Water Hydrogen-Bond Mediated Layer by Layer Alignment of Lipid Rafts as a Precursor of Intermembrane Processes.

Lipid rafts, which are dynamic nanodomains in the plasma membrane, play a crucial role in intermembrane processes by clustering together and growing in size within the plane of the membrane while also aligning with each other across different membranes. However, the physical origin of layer by layer alignment of lipid rafts remains to be elucidated. Here, by using fluorescence imaging and synchrotron X-ray reflectivity in a phase-separated multilayer system, we find that the alignment of raft-mimicking Lo domains is regulated by the distance between bilayers. Molecular dynamics simulations reveal that the aligned state is energetically preferred when the intermembrane distance is small due to its ability to minimize the volume of surface water, which has fewer water hydrogen bonds (HBs) compared to bulk water. Our results suggest that water HB-driven alignment of lipid rafts plays a role as a precursor of intermembrane processes such as cell-cell fusion, virus entry, and signaling.

Complexes of tubulin oligomers and tau form a viscoelastic intervening network cross-bridging microtubules into bundles.

The axon-initial-segment (AIS) of mature neurons contains microtubule (MT) fascicles (linear bundles) implicated as retrograde diffusion barriers in the retention of MT-associated protein (MAP) tau inside axons. Tau dysfunction and leakage outside of the axon is associated with neurodegeneration. We report on the structure of steady-state MT bundles in varying concentrations of Mg2+ or Ca2+ divalent cations in mixtures containing αß-tubulin, full-length tau, and GTP at 37 °C in a physiological buffer. A concentration-time kinetic phase diagram generated by synchrotron SAXS reveals a wide-spacing MT bundle phase (Bws), a transient intermediate MT bundle phase (Bint), and a tubulin ring phase. SAXS with TEM of plastic-embedded samples provides evidence of a viscoelastic intervening network (IN) of complexes of tubulin oligomers and tau stabilizing MT bundles. In this model, αß-tubulin oligomers in the IN are crosslinked by tau's MT binding repeats, which also link αß-tubulin oligomers to αß-tubulin within the MT lattice. The model challenges whether the cross-bridging of MTs is attributed entirely to MAPs. Tubulin-tau complexes in the IN or bound to isolated MTs are potential sites for enzymatic modification of tau, promoting nucleation and growth of tau fibrils in tauopathies.

Regulation of Interfacial Anchoring Orientation of Anisotropic Nanodumbbells.

Nanoparticles exhibiting geometrical and chemical anisotropies hold promise for environmentally responsive materials with tunable mechanical properties. However, a comprehensive understanding of their interfacial behaviors remains elusive. In this paper, we control the interfacial anchoring orientation of polystyrene nanodumbbells by adjusting interparticle forces. The film nanostructure is characterized by the orientation angle analysis of individual dumbbells from cross-sectional EM data: dumbbells undergo orientation transitions from a distinctive horizontal bilayer to an isotropic anchoring when electrostatic repulsion is suppressed by either an ionic strength increase or surface amine-modification. This anchoring orientation influences the film's mechanical properties and foam stability, as investigated by a 2D isotherm and dark/bright-field microscopy measurements. Our findings highlight the potential for precise control of supra-colloidal structures by modulating particle alignment, paving the way for smart delivery systems.

Synchrotron X-ray study of intrinsically disordered and polyampholytic Tau 4RS and 4RL under controlled ionic strength.

Aggregated and hyperphosphorylated Tau is one of the pathological hallmarks of Alzheimer's disease. Tau is a polyampholytic and intrinsically disordered protein (IDP). In this paper, we present for the first time experimental results on the ionic strength dependence of the radius of gyration (Rg) of human Tau 4RS and 4RL isoforms. Synchrotron X-ray scattering revealed that 4RS Rg is regulated from 65.4 to 58.5 Å and 4RL Rg is regulated from 70.9 to 57.9 Å by varying ionic strength from 0.01 to 0.592 M. The Rg of 4RL Tau is larger than 4RS at lower ionic strength. This result provides an insight into the ion-responsive nature of intrinsically disordered and polyampholytic Tau, and can be implicated to the further study of Tau-Tau and Tau-tubulin intermolecular structure in ionic environments.

Ionic contrast across a lipid membrane for Debye length extension: towards an ultimate bioelectronic transducer.

Despite technological advances in biomolecule detections, evaluation of molecular interactions via potentiometric devices under ion-enriched solutions has remained a long-standing problem. To avoid severe performance degradation of bioelectronics by ionic screening effects, we cover probe surfaces of field effect transistors with a single film of the supported lipid bilayer, and realize respectable potentiometric signals from receptor-ligand bindings irrespective of ionic strength of bulky solutions by placing an ion-free water layer underneath the supported lipid bilayer. High-energy X-ray reflectometry together with the circuit analysis and molecular dynamics simulation discovered biochemical findings that effective electrical signals dominantly originated from the sub-nanoscale conformational change of lipids in the course of receptor-ligand bindings. Beyond thorough analysis on the underlying mechanism at the molecular level, the proposed supported lipid bilayer-field effect transistor platform ensures the world-record level of sensitivity in molecular detection with excellent reproducibility regardless of molecular charges and environmental ionic conditions.

A bioinspired and hierarchically structured shape-memory material.

Shape-memory polymeric materials lack long-range molecular order that enables more controlled and efficient actuation mechanisms. Here, we develop a hierarchical structured keratin-based system that has long-range molecular order and shape-memory properties in response to hydration. We explore the metastable reconfiguration of the keratin secondary structure, the transition from α-helix to ß-sheet, as an actuation mechanism to design a high-strength shape-memory material that is biocompatible and processable through fibre spinning and three-dimensional (3D) printing. We extract keratin protofibrils from animal hair and subject them to shear stress to induce their self-organization into a nematic phase, which recapitulates the native hierarchical organization of the protein. This self-assembly process can be tuned to create materials with desired anisotropic structuring and responsiveness. Our combination of bottom-up assembly and top-down manufacturing allows for the scalable fabrication of strong and hierarchically structured shape-memory fibres and 3D-printed scaffolds with potential applications in bioengineering and smart textiles.

Tubulin Double Helix: Lateral and Longitudinal Curvature Changes of Tubulin Protofilament.

By virtue of their native structures, tubulin dimers are protein building blocks that are naturally preprogrammed to assemble into microtubules (MTs), which are cytoskeletal polymers. Here, polycation-directed (i.e., electrostatically tunable) assembly of tubulins is demonstrated by conformational changes to the tubulin protofilament in longitudinal and lateral directions, creating tubulin double helices and various tubular architectures. Synchrotron small-angle X-ray scattering and transmission electron microscopy reveal a remarkable range of nanoscale assembly structures: single- and double-layered double-helix tubulin tubules. The phase transitions from MTs to the new assemblies are dependent on the size and concentration of polycations. Two characteristic scales that determine the number of observed phases are the size of polycation compared to the size of tubulin (≈4 nm) and to MT diameter (≈25 nm). This work suggests the feasibility of using polycations that have scissor- and glue-like properties to achieve "programmable breakdown" of protein nanotubes, tearing MTs into double-stranded tubulins and building up previously undiscovered nanostructures. Importantly, a new role of tubulins is defined as 2D shape-controllable building blocks for supramolecular architectures. These findings provide insight into the design of protein-based functional materials, for example, as metallization templates for nanoscale electronic devices, molecular screws, and drug delivery vehicles.

Tubulin-Based Nanotubes as Delivery Platform for Microtubule-Targeting Agents.

Tubulin-based nanotubes (TNTs) to deliver microtubule-targeting agents (MTAs) for clinical oncology are reported. Three MTAs, docetaxel (DTX), laulimalide (LMD), and monomethyl auristatin E (MMAE), which attach to different binding sites in a tubulin, are loaded onto TNTs and cause structural changes in them, including shape anisotropy and tubulin layering. This drug-driven carrier transformation leads to changes in the drug-loading efficiency and stability characteristics of the carrier. TNTs coloaded with DTX and LMD efficiently deliver dual drug cargoes to cellular tubulins by the endolysosomal pathway, and results in synergistic anticancer and antiangiogenic action of the drugs in vitro. In in vivo tests, TNTs loaded with a microtubule-destabilizing agent MMAE suppress the growth of tumors with much higher efficacy than free MMAE did. This work suggests a new concept of using a drug's target protein as a carrier. The findings demonstrate that the TNTs developed here can be used universally as a delivery platform for many MTAs.

Aggregation and Cellular Toxicity of Pathogenic or Non-pathogenic Proteins.

More than 20 unique diseases such as diabetes, Alzheimer's disease, Parkinson's disease are caused by the abnormal aggregations of pathogenic proteins such as amylin, ß-amyloid (Aß), and α-synuclein. All pathogenic proteins differ from each other in biological function, primary sequences, and morphologies; however, the proteins are toxic when aggregated. Here, we investigated the cellular toxicity of pathogenic or non-pathogenic protein aggregates. In this study, six proteins were selected and they were incubated at acid pH and high temperature. The aggregation kinetic and cellular toxicity of protein species with time were characterized. Three non-pathogenic proteins, bovine serum albumin (BSA), catalase, and pepsin at pH 2 and 65 °C were stable in protein structure and non-toxic at a lower concentration of 1 mg/mL. They formed aggregates at a higher concentration of 20 mg/mL with time and they induced the toxicity in short incubation time points, 10 min and 20 min only and they became non-toxic after 30 min. Other three pathogenic proteins, lysozyme, superoxide dismutase (SOD), and insulin, also produced the aggregates with time and they caused cytotoxicity at both 1 mg/mL and 20 mg/mL after 10 min. TEM images and DSC analysis demonstrated that fibrils or aggregates at 1 mg/mL induced cellular toxicity due to low thermal stability. In DSC data, fibrils or aggregates of pathogenic proteins had low thermal transition compared to fresh samples. The results provide useful information to understand the aggregation and cellular toxicity of pathogenic and non-pathogenic proteins.

Minireview - Microtubules and Tubulin Oligomers: Shape Transitions and Assembly by Intrinsically Disordered Protein Tau and Cationic Biomolecules.

In this minireview, which is part of a special issue in honor of Jacob N. Israelachvili's remarkable research career on intermolecular forces and interfacial science, we present studies of structures, phase behavior, and forces in reaction mixtures of microtubules (MTs) and tubulin oligomers with either intrinsically disordered protein (IDP) Tau, cationic vesicles, or the polyamine spermine (4+). Bare MTs consist of 13 protofilaments (PFs), on average, where each PF is made of a linear stack of αß-tubulin dimers (i.e., tubulin oligomers). We begin with a series of experiments which demonstrate the flexibility of PFs toward shape changes in response to local environmental cues. First, studies show that MT-associated protein (MAP) Tau controls the diameter of microtubules upon binding to the outer surface, implying a shape change in the cross-sectional area of PFs forming the MT perimeter. The diameter of a MT may also be controlled by the charge density of a lipid bilayer membrane that coats the outer surface. We further describe an experimental study where it is unexpectedly found that the biologically relevant polyamine spermine (+4e) is able to depolymerize taxol-stabilized microtubules with efficiency that increases with decreasing temperature. This MT destabilization drives a dynamical structural transition where inside-out curving of PFs, during the depolymerization peeling process, is followed by reassembly of ring-like curved PF building blocks into an array of helical inverted tubulin tubules. We finally turn to a very recent study on pressure-distance measurements in bundles of MTs employing the small-angle X-ray scattering (SAXS)-osmotic pressure technique, which complements the surface-forces-apparatus technique developed by Jacob N. Israelachvili. These latter studies are among the very few which are beginning to shed light on the precise nature of the interactions between MTs mediated by MAP Tau in 37 °C reaction mixtures containing GTP and lacking taxol.

Inhibition of Human Amylin Aggregation and Cellular Toxicity by Lipoic Acid and Ascorbic Acid.

More than 30 human degenerative diseases result from protein aggregation such as Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM). Islet amyloid deposits, a hallmark in T2DM, are found in pancreatic islets of more than 90% of T2DM patients. An association between amylin aggregation and reduction in ß-cell mass was also established by post-mortem studies. A strategy in preventing protein aggregation-related disorders is to inhibit the protein aggregation and associated toxicity. In this study, we demonstrated that two inhibitors, lipoic acid and ascorbic acid, significantly inhibited amylin aggregation. Compared to amylin (15 µM) as 100%, lipoic acid and ascorbic acid reduced amylin fibril formation to 42.1 ± 17.2% and 42.9 ± 12.8%, respectively, which is confirmed by fluorescence and TEM images. In cell viability tests, both inhibitors protected RIN-m5f ß-cells from the toxicity of amylin aggregates. At 10:1 molar ratio of lipoic acid to amylin, lipoic acid with amylin increased the cell viability to 70.3%, whereas only 42.8% RIN-m5f ß-cells survived in amylin aggregates. For ascorbic acid, an equimolar ratio achieved the highest cell viability of 63.3% as compared to 42.8% with amylin aggregates only. Docking results showed that lipoic acid and ascorbic acid physically interact with amylin amyloidogenic region (residues Ser20-Ser29) via hydrophobic interactions; hence reducing aggregation levels. Therefore, lipoic acid and ascorbic acid prevented amylin aggregation via hydrophobic interactions, which resulted in the prevention of cell toxicity in vitro.

Comparison between 102k and 20k Poly(ethylene oxide) Depletants in Osmotic Pressure Measurements of Interfilament Forces in Cytoskeletal Systems.

The proliferation of successful, cell-free reconstitutions of cytoskeletal networks have prompted measurements of forces between network elements via induced osmotic pressure by the addition of depletants. Indeed, it was through osmotic pressurization that Tau, an intrinsically disordered protein found in neuronal axons, was recently discovered to mediate two distinct microtubule (MT) bundle states, one widely spaced and a second tightly packed, separated by an energy barrier due to polyelectrolyte repulsions between opposing Tau projection domains on neighboring MT surfaces. Here, we compare interfilament force measurements in Tau coated MT bundles using PEO20k (poly(ethylene oxide), Mw = 20000), a commonly used inert depletant, and recently published measurements with PEO102k. While force measurements with either depletant reveals the transition between the two bundled states, measurements with PEO20k cannot recapitulate the correct critical pressure (Pc) at which widely spaced MT bundles transition to tightly packed MT bundles due to depletant penetration into widely spaced bundles below Pc. Surprisingly, upon transitioning to the tightly packed bundle state data from both depletants are in quantitative agreement indicative of expulsion of the smaller PEO20k depletant, but only at distances comparable or less than the PEO20k radius of gyration, significantly smaller than the effective diameter of PEO20k. While PEO102k (with size larger than the wall-to-wall distance between MTs in bundles) can more accurately capture the force response behavior at low to intermediate pressures (<104 Pa), measurements with PEO20k, beyond the overlap regime with PEO102k, extend the achievable osmotic pressure range into the higher-pressure regime (∼5 × 104 Pa). The data underscores the importance of the use of polymeric depletants of different sizes to elucidate force response behavior of cytoskeletal filamentous networks over a more complete extended pressure range.

Synchrotron small-angle X-ray scattering and electron microscopy characterization of structures and forces in microtubule/Tau mixtures.

Tau, a neuronal protein known to bind to microtubules and thereby regulate microtubule dynamic instability, has been shown recently to not only undergo conformational transitions on the microtubule surface as a function of increasing microtubule coverage density (i.e., with increasing molar ratio of Tau to tubulin dimers) but also to mediate higher-order microtubule architectures, mimicking fascicles of microtubules found in the axon initial segment. These discoveries would not have been possible without fine structure characterization of microtubules, with and without applied osmotic pressure through the use of depletants. Herein, we discuss the two primary techniques used to elucidate the structure, phase behavior, and interactions in microtubule/Tau mixtures: transmission electron microscopy and synchrotron small-angle X-ray scattering. While the former is able to provide striking qualitative images of bundle morphologies and vacancies, the latter provides angstrom-level resolution of bundle structures and allows measurements in the presence of in situ probes, such as osmotic depletants. The presented structural characterization methods have been applied both to equilibrium mixtures, where paclitaxel is used to stabilize microtubules, and also to dissipative nonequilibrium mixtures at 37°C in the presence of GTP and lacking paclitaxel.

Holographic deep learning for rapid optical screening of anthrax spores.

Establishing early warning systems for anthrax attacks is crucial in biodefense. Despite numerous studies for decades, the limited sensitivity of conventional biochemical methods essentially requires preprocessing steps and thus has limitations to be used in realistic settings of biological warfare. We present an optical method for rapid and label-free screening of Bacillus anthracis spores through the synergistic application of holographic microscopy and deep learning. A deep convolutional neural network is designed to classify holographic images of unlabeled living cells. After training, the network outperforms previous techniques in all accuracy measures, achieving single-spore sensitivity and subgenus specificity. The unique "representation learning" capability of deep learning enables direct training from raw images instead of manually extracted features. The method automatically recognizes key biological traits encoded in the images and exploits them as fingerprints. This remarkable learning ability makes the proposed method readily applicable to classifying various single cells in addition to B. anthracis, as demonstrated for the diagnosis of Listeria monocytogenes, without any modification. We believe that our strategy will make holographic microscopy more accessible to medical doctors and biomedical scientists for easy, rapid, and accurate point-of-care diagnosis of pathogens.

Paclitaxel suppresses Tau-mediated microtubule bundling in a concentration-dependent manner.

BACKGROUND: Microtubules (MTs) are protein nanotubes comprised of straight protofilaments (PFs), head to tail assemblies of αß-tubulin heterodimers. Previously, it was shown that Tau, a microtubule-associated protein (MAP) localized to neuronal axons, regulates the average number of PFs in microtubules with increasing inner radius observed for increasing Tau/tubulin-dimer molar ratio ΦTau at paclitaxel/tubulin-dimer molar ratio ΛPtxl=1/1. METHODS: We report a synchrotron SAXS and TEM study of the phase behavior of microtubules as a function of varying concentrations of paclitaxel (1/32≤ΛPtxl≤1/4) and Tau (human isoform 3RS, 0≤Φ3RS≤1/2) at room temperature. RESULTS: Tau and paclitaxel have opposing regulatory effects on microtubule bundling architectures and microtubule diameter. Surprisingly and in contrast to previous results at ΛPtxl=1/1 where microtubule bundles are absent, in the lower paclitaxel concentration regime (ΛPtxl≤1/4), we observe both microtubule doublets and triplets with increasing Tau. Furthermore, increasing paclitaxel concentration (up to ΛPtxl=1/1) slightly decreased the average microtubule diameter (by ~1 PF) while increasing Tau concentration (up to Φ3RS=1/2) significantly increased the diameter (by ~2-3 PFs). CONCLUSIONS: The suppression of Tau-mediated microtubule bundling with increasing paclitaxel is consistent with paclitaxel seeding more, but shorter, microtubules by rapidly exhausting tubulin available for polymerization. Microtubule bundles require the aggregate Tau-Tau attractions along the microtubule length to overcome individual microtubule thermal energies disrupting bundles. GENERAL SIGNIFICANCE: Investigating MAP-mediated interactions between microtubules (as it relates to in vivo behavior) requires the elimination or minimization of paclitaxel.

Enhanced stability of freestanding lipid bilayer and its stability criteria.

We present a new strategy to dramatically enhance the stability of freestanding lipid bilayers. We found that an addition of a water in oil emulsion stabilizer, SPAN 80 to a solvent phase guarantees nearly millimeter-scale stable freestanding lipid bilayers. The water permeability, bilayer area, contact angle, and interfacial tension were measured as a function of time and SPAN 80-to-lipid weight ratio (ΦSPAN 80) with several different solvents. Surprisingly, the SPAN 80, instead of remaining in the bilayer, was moved out of the bilayer during the bilayer formation. Also we studied the effect of solvent on freestanding bilayer formation, and found that squalene was the only solvent that was not incorporated into the bilayer. The regime of stable bilayer formation was experimentally determined to be 3/1 < ΦSPAN 80 < 15/1, and we suggest general stability criteria for bilayer formation. This technique and the suggested stability criteria can be potentially helpful to many model membrane-based researches in life sciences, physical sciences and biomedical engineering fields.

Tau mediates microtubule bundle architectures mimicking fascicles of microtubules found in the axon initial segment.

Tau, an intrinsically disordered protein confined to neuronal axons, binds to and regulates microtubule dynamics. Although there have been observations of string-like microtubule fascicles in the axon initial segment (AIS) and hexagonal bundles in neurite-like processes in non-neuronal cells overexpressing Tau, cell-free reconstitutions have not replicated either geometry. Here we map out the energy landscape of Tau-mediated, GTP-dependent 'active' microtubule bundles at 37 °C, as revealed by synchrotron SAXS and TEM. Widely spaced bundles (wall-to-wall distance Dw-w≈25-41 nm) with hexagonal and string-like symmetry are observed, the latter mimicking bundles found in the AIS. A second energy minimum (Dw-w≈16-23 nm) is revealed under osmotic pressure. The wide spacing results from a balance between repulsive forces, due to Tau's projection domain (PD), and a stabilizing sum of transient sub-kBT cationic/anionic charge-charge attractions mediated by weakly penetrating opposing PDs. This landscape would be significantly affected by charge-altering modifications of Tau associated with neurodegeneration.

The effect of multivalent cations and Tau on paclitaxel-stabilized microtubule assembly, disassembly, and structure.

In this review we describe recent studies directed at understanding the formation of novel nanoscale assemblies in biological materials systems. In particular, we focus on the effects of multivalent cations, and separately, of microtubule-associated protein (MAP) Tau, on microtubule (MT) ordering (bundling), MT disassembly, and MT structure. Counter-ion directed bundling of paclitaxel-stabilized MTs is a model electrostatic system, which parallels efforts to understand MT bundling by intrinsically disordered proteins (typically biological polyampholytes) expressed in neurons. We describe studies, which reveal an unexpected transition from tightly spaced MT bundles to loose bundles consisting of strings of MTs as the valence of the cationic counter-ion decreases from Z=3 to Z=2. This transition is not predicted by any current theories of polyelectrolytes. Notably, studies of a larger series of divalent counter-ions reveal strong ion specific effects. Divalent counter-ions may either bundle or depolymerize paclitaxel-stabilized MTs. The ion concentration required for depolymerization decreases with increasing atomic number. In a more biologically related system we review synchrotron small angle x-ray scattering (SAXS) studies on the effect of the Tau on the structure of paclitaxel-stabilized MTs. The electrostatic binding of MAP Tau isoforms leads to an increase in the average radius of microtubules with increasing Tau coverage (i.e. a re-distribution of protofilament numbers in MTs). Finally, inspired by MTs as model nanotubes, we briefly describe other more robust lipid-based cylindrical nanostructures, which may have technological applications, for example, in drug encapsulation and delivery.

Direct force measurements reveal that protein Tau confers short-range attractions and isoform-dependent steric stabilization to microtubules.

Microtubules (MTs) are hollow cytoskeletal filaments assembled from αß-tubulin heterodimers. Tau, an unstructured protein found in neuronal axons, binds to MTs and regulates their dynamics. Aberrant Tau behavior is associated with neurodegenerative dementias, including Alzheimer's. Here, we report on a direct force measurement between paclitaxel-stabilized MTs coated with distinct Tau isoforms by synchrotron small-angle X-ray scattering (SAXS) of MT-Tau mixtures under osmotic pressure (P). In going from bare MTs to MTs with Tau coverage near the physiological submonolayer regime (Tau/tubulin-dimer molar ratio; ΦTau = 1/10), isoforms with longer N-terminal tails (NTTs) sterically stabilized MTs, preventing bundling up to PB ∼ 10,000-20,000 Pa, an order of magnitude larger than bare MTs. Tau with short NTTs showed little additional effect in suppressing the bundling pressure (PB ∼ 1,000-2,000 Pa) over the same range. Remarkably, the abrupt increase in PB observed for longer isoforms suggests a mushroom to brush transition occurring at 1/13 < ΦTau < 1/10, which corresponds to MT-bound Tau with NTTs that are considerably more extended than SAXS data for Tau in solution indicate. Modeling of Tau-mediated MT-MT interactions supports the hypothesis that longer NTTs transition to a polyelectrolyte brush at higher coverages. Higher pressures resulted in isoform-independent irreversible bundling because the polyampholytic nature of Tau leads to short-range attractions. These findings suggest an isoform-dependent biological role for regulation by Tau, with longer isoforms conferring MT steric stabilization against aggregation either with other biomacromolecules or into tight bundles, preventing loss of function in the crowded axon environment.

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer.

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after merging (FRAM). FRAM adopts the same principles as the fluorescence recovery after photobleaching (FRAP) technique, especially for measuring fluorescence recovery after bleaching a specific area, but FRAM uses a drop coalescence instead of photobleaching dye molecules to induce a chemical potential gradient of dye molecules. Our technique has several advantages over FRAP: it only requires a fluorescence microscope rather than a confocal microscope equipped with high power lasers; it is essentially free from the selection of fluorescence dyes; and it has far more freedom to define the measured diffusion area. Furthermore, FRAM potentially provides a route for studying the mixing or inter-diffusion of two different surfactants, when the monolayers at a surface of droplet and at a flat air/water interface are prepared with different species, independently.