fluorescence dyes severely limit the numbers of barcodes that can be detected simultaneously. We here demonstrate the use of single-molecule fluorescence resonance energy transfer (FRET) to encode virtual signals in DNA barcodes using conventional two-color fluorescence microscopy. By optimizing imaging and biochemistry conditions for weak DNA hybridization events, we markedly enhanced accuracy in our determination of the single-molecule FRET efficiency exhibited by each binding event between DNA barcode sequences. This allowed us to unambiguously differentiate six DNA barcodes encoding different FRET values without involving any probe sequence exchanges. Our method can be directly incorporated with previous DNA barcode techniques, and may thus be widely adopted to expand the signal space of DNA barcoding.

Targeted Solvatochromic Fluorescent Probes for Imaging Lipid Order in Organelles under Oxidative and Mechanical Stress.

Biomembranes constitute a basis for all compartments of live cells, and therefore, the monitoring of their lipid organization is essential for understanding cell status and activity. However, the sensing and imaging of lipid organization specifically in different organelles of live cells remain challenging. Here, we designed an array of solvatochromic probes based on Nile Red bearing ligands for specific targeting of the endoplasmic reticulum, mitochondria, lysosomes, Golgi apparatus, plasma membranes, and lipid droplets. These polarity-sensitive probes detected variations in the lipid order by changing their emission maximum, as evidenced by fluorescence spectroscopy in model membranes. In colocalization microscopy experiments with reference organelle markers, they exhibited good organelle selectivity. Using two-color fluorescence microscopy, the new probes enabled imaging of the local polarity of organelles in live cells. To exclude the biased effect of the probe design on the sensitivity to the membrane properties, we calibrated all probes in model membranes under the microscope, which enabled the first quantitative description of the lipid order in each organelle of interest. Cholesterol extraction/enrichment confirmed the capacity of the probes to sense the lipid order, revealing that organelles poor in cholesterol are particularly affected by its enrichment. The probes also revealed that oxidative and mechanical stresses produced changes in the local polarity and lipid order that were characteristic for each organelle, with mitochondria and lysosomes being particularly stress sensitive. The new probes constitute a powerful toolbox for monitoring the response of the cells to physical and chemical stimuli at the level of membranes of individual organelles, which remains an underexplored direction in cellular research.

4D Widefield Fluorescence Imaging of Appressorium Morphogenesis by Magnaporthe oryzae.

Fluorescence microscopy has become a widely used and indispensable tool for the M. oryzae research community, providing unique insight into appressorium formation and function. A common practice within the field is to acquire and present images of a number of different conidia, expressing a fluorescent fusion protein of interest, at various stages of infectious development, therein providing a representative "snapshot" of the population at a given point in time. Furthermore, these images typically show only a single focal plane through the specimen (2D) and therefore lack, often valuable, volumetric information. While this approach has its advantages, the continuous imaging of (multiple) single conidia in three dimensions (3D), and over time (4D), can provide additional insight into the spatial and temporal dynamics of fluorescent fusion proteins, and the subcellular structures and compartments they label, in living cells. Here we describe our typical workflow for the 4D live-cell imaging of appressorium morphogenesis in vitro using two-color widefield fluorescence microscopy and briefly outline some important considerations for strain construction, and downstream image processing and visualization.

Glycobiology of developing chicken kidney: Profiling the galectin family and selected β-galactosides.

The concept of the sugar code interprets the cellular glycophenotype as a rich source of information read by glycan-lectin recognition in situ. This study's aim is the comprehensive characterization of galectin expression by immunohistochemistry during chicken nephrogenesis along with mapping binding sites by (ga)lectin histochemistry. Light and two-color fluorescence microscopy were used. First, six plant/fungal lectins that are specific for galectin-binding parts of N- and O-glycans were applied. The spatiotemporally regulated distributions of these glycans in meso- and metanephros equip cells with potential binding partners for the galectins. Complete galectin profiling from HH Stage 20 (about 70-72 hr) onward revealed cell-, galectin-, and stage-dependent expression patterns. Representatives of all three types of modular architecture of the galectin family are detectable, and overlaps of signal distribution in light and two-color fluorescence microscopy illustrate a possibility for functional cooperation among them. Performing systematic galectin histochemistry facilitated comparisons between staining profiles of plant lectins and galectins. They revealed several cases for differences so that tissue lectins appear to be selective among the β-galactosides. Notably, selectivity is also disclosed in intrafamily comparison. Thus, combining experimental series with plant and tissue lectins is a means to characterize target populations of glycans presented by cellular glycoconjugates for individual galectins. Our results document the presence and sophisticated level of elaboration among β-galactosides and among the members of the family of galectins during organogenesis, using chicken galectins and kidney as model. Thus, they provide a clear guideline for functional assays using supramolecular tools, cells, and organ cultures.

Triggering Cation-Induced Contraction of Cytoskeleton Networks via Microfluidics.

The dynamic morphology and mechanics of the cytoskeleton is determined by interacting networks of semiflexible actin filaments and rigid microtubules. Active rearrangement of networks of actin and microtubules can not only be driven by motor proteins but by changes to ionic conditions. For example, high concentrations of multivalent ions can induce bundling and crosslinking of both filaments. Yet, how cytoskeleton networks respond in real-time to changing ion concentrations, and how actin-microtubule interactions impact network response to these changing conditions remains unknown. Here, we use microfluidic perfusion chambers and two-color confocal fluorescence microscopy to show that increasing magnesium ions trigger contraction of both actin and actin-microtubule networks. Specifically, we use microfluidics to vary the Mg2+ concentration between 2 and 20 mM while simultaneously visualizing the triggered changes to the overall network size. We find that as Mg2+ concentration increases both actin and actin-microtubule networks undergo bulk contraction, which we measure as the shrinking width of each network. However, surprisingly, lowering the Mg2+concentration back to 2 mM does not stop or reverse the contraction but rather causes both networks to contract further. Further, actin networks begin to contract at lower Mg2+ concentrations and shorter times than actin-microtubule networks. In fact, actin-microtubule networks only undergo substantial contraction once the Mg2+ concentration begins to lower from 20 mM back to 2 mM. Our intriguing findings shed new light on how varying environmental conditions can dynamically tune the morphology of cytoskeleton networks and trigger active contraction without the use of motor proteins.

Smartphone-assisted detection of nucleic acids by light-harvesting FRET-based nanoprobe

Point-of-care assays for optical detection of biomolecular markers attract growing attention, because of their capacity to provide rapid and inexpensive diagnostics of cancer and infectious diseases. Here, we designed a nanoprobe compatible with a smartphone RGB camera for detection of nucleic acids. It is based on light-harvesting polymeric nanoparticles (NPs) encapsulating green fluorescent donor dyes that undergo efficient Förster Resonance Energy Transfer (FRET) to red fluorescent acceptor hybridized at the particle surface. Green-emitting NPs are based on rhodamine 110 and 6G dyes paired with bulky hydrophobic counterions, which prevent dye self-quenching and ensure efficient energy transfer. Their surface is functionalized with a capture DNA sequence for cancer marker survivin, hybridized with a short oligonucleotide bearing FRET acceptor ATTO647N. Obtained 40-nm poly(methyl methacrylate)-based NP probe, encapsulating octadecyl rhodamine 6G dyes with tetrakis(perfluoro-tert-butoxy)aluminate counterions (~6000 dyes per NP), and bearing 65 acceptors, shows efficient FRET with >20% quantum yield and a signal amplification (antenna effect) of 25. It exhibits ratiometric response to the target DNA by FRET acceptor displacement and enables DNA detection in solution by fluorescence spectroscopy (limit of detection 3 pM) and on surfaces at the single-particle level using two-color fluorescence microscopy. Using a smartphone RGB camera, the nanoprobe response can be readily detected at 10 pM target in true color and in red-to-green ratio images. Thus, our FRET-based nanoparticle biosensor enables detection of nucleic acid targets using a smartphone coupled to an appropriate optical setup, opening the way to simple and inexpensive point-of-care assays.

SNARE-Mediated Fusion of Single Chromaffin Granules with Pore-Spanning Membranes

Pore-spanning membranes (PSMs) composed of supported membrane parts as well as freestanding membrane parts are shown to be very versatile to investigate SNARE-mediated fusion on the single-particle level. They provide a planar geometry readily accessible by confocal fluorescence microscopy, which enabled us for the first time, to our knowledge, to investigate the fusion of individual natural secretory granules (i.e., chromaffin granules (CGs)) on the single-particle level by two-color fluorescence microscopy in a time-resolved manner. The t-SNARE acceptor complex ΔN49 was reconstituted into PSMs containing 2 mol % 1,2-dipalmitoyl-sn-glycero-3-phosphatidylinositol-4,5-bisphosphate and Atto488-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, and CGs were fluorescently labeled with 2-((1E,3E)-5-((Z)-3,3-dimethyl-1-octadecylindolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-1-octadecyl-3H-indol-1-ium perchlorate. We compared the dynamics of docked and hemifused CGs as well as their fusion efficacy and kinetics with the results obtained for synthetic synaptobrevin 2-doped vesicles fusing with PSMs of the same composition. Whereas the synthetic vesicles were fully immobile on supported PSMs, docked as well as hemifused CGs were mobile on both PSM parts, which suggests that this system resembles more closely the natural situation. The fusion process of CGs proceeded through three-dimensional post-lipid-mixing structures, which were readily resolved on the gold-covered pore rims of the PSMs and which are discussed in the context of intermediate states observed in live cells.

Dip1 Co-opts Features of Branching Nucleation to Create Linear Actin Filaments that Activate WASP-Bound Arp2/3 Complex

When activated by Wiskott-Aldrich syndrome proteins (WASP), Arp2/3 complex nucleates branched actin filaments important for processes like cellular motility and endocytosis [1]. WASP-mediated activation of Arp2/3 complex requires a preformed actin filament, ensuring that activation by WASP creates branched instead of linear filaments. However, this biochemical requirement also means that assembly of branched actin networks must be primed with an initial seed filament [2-4]. We recently described a class of activators called WISH/DIP/SPIN90 (WDS) proteins, which, unlike WASP, activate Arp2/3 complex without a preformed filament [4]. Although this property may allow WDS proteins to serve as seed filament generators, it is unknown whether actin filaments nucleated by WDS-activated Arp2/3 complex can activate WASP-bound Arp2/3 complex. Further, despite their potential importance as branched actin network initiators, little is known about how WDS proteins turn on Arp2/3 complex. Here, we use two-color single-molecule total internal reflection fluorescence (TIRF) microscopy to show that Dip1, the S.pombe WDS protein [5], co-opts features of branching nucleation to activate Arp2/3 complex. Specifically, it activates Arp2/3 complex to nucleate linear filaments analogous to the branch created by WASP-mediated activation. The barbed ends of Dip1-Arp2/3 nucleated filaments are free to elongate, and their pointed ends remain anchored to Dip1-bound Arp2/3 complex. The linear filaments nucleated by Dip1-bound Arp2/3 complex activate WASP-bound Arp2/3 complex as potently as spontaneously nucleated or branched actin filaments. These observations provide important insights into the regulation of Arp2/3 complex by its activators and the molecular basis for initiation of branched actin networks.

Syntaxins on granules promote docking of granules via interactions with munc18

SNAREs and SNARE-binding accessory proteins are believed to be central molecular components of neurotransmitter release, although the precise sequence of molecular events corresponding to distinct physiological states is unclear. The mechanism of docking of vesicles to the plasma membrane remains elusive, as the anchoring protein residing on vesicles is unknown. Here I show that targeting small amounts of syntaxin to granules by transmembrane domain alteration leads to a substantial enhancement of syntaxin clustering beneath granules, as well as of morphological granule docking. The effect was abolished without munc18 and strongly reduced by removal of the N-terminal peptide in the syntaxin mutant. Thus, in contrast to the current paradigm, I demonstrate that syntaxin acts from the vesicular membrane, strongly facilitating docking of vesicles, likely via interaction of its N-peptide with munc18. Docking was assayed by quantifying the syntaxin clusters beneath granules, using two-color Total Internal Reflectance Fluorescence microscopy in live PC-12 cells and confirmed by electron microscopy. Hereby, I propose a new model of vesicle docking, wherein munc18 bridges the few syntaxin molecules residing on granules to the syntaxin cluster on the plasma membrane, suggesting that the number of syntaxins on vesicles determines docking and conceivably fusion probability.

Two-Color Total Internal Reflection Fluorescence Microscopy of Exocytosis in Endocrine Cells.

We describe a comprehensive method for imaging and analysis of local protein dynamics at single sites of exocytosis in living cultured endocrine cells. This method is well suited to quantitatively map the complex dynamics of individual molecules at single sites of vesicle fusion in live cells.

Imaging the recruitment and loss of proteins and lipids at single sites of calcium-triggered exocytosis

How and when the dozens of molecules that control exocytosis assemble in living cells to regulate the fusion of a vesicle with the plasma membrane is unknown. Here we image with two-color total internal reflection fluorescence microscopy the local changes of 27 proteins at single dense-core vesicles undergoing calcium-triggered fusion. We identify two broad dynamic behaviors of exocytic molecules. First, proteins enriched at exocytic sites are associated with DCVs long before exocytosis, and near the time of membrane fusion, they diffuse away. These proteins include Rab3 and Rab27, rabphilin3a, munc18a, tomosyn, and CAPS. Second, we observe a group of classical endocytic proteins and lipids, including dynamins, amphiphysin, syndapin, endophilin, and PIP2, which are rapidly and transiently recruited to the exocytic site near the time of membrane fusion. Dynamin mutants unable to bind amphiphysin were not recruited, indicating that amphiphysin is involved in localizing dynamin to the fusion site. Expression of mutant dynamins and knockdown of endogenous dynamin altered the rate of cargo release from single vesicles. Our data reveal the dynamics of many key proteins involved in exocytosis and identify a rapidly recruited dynamin/PIP2/BAR assembly that regulates the exocytic fusion pore of dense-core vesicles in cultured endocrine beta cells.

The F-actin bundler α-actinin Ain1 is tailored for ring assembly and constriction during cytokinesis in fission yeast.

The actomyosin contractile ring is a network of cross-linked actin filaments that facilitates cytokinesis in dividing cells. Contractile ring formation has been well characterized in Schizosaccharomyces pombe, in which the cross-linking protein α-actinin SpAin1 bundles the actin filament network. However, the specific biochemical properties of SpAin1 and whether they are tailored for cytokinesis are not known. Therefore we purified SpAin1 and quantified its ability to dynamically bind and bundle actin filaments in vitro using a combination of bulk sedimentation assays and direct visualization by two-color total internal reflection fluorescence microscopy. We found that, while SpAin1 bundles actin filaments of mixed polarity like other α-actinins, SpAin1 has lower bundling activity and is more dynamic than human α-actinin HsACTN4. To determine whether dynamic bundling is important for cytokinesis in fission yeast, we created the less dynamic bundling mutant SpAin1(R216E). We found that dynamic bundling is critical for cytokinesis, as cells expressing SpAin1(R216E) display disorganized ring material and delays in both ring formation and constriction. Furthermore, computer simulations of initial actin filament elongation and alignment revealed that an intermediate level of cross-linking best facilitates filament alignment. Together our results demonstrate that dynamic bundling by SpAin1 is important for proper contractile ring formation and constriction.

Conventional fluorescence microscopy below the diffraction limit with simultaneous capture of two fluorophores in DNA origami.

A conventional fluorescence microscope was previously constructed for simultaneous imaging of two colors to gain subdiffraction localization. The system is predicated on color separation of overlapping Airy discs, construction of matrices of Cartesian coordinates to determine locations as well as centers of the point spread functions of fluorophores. Quantum dots that are separated by as little as 10 nm were resolved in the x-y coordinates. Inter-fluorophore distances that vary by 10 nm could also be distinguished. Quantum dots are bright point light source emitters that excite with a single laser and can serve as a label for many biomolecules. Here, alterations in the method are described to test the ability to resolve Atto 488 and Atto 647 dyes attached to DNA origami at ~40 nm spacing intervals. Dual laser excitation is used in tandem with multi-wavelength bandpass filters. Notwithstanding challenges from reduced intensity in Atto labeled DNA origami helical bundles compared to quantum dots, preliminary data show a mean inter-fluorophore distance of 56 nm with a range (14-148 nm). The range closely matches published results with DNA origami with other methods of subdiffraction microscopy. Sub-diffraction simultaneous two-color imaging fluorescence microscopy acronymically christened (SSTIFM) is a simple, readily accessible, technique for measurement of inter-fluorophore distances in compartments less than 40 nm. Preliminary results with so called nanorulers are encouraging for use with other biomolecules.

Class A Plexins Are Organized as Preformed Inactive Dimers on the Cell Surface

Plexins are single-pass transmembrane receptors that bind the axon guidance molecules semaphorins. Single-pass transmembrane proteins are an important class of receptors that display a wide variety of activation mechanisms, often involving ligand-dependent dimerization or conformational changes. Resolving the activation mechanism and dimerization state of these receptors is extremely challenging, especially in a live-cell environment. Here, we report on the dimerization state of PlexinA4 and its response to activation by semaphorin binding. Semaphorins are dimeric molecules that activate plexin by binding two copies of plexin simultaneously and inducing formation of a specific active dimer of plexin. An open question is whether there are preexisting plexin dimers that could act as autoinhibitory complexes. We address these questions with pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS is a two-color fluorescence microscopy method that is directly sensitive to protein dimerization in a live-cell environment. With PIE-FCCS, we show that inactive PlexinA4 is dimerized in the live-cell plasma membrane. By comparing the cross correlation of full-length PlexinA4 to control proteins and plexin mutants, we show that dimerization of inactive PlexinA4 requires the Sema domain, but not the cytoplasmic domain. Ligand stimulation with Sema6A does not change the degree of cross correlation, indicating that plexin activation does not lead to higher-order oligomerization. Together, the results suggest that semaphorin activates plexin by disrupting an inhibitory plexin dimer and inducing the active dimer.

Multispectral Bayesian reconstruction technique for real-time two color fluorescence microscopy

We have developed a real-time imaging method for two-color widefield fluorescence microscopy using a combined approach that integrates multi-spectral imaging and Bayesian image reconstruction technique.

Two-Color Imaging using Spectral Variants of iRFP670 and iRFP682 Near-Infrared Fluorescent Proteins

The new class of fluorescent proteins (FPs) engineered from bacterial phytochromes (BphPs) attracts much attention for in vivo imaging due to their near-infrared (NIR) fluorescence spectra. These FPs utilize widely available in mammalian cells biliverdin (BV), a product of heme degradation, as a chromophore and, therefore, are as easy to use as common GFP-like proteins. We recently reported five NIR FPs, called iRFPs, with different fluorescence and biochemical properties. Interestingly, two of them, iRFP670 and iRFP682, exhibited the twice higher molecular brightness, as well as the blue-shifted absorbance (643 nm and 663 nm) and fluorescence (670 nm and 682 nm) compared to other iRFPs. Here we characterized the unusual properties of these NIR FPs in detail. Our biochemical and biophysical analysis showed that iRFP670 and iRFP682 incorporate the BV chromophore in two distinct confirmations. A single amino acid mutation resulted in a depletion of one BV confirmations in the protein binding pocket and, consequently, in 30 nm red-shifts of both absorbance and fluorescence. The point mutation also caused a slight decrease in the molecular brightness and an increase in the pH stability of the obtained red-shifted variants, which we named iRFP670-red and iRFP682-red. The effective brightness of the iRFP670-red and iRFP682-red in live mammalian cells was comparable to that of the parental proteins, suggesting that the high efficiency and high specificity of the incorporation of endogenous BV chromophore was not affected. Spectrally resolvable fluorescence of the iRFP670 and iRFP670-red pair, as well as of the iRFP682 and iRFP682-red pair, allowed easy separation of two cellular populations using FACS cytometry and straightforward two-color fluorescence microscopy of live cells, thus making them the probes of choice for cell labeling in the NIR region.

ME-09 * DYNAMIC EVIDENCE OF TUMOR INDUCED MICROGLIA ACTIVATION AT THE INFILTRATIVE MARGINS OF GLIOMA

PURPOSE: Microglia are a major cellular component of malignant glioma, and in some cases, compose up to 40% of the mass of the tumor. Previous studies have shown that microglia can decrease T-cell response to glioma, and their abundance is correlated with increased histologic grade. These studies suggest that microglia facilitate the progression and infiltration of glioma, however the dynamics of the relationship between tumor cells and microglia are not well characterized. METHODS: In this work, we examined the dynamic migratory behavior of glioma and microglia cells using two-color time-lapse fluorescence microscopy of brain slices from a PDGF-driven rat model of glioma in which glioma cells and microglia were labeled with separate fluorescent markers. We quantified glioma cells and microglia motility through single cell tracking and particle image velocimetry. RESULTS: We found that microglia were predominately abundant within the tumor mass and that microglia motility was strongly correlated with the presence of glioma cells. This provides the first dynamic evidence that glioma induces microglial motility. We found that motility of glioma cells and microglia were variably correlated. Our results also show that microglia and glioma cells exhibit stark differences in migratory behavior. Microglia move by a simple random walk, while glioma cells exhibit highly persistent motion, characterized as super diffusion, within the same microenvironment indicating intrinsic differences in response to migratory cues. CONCLUSION: These results provide the first dynamic evidence of glioma cells stimulating the activation of microglia, by means of increasing motility and localization in and around the infiltrative edge of glioma. Further, these results show dynamic interactions between glioma and microglia and suggest that glioma cells and microglia are either responding to different migratory cues, or are responding to the same cues in different ways.

Abstract 4942: Metastatic tumor migration in lymphatic vessels revealed by real-time intravital imaging

Abstract Distant tumor metastases and their confounding symptoms are often responsible for reduced patient quality-of-life and decreased overall survival. The main route of metastasis for many solid tumors is the lymphatic vasculature. The mechanisms of metastatic spread of tumor cells through the lymphatic vessels remain poorly understood. We have developed a novel transgenic mouse model and customized real-time intravital microscopy platforms to study the migration of tumor cells into lymphatic vessels in live animals over the course of several days. TdTomato fluorescent protein was expressed in lymphatic endothelial cells using an inductive CRE recombinase approach. This transgenic system coupled with the use of fluorescent stereomicroscopy techniques allowed us to visualize the cutaneous lymphatic vessel network with remarkable anatomic detail in living mice. We tracked the fate of CFSE-labeled B16 tumor cells administered cutaneously using two-color intravital fluorescence microscopy. This strategy allowed us to simultaneously visualize the lymphatic vessel network (red) and the B16 tumor cells (green) at single-cell resolution. We clearly visualized and documented several key behaviors of metastasizing tumor cells including single-cell intravasation, movement of tumor cells within vessels, clearing from vessels, and the accumulation of tumor cells in regional lymph nodes. These events occurred at a low frequency over the course of days in conditions of tissue homeostasis. Current studies are designed to define the anatomic site of tumor intravasation into lymphatic vessels and investigate how the inflamed tumor microenvironment affects tumor migration into the lymphatic vasculature. Citation Format: Darci M. Fink, Alicia L. Connor, Philip M. Kelley, Richard M. Tempero, Michael A. Hollingsworth. Metastatic tumor migration in lymphatic vessels revealed by real-time intravital imaging. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4942. doi:10.1158/1538-7445.AM2014-4942