Neutron Reflectivity Measurements Research Articles

Membrane-Associated Conformation of HIV-1 Nef Investigated with Hydrogen Exchange Mass Spectrometry at a Langmuir Monolayer.

In the companion paper to this work, we described development of a new type of hydrogen exchange (HX) mass spectrometry (MS) measurement that integrates Langmuir monolayers. With Langmuir monolayers, the lipid packing density can be reproducibly controlled and changed as desired. Analysis of HX in proteins that may undergo conformational changes as a function of lipid packing (for example, conformational rearrangements after insertion into a lipid layer) are then possible. We previously used neutron reflection to characterize just such a conformational change in the myristoylated HIV-1 Nef protein (myrNef): at high lipid packing density, myrNef could not insert into the lipids and maintained a compact conformation adjacent to the monolayer, whereas at lower lipid packing density, myrNef was able to insert N-terminal arm residues, causing displacement of the core domain away from the monolayer. In order to locate where conformation may have been altered by lipid association, we applied the HX MS Langmuir monolayer method to myrNef associated with monolayers of packing densities identical to those used for the prior neutron reflection measurements. The results show that the N-terminal region and the C-terminal unstructured loop undergo conformational changes when associated with a low density lipid monolayer. The results are not consistent with the hypothesis of myrNef dimerization upon membrane association in the absence of other myrNef binding partners. The HX MS Langmuir monolayer method provides new and meaningful information for myrNef that helps explain necessary conformational changes required for function at the membrane.

Dynamics of silver photo-diffusion into Ge-chalcogenide films: time-resolved neutron reflectometry

Silver diffuses into an amorphous (a-) chalcogenide layer while visible light illuminates Ag/a-chalcogenide films and neutron reflectometry is a suitable technique probing time evolution of the depth profiles without damaging the sample by the probe beam itself. In this paper, we report the results of time-resolved neutron reflectivity measurements of a-Ge40Se60/Ag/ Si films taken while the films are exposed to visible light. From the measurements, we found enormous changes in the neutron reflectivity profile, including a loss of total reflection region, with continuous illumination even after forming one homogeneous layer, which occurred about 50 min after starting illumination. At this stage, a clear off-specular scattering was observed by a linear detector and a surface roughness was observed with naked eyes.

Gas expanded polymer process to anneal nanoparticle dispersion in thin films

Organic photovoltaic (OPV) active layers made by spin-coating a solution comprising poly(3-hexylthiophene) (P3HT) and phenyl-C61-butyric acid methyl ester (PCBM) nanoparticles have been shown to adopt a non-uniform concentration profile across the thin film dimension. This inhomogeneous distribution can reduce the efficiency of the device. In the present work a new process, gas expanded polymer (GXP) annealing, is applied to P3HT/PCBM thin film blends, enabling the distribution of the PCBM nanoparticles to be manipulated by varying the GXP processing conditions. Films of 50nm thickness (nominally) created by spin casting a blend of P3HT mixed with PCBM were annealed by oscillatory GXP and GXP at constant pressure using high pressure CO2. An increase in P3HT crystallinity (detected by X-ray diffraction and UV–vis spectroscopy) along with a more uniform distribution of PCBM nanoparticles in the thickness dimension, as interpreted from neutron reflectivity measurements, were observed after oscillatory GXP annealing. In addition, static water contact angles suggest that the film/air interface is enriched in PCBM relative to the as-cast film. These results demonstrate that GXP annealing, which is commercially scalable, can be successfully used to create a uniform distribution of PCBM nanoparticles across the thickness dimension in a P3HT thin film.

Poly-acrylic Acid Brushes and Adsorbed Proteins

Abstract Planar polyelectrolyte brushes are prepared by Langmuir–Schaefer based grafting of perdeuterated (styrene)49-b-(acrylic acid)222 block copolymer (dPS-b-PAA) to dPS pre-coated silicon supports with grafting density σ PAA from 0.07 to 0.11 nm –2. The structure of the solvent-swollen brushes, i. e. the volume fraction profile of polymer segments, ϕ PAA, as a function of altitude z from the grafting plane into the liquid phase is extracted from neutron reflectivity measurements. We find that for all cases investigated ϕ PAA(z) resembles a Gaussian profile. Although very condensed, the PAA brushes can be loaded with bovine serum albumin (BSA). The integral amount of adsorbed BSA scales linearly with grafting density. We compare our z-resolved volume fraction profile ϕ BSA(z) of BSA on PAA brushes with existing literature on that system. It is found that a cross-over takes place in the adsorption scheme from ternary compressive, where proteins can approach the grafting surface only by compressing the brush, to ternary insertive, where proteins enter the brush with only local perturbation of the concentration profile, as a function of R P/H max, where R P is the Stokes-Radius of the protein, and H max is the experimentally determined maximum height of the brush.

Density Profiles of Well‐Defined Poly(methyl 2‐propenyl ether) (PMPE) Near Water Interface

SummaryPoly(methyl 2‐propenyl ether) (PMPE) is structually different in terms of the presence of an α‐methyl group on the main chain from poly(vinyl methyl ether) (PVME), the phase behaviors of which have been widely characterized with polystyrene (PS) and water. Due to the incorporation of the α‐methyl group, PMPE is immiscible with both PS and water. These results encouraged us to investigate a PMPE/water interface more closely. Thus, the density profiles of PMPE in the vicinity of the water interface were probed by neutron reflectivity (NR) measurement. Even in the presence of the α‐methyl group on the main chain, PMPE slightly swelled near the water interface. The obtained results will give us an insight into a better design and construction of the polymers for several bioapplications.

Neutron reflection study of the adsorption of the phosphate surfactant NaDEHP onto alumina from water.

The adsorption of a phosphorus analogue of the surfactant AOT, sodium bis(2-ethylhexyl) phosphate (NaDEHP), at the water/alumina interface is described. The material is found to adsorb as an essentially water-free bilayer from neutron reflection measurements. This is similar to the behavior of AOT under comparable conditions, although AOT forms a thicker, more hydrated layer. The NaDEHP shows rather little variation with added salt, but a small thickening of the layer on increasing the pH, in contrast to the behavior of AOT.

Towards neutron scattering experiments with sub-millisecond time resolution

Neutron scattering techniques offer several unique opportunities in materials research. However, most neutron scattering experiments suffer from the limited flux available at current facilities. This limitation becomes even more severe if time-resolved or kinetic experiments are performed. A new method has been developed which overcomes these limitations when a reversible process is studied, without any compromise on resolution or beam intensity. It is demonstrated that, by recording in absolute time the neutron detector events linked to an excitation, information can be resolved on sub-millisecond timescales. Specifically, the concept of the method is demonstrated by neutron reflectivity measurements in time-of-flight mode at the Liquids Reflectometer located at the Spallation Neutron Source, Oak Ridge National Laboratory, Tennessee, USA, combined within siturheometry. The opportunities and limitations of this new technique are evaluated by investigations of a micellar polymer solution offering excellent scattering contrast combined with high sensitivity to shear.

Activity of Antimicrobial Peptide Protegrin-1 is Tuned by Membrane Cholesterol Content

The ability of antimicrobial peptides (AMPs) to target and lyse the harmful microbial membrane over that of a host's is a unique characteristic, making these innate immune effectors promising candidates to fill a growing therapeutic void resulting from antibiotic drug resistance. This selectivity is believed to depend on the chemical and structural properties of the lipids that comprise the cell membrane. The selectivity of AMPs can be based on the electrostatic attraction of these predominately cationic peptides for the bacterial membrane surface heavily populated with negatively charged lipid components. We have previously shown with atomic force microscopy that zwitterionic dimyristoylphosphatidylcholine (DMPC) bilayers display concentration-dependent structural transformations induced by protegrin-1 (PG-1) that progress from finger-like instabilities at bilayer edges, to the formation of pores, and finally to a network of worm-like micelles. The increasing degree of membrane disruption in charge-neutral membranes demonstrates that a more complex interaction than that suggested by a simple electrostatic argument is needed to explain AMP selectivity. We propose that in addition to an electrostatic element, specific membrane compositional differences between host and pathogen tunes AMP activity to selectively disrupt microbial membranes. We have tailored our investigations to utilize membrane components which eukaryotes and prokaryotes contain in drastically different proportions, specifically the presence and absence of cholesterol. In these results we have employed a variety of biophysical techniques to elucidate how increasing cholesterol content in both phospholipid monolayers and bilayers attenuates the ability of PG-1 to induce membrane disruption. Atomic force microscopy and isothermal titration calorimetry were used to assess the propensity for peptide insertion and pore formation. X-ray and neutron reflectivity measurements were advantageous in providing molecular level detail on the location and orientation of PG-1 with respect to the membrane.

Structure of a liquid/liquid interface during solvent extraction combining X-ray and neutron reflectivity measurements.

We have resolved the molecular structure of a bulk oil/water interface that contains amphiphilic ligand molecules using a combination of X-ray and neutron reflectivity measurements for the first time. This new capability can greatly impact future work in the field of ion separation by phase transfer, i.e. liquid/liquid extraction.

Bimodal crystallization at polymer-fullerene interfaces.

The growth-kinetics of [6,6]-phenyl C61-butyric acid methyl ester (PCBM) crystals, on two different length-scales, is shown to be controlled by the thickness of the polymer layer within a PCBM-polymer bilayer. Using a model amorphous polymer we present evidence, from in situ optical microscopy and grazing-incidence X-ray diffraction (GIXD), that an increased growth-rate of nanoscale crystals impedes the growth of micron-sized, needle-like PCBM crystals. A combination of neutron reflectivity and GIXD measurements, also allows us to observe the establishment of a liquid-liquid equilibrium composition-profile between the PCBM layer and a polymer-rich layer, before crystallization occurs. While the interfacial composition-profile is independent of polymer-film-thickness, the growth-rate of nanoscale PCBM crystals is significantly larger for thinner polymer films. A similar thickness-dependent behavior is observed for different molecular weights of entangled polymer. We suggest that the behavior may be related to enhanced local-polymer-chain-mobility in nanocomposite thin-films.

Effect of Lipid Composition on the Affinity and Binding of Dimeric Tubulin to Membranes Studied using Surface Plasmon Resonance, Neutron Reflectivity, Electrophysiology, and AC Electrical Methods

Dimeric tubulin has emerged as an important regulatory factor of the permeability of voltage-dependent anion channel (VDAC) in the mitochondrial outer membrane, with implications for mitochondrial energetics as well as the Warburg effect observed in cancers. Previously, single-channel studies revealed that the on-rate of the VDAC-tubulin interaction is strongly dependent on the lipid environment. To understand the nature of the binding of this abundant cytosolic water-soluble protein to lipid membranes, we have employed an array of biophysical techniques using unsupported planar lipid membrane and tethered bilayer lipid membrane (tBLM) platforms. Surface plasmon resonance (SPR) of tBLMs shows that tubulin at concentrations less than 100 nM binds irreversibly to DPhPC and 1:1 DOPC:DOPE membranes. The binding rate is significantly larger in high salt concentrations, suggesting a hydrophobic interaction between tubulin and lipid membrane. No binding to DOPC membranes is observed under the same conditions. Electrochemical impedance spectroscopy (EIS) of tBLMs reveals that only DPhPC membranes are strongly perturbed by the binding of tubulin. Neutron reflectivity (NR) measurements on tBLMs give structural information regarding the penetration of the tubulin into the membranes. Electrophysiological measurements of the tubulin/VDAC interaction on the unsupported bilayers confirm the irreversibility of tubulin binding to DPhPC membranes and membranes with significant DOPE content. Second harmonic analysis of planar lipid membranes’ response to ac excitation suggests that tubulin binding causes significant rearrangements of the lipid headgroups. We conclude that tubulin binds to and modifies the structure of lipid membranes even at nanomolar concentrations. Considering that there is up to 10 μM of free dimeric tubulin in cells, our results suggest a new broad regulatory role of dimeric tubulin in vivo.

環状及び線状高分子の界面物質拡散現象に関する研究

Diffusion of cyclic and linear polymers at interfaces was studied. Interdiffusion of cyclic polystyrene/cyclic deuterated polystyrene was measured by dynamic secondary ion mass spectroscopy as functions of temperature and molecular weight. Diffusion coefficient of cyclic polystyrene with weight-average molecular weight of 113k is twice as large as that of the corresponding linear one at all the temperatures employed. This result clearly shows that both the linear and cyclic polystyrenes have the same temperature dependence of segmental frictional coefficient. At an iso-free volume condition, diffusion coefficient of cyclic polystyrene is larger than that of the linear one at a given molecular weight. The short-time interdiffusion at an interface of linear polystyrene / linear deuterated polystyrene having different molecular weights was examined by time-resolved neutron reflectivity measurements. The model scattering length density profiles obtained by solving a partial differential equation for the diffusion process were used to analyze the data. The results indicate that even though the molecular weights of both components are larger than the critical molecular weight for entanglement, the initial interfacial broadening of bilayer films with different molecular weight proceeds with asymmetric mobility being inversely proportional to the molecular weight.

Diffusion of nitroaromatic vapours into fluorescent dendrimer films for explosives detection

Fluorescence-based sensing with organic semiconductors is a powerful method for the detection of a broad range of analytes including explosives, chemical weapons and drugs. Diffusion of an analyte into an organic semiconductor thin film, and its subsequent interaction with the chromophore are key factors that govern the sensing performance of a chemosensor. In this study the diffusion behaviour of an explosive analyte analogue into a sensing film of a conjugated dendrimer was investigated using a quartz crystal microbalance (QCM) and correlated with neutron reflectivity measurements. The mechanistic insights of para-nitrotoluene (pNT) sorption in the films of different thicknesses of a first generation dendrimer with fluorenyl surface groups, carbazole dendrons and a spirobifluorene core were studied and interpreted in terms of the underlying kinetics and thermodynamics. Sorption measurements suggest that the process of diffusion of pNT vapour into the dendrimer films is Super Case II, which involves swelling of the film. Swelling of the film was confirmed by neutron reflectometry, which also showed uniform distribution of the pNT molecules throughout the film. The activation energy barrier and change in Gibbs free energy in the sorption process were calculated from the QCM responses. The sorption process was found to be thermodynamically (not kinetically) controlled and independent of film thickness. This work sheds insight into the structure–property relationships that govern the performance of organic semiconductor fluorescence-based chemosensors.

Surface adsorption of oppositely charged C14TAB-PAMPS mixtures at the air/water interface and the impact on foam film stability.

We have studied the oppositely charged polyelectrolyte/surfactant mixture of poly(acrylamidomethylpropanesulfonate) sodium salt (PAMPS) and tetradecyl trimethylammonium bromide (C14TAB) using a combination of neutron reflectivity and ellipsometry measurements. The interfacial composition was determined using three different analysis methods involving the two techniques for the first time. The bulk surfactant concentration was fixed at a modest value while the bulk polyelectrolyte concentration was varied over a wide range. We reveal complex changes in the surface adsorption behavior. Mixtures with low bulk PAMPS concentrations result in the components interacting synergistically in charge neutral layers at the air/water interface. At the bulk composition where PAMPS and C14TAB are mixed in an equimolar charge ratio in the bulk, we observe a dramatic drop in the surfactant surface excess to leave a large excess of polyelectrolyte at the interface, which we infer to have loops in its interfacial structure. Further increase of the bulk PAMPS concentration leads to a more pronounced depletion of material from the surface. Mixtures containing a large excess of PAMPS in the bulk showed enhanced adsorption, which is attributed to the large increase in total ionic strength of the system and screening of the surfactant headgroup charges. The data are compared to our former results on PAMPS/C14TAB mixtures [Kristen et al. J. Phys. Chem. B, 2009, 23, 7986]. A peak in the surface tension is rationalized in terms of the changing surface adsorption and, unlike in more concentrated systems, is unrelated to bulk precipitation. Also, a comparison between the determined interfacial composition with zeta potential and foam film stability data shows that the highest film stability occurs when there is enhanced synergistic adsorption of both components at the interface due to charge screening when the total ionic strength of the system is highest. The additional contribution to the foam stability of the negatively charged polyelectrolyte within the film bulk is also discussed.

Neutron reflectivity study of the swollen structure of polyzwitterion and polyeletrolyte brushes in aqueous solution

The swollen brush structures of polycation and zwitterionic polymer brushes, such as poly(2-methacryloyloxyethyltrimethylammonium chloride) (PMTAC), poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), and poly[3-(N-2-methacryloyloxyethyl-N,N-dimethyl)ammonatopropanesulfonate] (PMAPS), in aqueous solutions of various ionic strengths were characterized by neutron reflectivity (NR) measurements. A series of the polyelectrolyte brushes were prepared by surface-initiated controlled radical polymerization on silicon substrates. A high-graft-density PMTAC brush in salt-free water (D2O) adopted a two-region step-like structure consisting of a shrunk region near the Si substrate surface and a diffuse brush region with a relatively stretched chain structure at the solution interface. The diffuse region of PMTAC was reduced with increase in salt (NaCl) concentration. The PMAPS brush in D2O formed a collapsed structure due to the strong molecular interaction between betaine groups, while significant increase in the swollen thickness was observed in salt aqueous solution. In contrast, no change was observed in the depth profile of the swollen PMPC brush in D2O with various salt concentrations. The unique solution behaviors of zwitterionic polymer brushes were described.

Determining the structure of interfacial peptide films: comparing neutron reflectometry and molecular dynamics simulations.

The peptides AM1 and Lac21E self-organize into switchable films at an air-water interface. In an earlier study, it was proposed that both AM1 and Lac21E formed monolayers of α-helical peptides based on consistency with neutron reflectivity data. In this article, molecular dynamics simulations of assemblies of helical and nonhelical AM1 and Lac21E at an air-water interface suggest some tendency for the peptides to spontaneously adopt an α-helical conformation. However, irrespective of the structure of the peptides, the simulations reproduced not only the structural properties of the films (thickness and distribution of the hydrophobic and hydrophilic amino acids) but also the experimental neutron reflectivity measurements at different contrast variations. This suggests that neutron reflectometry alone cannot be used to determine the structure of the peptides in this case. However, together with molecular dynamics simulations, it is possible to obtain a detailed understanding of peptide films at an atomic level.

Impact of the degree of ethoxylation of the ethoxylated polysorbate nonionic surfactant on the surface self-assembly of hydrophobin-ethoxylated polysorbate surfactant mixtures.

Neutron reflectivity measurements have been used to study the surface adsorption of the polyethylene sorbitan monostearate surfactant, with degrees of ethoxylation varying from 3 to 20 ethylene oxide groups, with the globular protein hydrophobin. The surface interaction between the ethoxylated polysorbate nonionic surfactants and the hydrophobin results in self-assembly at the air-solution interface in the form of a well-defined layered surface structure. The surface interaction arises from a combination of the hydrophobic interaction between the surfactant alkyl chain and the hydrophobic patch on the surface of the hydrophobin, and the hydrophilic interaction between the ethoxylated sorbitan headgroup and the hydrophilic regions on the surface of the hydrophobin. The results presented show that varying the degree of ethoxylation of the polysorbate surfactant changes the interaction between the surfactant and the hydrophobin and the packing, and hence the evolution in the resulting surface structure. The optimal degree of ethoxylation for multilayer formation is over a broad range, from of order 6 to 17 ethylene oxide groups, and for degrees of ethoxylation of 3 and 20 only monolayer adsorption of either the surfactant or the hydrophobin is observed.

In-Situ Neutron Reflectometry on Polymer Thin Films in Different Environments

Neutron reflectometry is indispensable for the studies on material interfaces and thin films, since it can probe the interfaces non-destructively with a quite high depth resolution. Here, the interfacial structures of polymer thin films in different environments: hot and wet, were examined by means of in-situ neutron reflectometry. The neutron reflectivity measurements were performed on a time-of-flight reflectometer SOFIA [1, 2] at the Japan Proton Accelerator Research Complex (J-PARC). The depth distribution of components was investigated for binary blend thin films of polystyrenes with different molecular weights to clarify the mechanism of de-wetting suppression effect by blending a small amount of the longer homologue. It was found by static measurements that the longer chains deplete from the air surface of the film, while they slightly localize at the interface with the substrate. The time evolution of the component distribution was also examined by in-situ measurements above the glass transition temperature in a vacuum. The structural change of lamellarly-ordered styrene-2-vinylpyridine (PS-P2VP) diblock copolymer thin film in contact with solvent was explored by in-situ neutron reflectometry with a conventional solid/liquid cell. During the block copolymer thin film was in contact with methanol, which is selective for P2VP, some of methanol penetrated into the film causing the structural change. However, the original structure was almost recovered after drying, though annealing effect was slightly observed. When toluene was made contact, which is selective for PS, the block copolymer thin film was immediately dissolved into, and never recovered.

SLDMOL: A tool for the structural characterization of thermally disordered membrane proteins

SLDMOL is a program for modeling the 1-D scattering length density (SLD) profile of proteins at the lipid membrane–solution interface or adsorbed to other surfaces. The program reads experimental SLD data from neutron or X-ray reflectivity measurements and compares the results to a trajectory of protein structures, finding the conformation and orientation that best fits the experimental data. SLDMOL is a freely distributed open source program written in python that can be run independently using command lines or a GUI. SLDMOL has also been integrated into the larger SASSIE package extending molecular modeling capabilities. Sample environment conditions can be replicated including H2O/D2O solvent contrasts, specific amino acid deuteration and complex molecular assemblies. Ensembles of protein conformations can be generated independently (e.g. molecular dynamics simulations) or with SASSIE. For each individual structure a best-fit SLD profile is outputted along with a goodness of fit parameter, protein depth penetration and surface coverage. In addition to individual comparisons SLD profiles can be calculated over ensemble averages of protein structures. As a result, SLDMOL provides a detailed molecular interpretation of reflectivity data or conversely can be used to predict experimental outcomes for different protein conformation and specific deuteration schemes prior to measurements. Program summaryProgram title: SLDMOLCatalogue identifier: AETX_v1_0Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AETX_v1_0.htmlProgram obtainable from: CPC Program Library, Queen’s University, Belfast, N. IrelandLicensing provisions: GNU General Public License, version 3No. of lines in distributed program, including test data, etc.: 3302107No. of bytes in distributed program, including test data, etc.: 125715760Distribution format: tar.gzProgramming language: Python.Computer: PC/Mac.Operating system: 32- and 64-bit Linux (Ubuntu 10.04, Centos 5.6) and Mac OS X (10.7–10.9).RAM: 1 GBClassification: 3.External routines: Python 2.6.5, numpy 1.4.0, scipy 0.8.0, Tcl 8.5, Tk 8.5, Mac installation requires Xcode 3 development tools.Subprograms used:CatIdTitleReferenceAEKL_v1_0SASSIECPC 183(2012)382Nature of problem:Reflectivity is a powerful technique to study the conformation of biological molecules at surface and interfaces. Open source software that facilitates computational modeling and interpretation of experimental results in terms of detailed molecular structure is currently lacking.Solution method:SLDMOL takes one or an ensemble of atomistic configurations of proteins and compares them to 1-D scattering length density (SLD) profiles determined from reflectivity experiments. Protein structures can be generated independently (e.g. molecular dynamics simulations) or through the SASSIE software package. SLDMOL performs an optimization to determine which structure best fits the SLD profile. Structures can also be averaged either directly or through a Monte-Carlo weighting algorithm. Finally SLDMOL allows the modeling of reflectivity experiments through the in silico selective deuteration of amino acids and the calculation of SLD profiles in a number of different aqueous solvent contrasts.Additional comments:!!!!Due to the large file size, SLDMOL is not delivered directly when download or Email is requested. Instead an html file giving details of how the program can be obtained is sent.!!!!Running time:Varies depending on application. Typically 10 min to 24 h depending on the number of structures to be evaluated.

Probing opto-mechanical stresses within azobenzene-containing photosensitive polymer films by a thin metal film placed above.

Azo-modified photosensitive polymers offer the interesting possibility to reshape bulk polymers and thin films by UV-irradiation while being in the solid glassy state. The polymer undergoes considerable mass transport under irradiation with a light interference pattern resulting in the formation of surface relief grating (SRG). The forces inscribing this SRG pattern into a thin film are hard to assess experimentally directly. In the current study, we are proposing a method to probe opto-mechanical stresses within polymer films by characterizing the mechanical response of thin metal films (10 nm) deposited on the photosensitive polymer. During irradiation, the metal film not only deforms along with the SRG formation but ruptures in a regular and complex manner. The morphology of the cracks differs strongly depending on the electrical field distribution in the interference pattern, even when the magnitude and the kinetics of the strain are kept constant. This implies a complex local distribution of the opto-mechanical stress along the topography grating. In addition, the neutron reflectivity measurements of the metal/polymer interface indicate the penetration of a metal layer within the polymer, resulting in a formation of a bonding layer that confirms the transduction of light-induced stresses in the polymer layer to a metal film.