Efforts in the research of tensegrity structures applied to mobile robots have recently been focused on a purely tensegrity solution to all design requirements. Locomotion systems based on tensegrity structures are currently slow and complex to control. Although wheeled locomotion provides better efficiency over distances there is no literature available on the value of wheeled methods with respect to tensegrity designs, nor on how to transition from a tensegrity structure to a fixed structure in mobile robotics. This paper is the first part of a larger study that aims to combine the flexibility, light weight, and strength of a tensegrity structure with the efficiency and simple control of a wheeled locomotion system. It focuses on comparing different types of tensegrity structure for applicability to a mobile robot, and experimentally finding an appropriate transitional region from a tensegrity structure to a conventional fixed structure on mobile robots. It applies this transitional structure to what is, to the authors’ knowledge, the design of the world’s first wheeled tensegrity robot that has been designed with the goal of traversing air ducts. A tensegrity structure is comprised of elastic elements and rigid elements that interact so the structure extends over a space larger than the space of its individual parts. The sum of the forces between all its parts is zero; therefore, the structure is in equilibrium without the need of external forces. All elastic elements bear exclusively tensional force, but not all tensional elements are elastic. The most spread-out use of rigid and elastic elements is struts and cables, respectively. The “Needle Tower” Fig.  is made Tensegrity structures eventually expanded beyond art into engineering. Many studies have been done in statics, kinematics, dynamics, and biology. The main fields focused in tensegrity are civil engineering and robotics. The latter have seen more than one thousand peer reviewed articles in the last 10 years. Research efforts in robotics span in all sub-categories of study inside tensegrity: form-finding, kinematics, dynamics, control, and locomotion. Form-finding is the search for new topologies and morphologies of tensegrity structures. It also explores new methodologies to be used for the search itself. One of the sub-areas of exploration is biotensegrity. This is the realm of tensegrity structures inspired by biology.Most research papers focus on implementing tensegrity to address all design specification requirements. Tensegrity and biotensegrity structures are light weight, strong, flexible, compliant, and distribute the loads to every single element. Yet, when it comes to locomotive efficiency, these structures do not perform as well. A man with a bicycle is capable of travelling much faster and farther than a man running in spite of using the same source of power: the muscles in the legs. This work aims to join the advantages of tensegrity structures and wheeled locomotion. As a practical application of this new concept, it presents a biotensegrity structure that is able to climb through ducting by pressing wheels against the walls. The tensegrity structure is to provide the robot structural integrity as well as control of direction. The tensegrity structure, therefore, will need to provide enough flexibility to allow the robot to turn right, left, up, down, and in any other intermediate direction so the robot can change its path into other duct branches.This paper is the first part of the above-mentioned study into wheeled tensegrity robotics. It focuses on the transitional region from tensegrity to a standard fixed mobile frame for wheeled locomotion. To the authors’ knowledge, there is no formal research work done on this area. It firstly introduces criteria of evaluation for a transitional region. Secondly, it reviews some of the available regular configurations of tensegrity structures as a preliminary study. Thirdly, it proposes new conceptual transitional regions to be validated later in the paper. Fourthly, it shows the design and prototype of a novel wheeled tensegrity robot. Finally, it presents discussion and conclusions. As a representative problem that can be solved by a tensegrity-based wheeled robot, we have chosen the mechanical traversal and navigation of ducting networks. Ducting networks in buildings and industrial facilities can span and branch out in any direction in space. Most mobile robots are designed to travel only in a horizontal plane and have difficulty turning in arbitrary directions in three dimensions. Robotic duct climbers are only able to travel in one line and maybe follow the duct mainstream through wide bends. They currently do not have the ability to turn out of the duct into a branch. A robot able to transit through air ducts in houses and buildings will reduce time and number of personnel required as well as increasing flexibility and scope of work done. Due to the geometry and structural integrity of air ducts, flexibility and light weight are required key features. A hybrid wheeled tensegrity design would add speed and efficiency to the features of the robot. Basic work currently carried out in air ducts includes inspection and maintenance. However, in the near future, home automation, pioneered by companies like Apple, Google, and Amazon, will require the installation of cable terminals to power automated air vents. Being able to access areas through complex duct networks will continue to become more relevant.Tensegrity structures have advantages and benefits. A list by Skelton et al.  includesTensegrity is an ever-expanding field. Studies on tensegrity structures have been “widely carried out since 1970s, mainly about form-finding, static stability, and dynamic and control” . WilliamAfter the topology and morphology are defined, mathematical models can be created. A static analysis review was carried out by Hernàndez and Mirats . HoweverWhen efficiency is mentioned as one of the features of tensegrity robots, it refers to this potential energy stored as tension inside the structure itself. However, this efficiency does not relate to locomotive movement. All research papers studying locomotion by means of tensegrity do not measure efficiency in terms of distance covered over energy consumed. However, at least velocity is mentioned in the works of Friesen et al.  who statAlthough there is no mention of energy consumption. Paul et al.  introducDesigning a hybrid wheeled tensegrity robot, to the authors’ knowledge, has never been attempted before. Therefore, some criteria of evaluation need to be established beforehand in order to be able to interpret the results of this study.The first criteria developed were meant to classify tensional elements in two groups: elements that produce contraction of the structure and elements that produce Expansion.1 and m2 joined by cable A and attached to fixed points by cables B1 and B2 at each side of the masses. The tension in cable A is Ta, and the tension in cables B1 and B2 is Tb.Consider the system shown in Fig. 1 and B2 is increased by a magnitude F. If the cables are elastic and force F big enough to stretch them, force F would bring masses m1 and m2 closer together. Force F is replacing cable A in bringing the masses together. Thus, it can be said that cable A brings masses m1 and m2 together. Tension in cable A is a tension of Contraction.The first situation presented Fig.  is the s1 and B2 is reduced by a magnitude F and tension in cable A is increased by a magnitude F. If the cables are elastic and force F big enough to stretch them, F would take masses m1 and m2 apart from each other. Force F is replacing cables B1 and B2 in separating masses m1 and m2. Tension in cables B1 and B2 is a tension of Expansion.The second situation presented Fig.  is the s1 is in stable equilibrium in the horizontal direction due to a tension of contraction TA and a tension of expansion TB. Creating a stable equilibrium through tension is the central concept of tensegrity.In Fig. Tensegrity systems are made rigid by tension only, and therefore, the direction that tension is applied is central to their design. The system topology shown in Fig. at least three cables in tension attached for true three-dimensional stability.When designing a tensegrity system or a transitional region, this criterion has to be taken into consideration in order to have a stable system in all directions. In general, each structural element must be held by tensions that, considered together, have balanced projections on to all basis axes of the coordinate system. This implies a design principle that we will see is repeated throughout tensegrity structures: each structural node of the system must have An extensive study of different topologies and morphologies was carried out as part of the research work needed to design a hybrid wheeled tensegrity robot. This was an essential step in order to select the tensegrity structures that best fulfilled the design requirements. All tensegrity systems shown in this chapter were built by the author in order to gain a deep understanding of the capabilities, advantages, and disadvantages of each of them relative to a duct travelling robot.The area of tensegrity that studies the use of linear elements to form three-dimensional tensegrity structures is what the author calls Linear Tensegrity. This is where linear rigid elements, normally called struts or rods, and linear elastic elements, normally called strings or cables, are combined to form structures in two and three dimensions. Three-dimensional structures built with linear elements are the most common area of study in tensegrity. Planar tensegrity would include the use of two-dimensional elements; this was not covered in this exploration due to the short time available.The first half of this chapter is introductory to the mechanics of tensegrity systems. It is focused on the study of the triangular prism. “Almost all tensegrities can be seen as variations on tensegrity prisms” . It starts with a line segment, and it builds up a structure in steps until a triangular tensegrity prism is achieved. It is important to understand these basic structures in order to be acquainted with the more complex ones presented later in the chapter.The second half of this chapter continues dissecting tensegrity structures as it increments the complexity of them. It also shows how these structures relate to a potential design for a robot and discusses about advantages and disadvantages of each one.This is the basic building block for most regular structures. It is made with one strut and two cables on opposite sides. It should be noted that two cables are needed to have a properly balanced system with two cables under tension and one strut under compression. If it only had one cable, the system would generate momentum and shear forces. All tensegrity systems in this exploratory exercise will be built from this basic arrangement. Figure A square is made from two tensegrity line segments. It is constructed by inserting one tensegrity segment into another. Segment “B” is inserted in such a way that the ends of the strut are connected to the midpoint of the cables in segment “A”. Similarly, cables from segment “B” connect by the midpoint to the ends of the strut in segment “A”. The original cables in each segment are now considered split in two cables. Therefore, a square comprises two struts and four cables as shown in Fig. A triangular prism is constructed by inserting a third tensegrity segment “C” into the previously seen square. Also known as 3-strut prism, it is the simplest three-dimensional tensegrity structure and the very first one created and exhibited in 1921. It has three struts and nine cables. It also exhibits strong stability in three dimensions as the tension cables for each strut considered together have projections on all three-dimensional basis vectors, and three cables are attached to each node.1, B1, and C1. At the bottom of the prism, there are three more nodes A2, B2, and C2. Cables are named in relation to the nodes. At the top, there are three horizontal cables marked in cyan forming a triangle: A1B1, B1C1, and C1A1. Notice that the order is not important; A1B1 and B1A1 is the same cable. There are three vertical cables marked in yellow: A1B2, B1C2, and C1A2. At the bottom, there are three more horizontal cables marked in magenta forming another triangle: A2B2, B2C2, and C2A2. The connection of the six nodes is illustrated by the graph structure in Fig. There is a particular nomenclature used in this analysis shown in Fig. Single plane This intermediate state topology, shown in Fig. Dual plane with three intersecting struts This intermediate state topology, shown in Fig. Dual plane with two intersecting struts This intermediate state topology, shown in Fig. As part of this study, three intermediate structures between the tensegrity square and the triangular prism were devised to help investigate the properties of a three-dimensional tensegrity structure and show what results if the symmetry mentioned above is broken. In other words, these are artificial transitioning state topologies between a 2D and a 3D tensegrity structure that help understand how a 3D tensegrity structure is able to stand; its internal mechanics and the relationship between its elements. The mechanics of such structures are integral to the performance of a tensegrity-based robot and also illustrate how a robot can be manufactured and transformed between different topologies.A square prism is comprised of four struts and twelve cables. A square prism and any other prism follows the same relationship found for the triangular prism. Top horizontal cables are marked in cyan, vertical cables are marked in yellow, and bottom horizontal cables are marked in magenta as shown in Figs. In the context of a duct travelling robot, the square prism is better fit for a square duct cross section where each strut could be pressed against each corner of the square section in the duct. The morphology shown has the bottom square with larger dimensions than the top square. This could be beneficial for expanding a narrow axle into a much bigger structure, as a joint between a square bar and another tensegrity structure or to reach out to the walls of a bigger ducting. A graph of the structural connections is given in Fig. Comprised of eight struts and twenty-four cables, the octagonal prism, shown in Figs. The expanded octahedron or icosahedron has six struts and twenty-four cables. All 6 struts can be organised in three groups: A, B, and C. Each group has of two parallel struts R and L (right and left). Therefore, all struts can be labelled AR, AL, BR, BL, CR, and CL as shown in Fig. The morphology of this topology can be changed into a truncated tetrahedral Fig.  by simplThe icosahedron resembles a ball when all struts and cables are the same length. NASA is developing a robot with this morphology that is capable of rolling as method of locomotion . In relaFormed by joining the parallel struts of the icosahedron together, Fig. This is a class on its own right. The number of struts required is the same as the number of edges, and the number of cables required is equal to three times the number of vertices plus the number of edges.The truncated tetrahedral, Fig. This structure could be used as flexible chassis for a three-wheeled robot. In a duct travelling robot, this structure could be used as a nose at the front of the robot to carry an instrument. The nose can be pointed in different directions by changing the tension in the cables. It can also be used as a tail to stabilise the robot when it needs to change direction into a new branch.A truncated cube, Fig. When the ends of the struts on each side of the cube are joined closer together a different morphology comprised of four intersecting triangles is made as shown in Fig. Following the same pattern, a truncated dodecahedral, Fig. While the structures considered up until now are illustrative of tensegrity principles and show how such structures could be of use in robotics, we now consider structures that are less based on geometric primitives and more on repeated structures found in nature. The first structure we consider in this area, the spiral vertebral mast, is built in stages. In this model, each stage has three struts that attach to the cable midpoint of the previous stage. The orientation of each stage changes, if the first stage is oriented clockwise, the second stage is oriented counter clockwise. This structure, designed by Tom Flemons, can be built to be as long as desired following the same pattern. At its minimal topology, it only has tensile elements of contraction as shown in Fig. The mast presented in Fig. This structure, in a way, is a simplification of the vertebral column shown in Fig. There is no representation for any of the four articular processes. They are omitted in this model which is one of the biggest differences. The forces produced by the vertebral body and articular processes are replaced by horizontal cables . Bones are the only elements to transcend those limits. It can be observed that the humerus extends beyond the shoulder and, similarly, the muscles in the shoulder area a located on one end of the humerus and do not extend up to the other end of it. In this case it can be considered that the rigid body acts as the transitional region between two localised tensegrity regions.There are few exceptions to this paradigm that are starting to appear. Hustig et al.  did applIt is a transition made exclusively of rigid elements. This type of transition is linked to the concept of fulfilled pattern described below.It is a transition made exclusively of elastic elements. In order to be stable, it needs to have an antagonistic tensional setting: elements of contraction and elements of expansion. This type of transition is linked to the concept of unfulfilled pattern described below.The lumbar vertebrae are connected among themselves by three surfaces. One located at the vertebral body and two at the articular process. In total, a lumbar vertebra has six joints: three at the top and three at the bottom. This means that the fifth lumbar vertebra, the last one at the bottom, needs three surfaces of connection as well when joining the sacrum. The sacrum at birth is composed of five separate sacral vertebrae. Postnatally they fuse to form a single bone that is flattened anteroposteriorly and has a triangular shape when viewed from the front .This is where the principle of fulfilled pattern takes place. When connecting a chain of rigid elements of the same morphology to another element of different morphology, the morphology of the first needs to be satisfied by the second. That is, the sacrum needs to have the same top three connecting surfaces to receive the bottom three connecting surfaces of the fifth lumbar vertebra. Figure This concept can be observed in the connection of the scapula to the axial skeleton in the musculoskeletal system of the human body. The scapula attaches to the axial skeleton only with soft tissue. The scapula (shoulder blade) is a flat, triangular bone located along the posterior aspect of the thoracic cage. Seventeen muscles attach to the scapula; therefore, this bone is usually resistant to fractures . Since tStatic analysis of tensegrity structures Find a stable configuration from a given topology or even design new topologies to achieve some desired results.Form-finding methodsStatic behaviour of tensegrity.Rigidity and stability.Dynamics and control of tensegrity Plan trajectories and motions taking into account the advantages that tensegrity structures offer.Traditional path planning algorithms.Dynamic characterisation of tensegrity structures.Node trajectory planning.When using tensegrity structures to design mobile robots, designers face the following challenges :Static aThis paper is meant to cover the first three items from the list. As there is no previous academic work in robotics that uses coupling between solid bodies and tensegrity structures, it was necessary to first perform the introductory study shown previously.The design of this prototype focuses on achieving two main requirements: (1) unprecedented flexibility for an air duct travelling robot and (2) unprecedented locomotive performance for a tensegrity robot. This section illustrates the development of the prototype and compares the features of this robot with the current state of the art.Regarding geometric characteristics of air ducts, most common rectangular air ducts range from 100 mm × 200 mm to 150 mm × 300 mm and circular ducts, from Ø100 to Ø200 mm in households. The layout of ducts is mainly in a horizontal plane with turns right and left. However, there are also branches that go upward and downward. There are two requirements here: (1) a robot must adapt to the range of ducting sizes and (2) the robot must be flexible enough to turn not only left and right but also up and down or any other intermediate direction.While requirement number one has already been solved by several designs, requirement number two has not. There are no robots in the market, to the authors’ knowledge, that can turn in any direction and are able to vertically climb. Therefore, requirement number two was the main focus of the design in this paper. The development process begun with the selection of the main tensegrity structure of the robot, followed by the design of the coupling from tensegrity to a solid body, and, finally, a model for the robot.Taking advantage of the flexibility of tensegrity structures to allow the robot to turn in every direction was the main requirement. All tensegrity structures are flexible, but not all of them have special flexibility in particular directions. Most of the structures tend to be omnidirectional and resemble the shape of a sphere.However, there are two topologies that fit the needs of a duct traveller robot: The spiral vertebral mast and tetrahedral vertebral mast. They were chosen for reasons explained in “Initially, one prototype was made for each structure following the criteria of a hard transitional region and fulfilled pattern. For the tetrahedral vertebral mast , it was necessary to build all four struts and not only two as it was originally thought as shown in Fig. As can be seen in Figs. Two more prototypes of the spiral mast transitional region were made following the concept of soft transitional regions and unfulfilled patterns. However, the design was not tensional antagonistic and it had a weak tensional morphology. Therefore, the structure was unstable. More development would be needed to achieve both requirements in future prototyping iterations of this principle.The final prototype is the first wheeled tensegrity robot (WTR) ever built to the authors’ knowledge. It was made with readily available and recycled parts in the design laboratory at the University of Strathclyde. The final version comprises two motorised modules and one non-motorised module which are shown in Figs. The robot has five sections. The first one is the front car, the second one is the front tetrahedral vertebral mast, the third one is the middle car, the fourth one is rear tetrahedral vertebral mast, and the fifth one is the rear car, as shown in Fig. Having finalised the design and before testing the mechanical characteristics of the robot, we present a brief discussion regarding its features. They are introduced after setting up a frame of reference for this comparison.Duct travelling robots can be classified in four categories: (1) conventional wheeled robots, (2) expanding robots, (3) tensegrity robots, and (4) hybrid tensegrity wheeled robots.Conventional wheeled robots are the most common. Sometimes they use tracks to add more traction and climb ducts with a small slope. Expanding robots have arms that extend to reach for the walls of the ducting and press against them to gain the traction needed for vertical climbing; there are few of these robots in the market. Regarding tensegrity robots that travel in air ducts, to the authors’ knowledge there is only one created as part of a research project and it is DuCTT which is included in the literature review. The fourth category is what is presented in this paper: a hybrid tensegrity structure and solid-body wheeled robot that we refer to as a wheeled tensegrity robot (WTR).This comparison is based on eight key mobility features that an air duct travelling robot should ideally have: (1) move horizontally inside a duct, (2) move vertically inside a duct, (3) turn right or left into a new branch (as opposed to just follow through a bend in the duct), (4) turn up or down into a new branch, (5) horizontal u-turn into a parallel adjacent branch with a narrow radius (6) vertical u-turn into a parallel adjacent branch in a narrow radius, (7) travel at standard speed for commercially available mobile robots and (8) move in an open plane, when not inside a duct.Table This table provides a basic example of the mobility that a WTR is capable of. The compliant structure can provide many other capabilities, but this brief list is indicative of the robot’s mobility within ducts. With appropriate actuation, a WTR can turn into branches of any direction, not just vertical or horizontal, and also twist its body in the axial direction simultaneously. Figures To better understand the performance of the prototype, and by extension, future generations of similar WTMRs, three tests were performed to analyse the mechanical characteristics of the rubber band used to build the prototypes. In all three, 100 mm rubber band samples were used. Figure The results showed that the mechanical properties of the rubber band deteriorated after a 20% displacement. Therefore, in order to have consistent results any deformation further than 20% was avoided. There was no noticeable linear pattern in the 20% deformation region; therefore, a simple linear formula was avoided and the tension in the cable was determined from interpolation of the experimental results obtained in Fig. The first set of tests was performed on three stages of the tensegrity spine . The purpose of the tests was to find the tension supported by each cable and how much tension was needed to bend the spine when unloaded and when linked to a load. The tensegrity spine was made out of soft wood and bamboo sticks. Rubber bands were used as cables. Custom-made rubber band loops were made with specific lengths (gluing together the ends) to be used as horizontal cables. The actuator was simulated with fishing nylon thread attached on the one end to the first strut in the structure and on the other end to a digital scale. Figure A labelling system of two letters and two digits was used to identify each cable in this structure as shown in Fig. The test consisted on measuring final length of 10 mm marks made on each cable. By measuring the displacement, the tension on the cables could be approximated. Tension in the actuator was measured with a digital scale.Rubber band loops made were 120 mm long which means that each horizontal cable was 30 mm long. Each vertical cable was also 30 mm long. Displacement was measured with no actuation and no loads acting upon the structure. Cable T3H4  was the most loaded, showing a displacement of 70%. It should be noted that this cable shows more tension due to the fact that the rigid elements are not perfectly symmetric. In later tests, this was compensated by slightly varying the length of the cables. Table For this test, 152 mm long rubber bands were prepared. With a displacement of 20%, the final length of the band was 182 mm which covered the displacement seen in Test 1. Test 2 comprises a set of six different runs. The first two runs were done with no actuation nor load. Runs 3 to 6 have an actuator cable to bend the tensegrity spine up, down, left, and right. The tension in this cable was measured as well. Run 1 showed cable T3H3 with a displacement of 25%. The length of the cable was slightly adjusted before the second run. Run 2 shows that this displacement was reduced to less than 20%; right under operational range. The next runs were under actuation to bend the tensegrity spine in four different directions. Two major displacements have been highlighted in Table Rubber band loops of 160 mm were prepared for the third test. With a displacement of 20%, a final length of 192 mm will be covered. The first 2 runs were made with no load and no actuation just to compensate for any peaks in tension in any of the cables. Indeed, some of the cables needed adjustment like T2H4 and T3H4 that were adjusted from 16 to 6 and 13 to 8%, respectively. Runs 3–6 were performed with actuation and no load. All displacements were under 20% this time. Runs 7–10 were performed with the front car (94 g) attached to the tensegrity spine. The results in Table The use of rubber bands as a tension medium proved to be a limiting factor since the beginning of the experiments due to the quick degradation of its mechanical properties. Nevertheless, it was a quick and cheap method of prototyping and some useful information could still be drawn from the tests. It was determined that the working zone for this rubber band was under 20% displacement and 1.2 N of force. With these conditions, it was determined in Test 2 an optimal length of 160 mm for horizontal rubber band loops and 30 mm for vertical rubber bands. However, Test 3 demonstrated that the front car, weighing 94 g, was too big a load for the rubber bands. Additionally, it was noticed that in the runs with the 94 g load the tensegrity rigid elements started touching each other. This meant the tensegrity stopped behaving as such; cables in the tensegrity structure did not bear all loads anymore. The structure was overwhelmed by the load. Runs 3–6 in Test 3 can be used as an approximated method to calculate the loads on each cable based on the tension in the actuator, displacement, and Fig. We have detailed how the very first wheeled tensegrity robot was successfully designed and prototyped. It was proven that this robot addressed all design requirements for a duct traveller mobile robot. This novel design proved to have capabilities not found in just one robot. There are robot designs for vertical climbing, designs for facilitating turns to the right and left, and designs that focus on speed and efficiency of mobility, but none of them group all these requirements into a single structure like the wheeled tensegrity robot does.This research furthers the knowledge in the ever-growing field of tensegrity mobile robotics. It actually opens a new sub-category of hybrid wheeled tensegrity robotics. The project methodology was proven successful as it led to an optimised design that fulfilled all requirements. It had an advantage over simulation and analytical methods in these particular circumstances. It certainly was best to approach the research by broadly study structures and build models to assess their properties. The criteria of evaluation were validated by prototyping. Accepted designs by the criteria were stable. Rejected designs by the criteria were unstable. Furthermore, these criteria need to be expanded. The author sees many more possibilities of growth that were not pursued due to time constraints. Tensegrity robots do not need to address all design requirements with a tensegrity solution. Hybrid approaches have just been proved to be valid. Practical applications should start to appear more in research work. Transition areas should not be constrained to mobile robotics. In civil engineering, they can be applied to add tensegrity structures to fixed structures.Future work should include testing the locomotive efficiency of this design. The weight can be further lowered by reducing the number of cars from three to only two and adding a tail section at the end for stability. It could also include computer simulation to refine the topology and morphology created, building a prototype with metal springs and a metal body  and adding motorised actuators to control the structure. Flexinol was also tested on this prototype as actuator, but the amount of actuation was much smaller that the structure required. However, a pulley arrangement is a possibility to be tested. Future work could also include exploring other topologies applied to wheeled tensegrity robotics and continue developing the criteria and designs for soft transitional regions.


Grazing Incidence Neutron Spin Echo Spectroscopy (GINSES), where an evanescent neutron wave probes the fluctuations close to a rigid interface. With this method it is possible to access length scales in the nano to micrometer region as well as energies in the μeV range. Using a new neutron resonator structure we achieved the required intensity gain for this experiment. During our investigations we found an excitation mode of the phospholipid membrane that has not been reported previously and only became visible using the new methodology. We speculate that the energy transported by that undulation can also serve to distribute energy over a larger area of the membrane, stabilizing it. This new methodology has the capability to probe the viscoelastic effects of biological membranes, becoming a new tool for tribology on the nanoscale and has allowed the observation of the hitherto invisible property of phospholipid membranes using neutrons.Here we present an approach to measure dynamic membrane properties of phospholipid membranes close to an interface. As an example we show results of the membrane dynamics of a phospholipid membrane multilayer-stack on a solid substrate (silicon). On this sample we were able to measure local interaction and friction parameters using  The links between the cellular behaviour and properties of phospholipid membranes are treated in a review by Tanaka4. Investigations of such systems have been carried out using a wide range of methods, such as light-scattering3, atomic force microscopy (AFM)5, X-ray scattering12 as well as elastic13 and inelastic15 neutron scattering. An overview of neutron scattering methods used to investigate phospholipid membranes was presented by Fragneto and Rheinstädter16, while Salditt focused on X-ray scattering10. Neutron-Spin Echo (NSE) measurements in quasi-reflective mode with a stack of supported membranes were introduced by Rheinstädter et al.17. Extending this approach, using only a single supported membrane stack, here we present GINSES as a novel method that enables the investigation of the dynamics in complex fluids in the vicinity of a macroscopic solid surface on a molecular scale. Comparing the two approaches, using only a single membrane stack, both the background from the surrounding D2O as well as the error in angle due to spatial separation of the single wafers is lower. This is especially true for extremely small Q|| (here −1), allowing the investigation of dynamics with a longer spatial range.A detailed understanding of the rheology and friction at interfaces is of vital importance for a wide range of biological and medical applications, such as lubrication and coating in mammalian joints19, but on shorter length and time scales and with a different contrast, as given by the difference in interaction between matter and neutrons or photons. While GINSES as a method has been used before in our group22, only the use of the resonator enabled us to gain a clear enough signal in this special case and to properly distinguish for the first time between the in- and out-of-plane components of the scattering vector Q.The basic principle is analogue to evanescent wave scattering of lightα-phosphatidylcholin (SoyPC) membrane that shows surface modes, which previously could not be observed by other methods. The structure of the above membrane close to the surface has been studied previously20 where the system was shown to develop a multilayer structure. Apart from indicating a promising novel method offering insight into the dynamics of complex fluids at solid/liquid interfaces, the results are also of immediate interest for the understanding of bio-lubrication in joints, where phospholipid membranes at interfaces contribute to the lubrication of joints1.As an application, we show the direct measurement of the intra-membrane viscosity and the compressibility measurement of phospholipid membranes at the silicon/water interface. This was achieved by using resonance-enhanced GINSES to analyze the thermal fluctuations close to the solid interface on the scale of nanometers and nanoseconds. A multilayer structure (titanium-platinum) at the interface yields resonant enhancement of the evanescent neutron wave. This is essential to overcome the severe intensity limitation of the limited scattering volume  and reflectometry20. The pure SoyPC in contact with liquid D2O at T = 35 °C forms double layers with a repeat distance of d = 68.4 Å. That repeat interval gives rise to the pseudo Bragg-peaks at We performed a GINSES measurement on a stack of phospholipid layers at maximum swelling in D−1 (see method section for details). Here the difference between incoming and outgoing angle of reflection was 7°.In order to avoid influences on the dynamic measurement, such as de Gennes narrowing, the measurement was performed at This yielded the intermediate scattering function as shown in Fig. When compared to conventional NSE experiments on soft matter systems the most striking feature of the data presented in Fig. We attribute these oscillations to undulation modes of the SoyPC membrane stack close to a solid substrate, which is corroborated by a model computation . We attribute this to the fact that for extremely small Q|| the membrane is seen on a larger scale, where only movements of the complete membrane would be visible. Similarly for large Q|| the local over-damped motion of a few phospholipid molecules is dominant. Thus, only in a certain range of Q|| are the undulations visible.Looking at the simulated data in Fig. 24 for freely supported films25, which have been observed in X-ray photocorrelation experiments.All modes observed here occur at solid-liquid interfaces, where a membrane is parallel and in close proximity to a solid substrate. Although very similar in nature, they must not be confused with capillary modes on free surfaceset al.23 applying the basic equation to describe smectic systems by Poniewierski26.A theoretical model to describe the dynamics of a finite lamellar system on a solid support in terms of layer undulation modes has been formulated by Romanov N flexible layers in an equidistant stack on a solid support on the one end and a free surface at the other end of the stack. The layer-layer interaction is described in terms of the compression modulus B and the layer-bending elasticity in terms of a bending modulus K = κ/d ; both values are given as bulk properties and not for single layers. The model contains a surface tension γ for the free surface and the layer-layer sliding viscosity l of the model solutions in ref. n dependent displacements ωl. Each ωl always contains a viscosity-dependent dampening term f also depending on the model parameters.The model consists of f is negative. For a thick layer seen from below, where the evanescent wave is present, the surface tension γ only weakly influences the overall system, and intensity is contributed mainly by high l modes with sizeable displacement coefficients at the low-lying layers. With this (letting l = N) Eq. (2.19) used by Romanov et al.23 yields a condition for the occurence of oscillating modes:l isThe modes are overdamped if Q values of NSE but in the range of Q|| of GINSES experiments. Note, however, that q|| denotes a mode wavevector, which is different from the scattering wavevector Q||. Certainly, matching mode wavevectors make a significant contribution to the scattering intensity observed at a corresponding value of Q|| but there is no strict one-to-one correspondence and the coupling is such that other modes do contribute. This is also why the expression for the eigenfrequency cannot be employed directly, as only an integration over all possible eigenmodes will render a usable result for any given Q|| value.Here The GINSES geometry has another feature that favours the observation of undulation modes in the present case of lamellar stacked layers. The layer displacements B and the in-plane viscosity B is also accessible by other methods, such as X-ray reflectometry28 it has not been previously possible to directly measure the in-plane viscosity within this time and size regime (nanoseconds and nanometers) as well as possible deviations of the interface layer compression modulus from the bulk value in the vicinity of a solid substrate. Here it should also be mentioned, that using the Caillé exponent approach29 as used in some of the other publications is an indirect method, as compared to the method presented here. This is due to the fact that the Caillé theory is based on elastic energies, independent of the viscosity, whereas the modes presented here depend on both.The compression modulus ||, indicating that the surface modes detected here are indeed long range correlations.Comparing the simulations in Fig. 24 as well as internal modes of membranes17.Here, long-range in-plane modes of phospholipid membranes adjacent to a solid substrate-liquid interface are observed. While not completely dissimilar they should be clearly distinguished from surface modes at liquid-gas interfaces16, using a Langmuir trough with a tensiometer30 and driving a lipid vesicle through an aperture31, so they give a representative sample of both available techniques and possible values for the parameters.In Table et al. is performed for −1 the experiment shown here is performed for −1. This translates to length scales of ≤500 Å for Rheinstädter et al. and ≥1000 Å for the experiment shown here. This allows the conclusion that the local viscosity is higher than the one averaged over a larger area of the membrane.A more detailed comparison shows that the difference between the various modes of measurement is quite pronounced. While the bending properties signified by B can also be explained by similar considerations: a membrane may be much more rigid locally, than the same membrane averaged over a large area, because the necessary displacement for each single phospholipid molecule becomes increasingly smaller as the number of molecules participating the in motion increases. Additionally the vicinity of the hard substrate may inhibit fluctuations that would decrease the compression modulus B.The increase in the compression modulus et al.30, may fundamentally differ from the surface tension of a layer stack and that thus a variation is to be expected. Rheinstädter et al.16 argued that for such a case the surface tension might even be neglected completely. In addition, γ is only considered for the topmost layer of the membrane stack23.Surface tension was adapted manually to our fit. Here it should be stated that surface tension at the air-water interface as investigated by Brzozowska et al.32 also predicted a damped oscillating correlation function for a bulk microemulsion model on a different basis B, γ, κ and the relevant length scale. Our present result indicates that this predicted behaviour indeed occurs, but only at the Q-values probed here, conforming with the results of the simulation.In terms of a theoretical prediction of the observed surface modes, Gompper 1.The the analysis of these modes and the resulting knowledge of the rheological key parameters of phospholipid membranes may prove useful in applications in biotechnology and medical sciences. One very prominent example is in phospholipid membranes on a solid substrate in an aqueous environment as found in mammalian joints1. We did not encounter this behaviour in any softer systems we investigated previously, such as those in microemulsions22. From general observations it is clear that the membranes withing mammalian joints need to be durable , compared to those in emulsions, which exist for a time period of mere seconds or minutes. Thus a self-stabilizing mechanism must exist in these membranes, and such an undulation as reported here is an excellent candidate for dissipating energy throughout the system. The stability of a membrane is also linked to the stability as described by Salditt10; however in this case phase transitions due to temperature change probably play a major role.Any considerations given here about the interplay between the described mechanism and the stability of a given membrane are very general. The investigated membrane is structurally very similar to the membranes found in mammalian joints33. Neutron spin echo (NSE) spectroscopy provides the highest energy resolution in neutron scattering and measures the normalized time correlation function in reciprocal space, S. NSE uses a polarized neutron beam.Neutron scattering experiments were performed using neutron spin-echo spectrometer at the spallation neutron source SNS at the Oak Ridge National LaboratoryThe high resolution is achieved by encoding and decoding the neutron velocity before and after the scattering process by way of a number of spin precessions in a magnetic field. In grazing incidence geometry, the sample cell is very similar to that used in neutron reflectometry experiments. The incoming neutron beam hits the interface between a Si-block and the sample containing the phospholipid membranes at an angle below the critical angle, thus being totally reflected. Only an evanescent wave penetrates the sample and can be scattered by the membranes in the vicinity of the interface. Only the near-interface dynamics are probed in this geometry. The penetration depth of the evanescent wave can be varied by changing the incident angle and the contrast between sample and Si-block (by an appropriate deuteration of the sample). In order to overcome the huge intensity penalty due to the strongly collimated beam and the use of the evanescent wave only, a resonator structure on top of the Si-block, consisting of Pt-Ti layers at the interface, which provide a neutron waveguide for an appropriate incident angle, has been used. The intensity of the evanescent wave very close to the interface is increased in this improved GINSES setup.αi values. This is analogue to the use of dielectric layers in light optics34. The fluid sample is contained in a volume confined by this interface and a glass plate at a few 0.1 mm distance, sealed by O-rings20. The active area was 2 × 15 cm2, while the Si-block was 5 cm thick.The sample stage consists of a polished block of single crystalline silicon with a Ti-Pt multilayer between the silicon and the sample see Fig. . The Ti-ρ is the scattering length density difference between the sample and Si-block) so that there is only the narrow evanescent neutron wave on the sample side. In this case an incoming angle of αi = 0.02° was chosen. The direct beam is absorbed by a cadmium strip on the exit side of the Si-block. Evanescent field neutrons that are scattered by the sample reenter Si-block and are transmitted with an average angle of several degrees. They undergo the second part of the NSE-typical Larmor precession velocity encoding-decoding analysis and are detected from within a solid angle of ΔΩ = 4° × 4°. The intensities are evaluated to yield the intermediate scattering function S on detector slices with a solid angle of ΔΩ = 1° × 4°.The sample cell was used in a reflectometer-type geometry. Unlike in typical NSE experiments, the beam was narrowly collimated in the direction of the scattering plane down to a divergence of ±0.05° by a slit at the entry of the neutron beam to the secondary spectrometer and a slit attached to the front side of the Si-plate. The orientation of the Si-block was such that the collimated beam hit the interface at an angle below the critical total reflection angle 2 prism with an apex angle of 170° was used. This establishes an angle-wavelength correlation such that the penetration depth of the evanescent wave 22. We observe a finite number (on the order of 10) of double layers due to the finite penetration depth.Since the SNS-NSE instrument is located at a pulsed spallation source it uses a broad range A sketch of the used geometry is shown in Fig. The classical reflectometry condition of −3 Å−1. In the present experiment the deviation between the incoming and outgoing beam was 7°. This allows to calculate of the in-plane component of the Q-vector with the expression As the difference between the incoming and outgoing angle of just a few degrees is still small, this implies very small in-plane components of 21. Even under optimal conditions it was not always possible to achieve the necessary count rates and, by extension, the accuracy of the intermediate scattering function, for the analysis presented here. The intensity limitations can be alleviated by using a resonator structure on top of the Si-block, which acts as a neutron wave-guide and increases the field of the evanescent wave. Three double layers of titanium (130 Å) and platinum (320 Å) and a final layer of titanium (130 Å) provide a SLD profile that leads to three conditions of constructive interference for angles below the angle of total critical reflection. In Fig. In previous experiments we already performed GINSES measurements; however, the extremely low sample volume limited the experiments to strongly scattering samplesitanium 10 Å and pConstructive interferences were obtained while evanescent waves developed in the sample, so that the intensities were accumulated. For sharp conditions of resonance, amplification factors of 10 to several 100 can be obtained. Under conventional collimation conditions, the gain factors are still between 3 and 10. This gain was essential for the observations of the collective membrane modes observed here due to the small amplitudes of the individual phospholipid bilayer.20. 15 mL of SoyPC/isopropanol solution with a concentration of 174.6 mgmL−1 were prepared. The solution was shaken for at least 15 min. until a clear solution was obtained. This solution was subsequently deposited on top of a silicon block in a holding frame, which had been previously cleaned with a 2% Hellmanex solution and rinsed with Milipor water. Afterwards the sample was dried overnight at room temperature and a pressure of 2500 Pa in order to prevent bubbles. This preparation method yielded macroscopically thick films on the Si-block. To facilitate measurements, the sample cell was kept at 35 °C and was completely filled with D2O in order to obtain a completely hydrated multilayer sample.For the preparation of the sample we followed the protocol from our previous publicationAlthough the penetration depth of the neutrons into the sample is only a few hundred nanometers, we used a preparation protocol that renders a film thickness in the millimetre range. Earlier experiments with thinner films  have shown that this does not change the behaviour of the film close to the substrate, but it facilitates sample preparation and reproducibility.The standard neutron spin echo data evaluation consists in precisely determining the degree of polarization of the neutron beam at the detector and normalizing it with the degree of polarization of a reference measurement with an elastically scattering sample (graphene foil).τ), has been fitted with a model for the scattering function described by Romanov et al.23 extended to include the dynamics of the system as described previously.The intermediate scattering function determined in this way, S, is confined in the membrane, it will reduce the polarization of the echo measurement as well as for the elastic polarization measurement. Long wavelength undulations cannot be observed with the incoherent part of the scattering, since it represents only the self-correlation function, not the pair-correlation function. This part has been included as a static incoherent contribution in the model, which thus only accounts for the hydrogen in the phospholipid itself.2O) also contributes to the incoherent scattering (p(spin incoherent scattering) = τ < 0.5 ns. The relaxation time D = 190 Å2/ns which we would expect for pure deuterated water diffusion. The incoherent contribution from the solvent D2O seems to be slightly higher than expected compared to the phospholipid layer contribution, which might stem from some residual protonated solvent molecules from the sample preparation process.Secondly, the solvent (D


Glaucoma is the primary cause of irreversible blindness worldwide associated with high intraocular pressure (IOP). Elevated intraocular pressure will affect the normal aqueous humor outflow, resulting in deformation of iris. However, the deformation ability of iris is closely related to its material properties. Meanwhile, the passive deformation of the iris aggravates the pupillary block and angle closure. The nature of the interaction mechanism of iris deformation and aqueous humor fluid flow has not been fully understood and has been somewhat a controversial issue. The purpose here was to study the effect of IOP, localization, and temperature on the flow of the aqueous humor and the deformation of iris interacted by aqueous humor fluid flow.Based on mechanisms of aqueous physiology and fluid dynamics, 3D model of anterior chamber (AC) was constructed with the human anatomical parameters as a reference. A 3D idealized standard geometry of anterior segment of human eye was performed. Enlarge the size of the idealization geometry model 5 times to create a simulation device by using 3D printing technology. In this paper, particle image velocimetry technology is applied to measure the characteristic of fluid outflow in different inlet velocity based on the device. Numerically calculations were made by using ANSYS 14.0 Finite Element Analysis. Compare of the velocity distributions to confirm the validity of the model. The fluid structure interaction (FSI) analysis was carried out in the valid geometry model to study the aqueous flow and iris change.In this paper, the validity of the model is verified through computation and comparison. The results indicated that changes of gravity direction of model significantly affected the fluid dynamics parameters and the temperature distribution in anterior chamber. Increased pressure and the vertical position increase the velocity of the aqueous humor fluid flow, with the value increased of 0.015 and 0.035 mm/s. The results act on the iris showed that, gravity direction from horizontal to vertical decrease the equivalent stress in the normal IOP model, while almost invariably in the high IOP model. With the increased of the iris elasticity modulus, the equivalent strain and the total deformation of iris is decreased. The maximal value of equivalent strain of iris in high IOP model is higher than that of in normal IOP model. The maximum deformation of iris is lower in the high IOP model than in the normal IOP model.The valid model of idealization geometry of human eye could be helpful to study the relationship between localization, iris deformation and IOP. So far the FSI analysis was carried out in that idealization geometry model of anterior segment to study aqueous flow and iris change. Glaucoma is the primary cause of incurable blindness in the world . It geneAqueous humor is a kind of transparent fluid, which circulates through the anterior segment and provides nutrition for avascular ocular tissues, such as lens, anterior vitreous, cornea and trabecular meshwork Fig. . AqueousThe interest in comprehensive the iris mechanics develops from the influence of abnormal iris morphology on specific ocular disorders. For instance, in angle closure, the iris bows anteriorly, and the passively deformed shape and position of the iris restrict aqueous humor outflow, leading to a high intraocular pressure . A recenPrevious report carried out several simulations and mathematical models to research the mechanism of aqueous humor fluid flow in anterior chamber, such as the distribution of temperature and velocity –13. HeysThe goal of this research was to develop a 3D eye model based on idealization geometry of human eye and to establish the characteristics of aqueous humor flow under physiological and pathological conditions such as primary angle-closure glaucoma. This device will make a foundation for further simulations and evaluations of different influence factors of glaucoma.The model of the anterior segment studied here is similar to that of Repetto et al. . The geoPIV technology and 3D printing, as the new technology, are increasingly applied to the biomedical field, and helping to create a model for effectively experiments that cannot be achieved in animals , 19. In Compared with the idealization geometry, a cylindrical water sink with a 78 mm diameter was created outside the cornea of the simulation device to reduce the laser reflection on cornea surface. We also designed an inlet and an outlet channel to simulate the inflow and the outflow of aqueous humor. In order to make the fluid flow balance and uniform through the simulation device, outside of the inlet buffer space and the outlet buffer space are designed with many small holes. A column below the lens plays a supporting role, engendering a 10 µm gap between lens and iris. A silicon wafer, which to prevent the downward leakage of fluid flow, is provided substantially entirely on the back surface of the lens, connected under the holes of inlet.The simulation device of anterior segment is used to simulate the physiological environment of aqueous humor fluid flow. The flow field in anterior chamber is measured by particle image velocimetry technology. The pivotal device in our study is image velocimetry system, provided by Beijing Institute of Technology, which can be found in Yang et al. .Polystyrene particle is selected as the tracer particle, easily passing through the pupil with 10 microns in diameter. They can be isotropic spread and show a real following feature in intraocular fluid . DevicesCompared with the velocity calculated by ANSYS Fluent14.0, utilized the 5 times geometry model, to verify the validity of the finite element model.p represent pressure, ρ is fluid density, μ is fluid dynamic viscosity, and g is gravitational acceleration, with the magnitude g = |g|.The motion of aqueous humor in anterior segment is treated as a steady and incompressible Newtonian viscous fluid in this study, governed by the equations of momentum (Navier–Stokes) and continuity.T stands for temperature, k represents the fluid thermal conductivity, and Cp defines the specific heat at constant pressure. We have neglected the heat production due to mechanical energy dissipation in Eq. In the case of buoyancy-driven flow, the equations above are coupled to the energy equationρ0 stand for the density of fluid at a reference temperature α defines the fluid volume expansion coefficient. Values are summarized in Table The Boussinesq approximation was used to stand for the unfluence of buoyancy, because of the temperature variations. The approximation states that the density of fluid changes with temperature and has little correlation in pressure. The Boussinesq approximation was given by:mentclass2pt{minimThe cornea limbus, iris root, and lens are modeled as stationary boundaries, and impased no-slip boundary condition on the surfaces. The front and back surface of iris and the corneal endothelium are set as FSI surfaces. Set the plane XY to a plane of symmetry. The temperature at the cornea is assumed along the outer surface, generally considered to lie between 33 and 35 °C , and wasAqueous humor is simulated inflows from the ciliary body area at a rate of 10 e−8 kg/s (3 µl/min). The working IOP condition was defined by an IOP of 13.5 mmHg to represent the healthy model while the glaucomatous case was set of 27 mmHg of IOP. Thus, the static pressure forced at the trabecular meshwork was estimated 13.5 mmHg and 27 mmHg to simulate the healthy and the glaucomatous models. In our study, aqueous humor secreted from the ciliary body and the trabecular meshwork was assumed to be at 37 °C. The boundary conditions are presented in Fig. A volumetric mesh was developed by ICEM CFD software  and exported to ANSYS Fluent  for the fluid simulation. The 3D model of fluid structure and solid structures (iris and cornea) are depicted in Fig. The distributions of flow fields in anterior chamber were gained in different inlet velocity. Figure As shown in Figs. The increase of the intraocular pressure could affect the aqueous humor fluid flow in anterior chamber. The working IOP conditions are 13.5 and 27 mmHg in our study to simulate the healthy model and the glaucomatous case, respectively. The values of aqueous humor fluid flow with different intraocular pressure are shown in Table In Fig. Figure Distributions of velocities in the anterior chamber are similar in healthy and glaucomatous simulation calculations.The values of equivalent stress, equivalent strain, and the total deformation of cornea and iris are represented in Table We also show the corresponding equivalent strain values in Table Through the analysis of fluid–structure interaction, we can also see the presence of IOP makes some difference to the total deformation of iris. The maximum deformation with a value of 0.33917 mm ,,Q. Volumvm and v could be neglect in Eq. (Qm (15 µl/min) in PIV experiment. And we will achieve this low speed measurement in the following experiments.Due to the low rate of aqueous humor fluid flow, the difference in t in Eq. . The simThe study on the visualization of the flow field distribution, the experimental model will be amplified to a certain degree based on the Reynolds number similarity principle. Because the human eye is small, and the fluid flow rate of aqueous humor is relatively low, there is very little experimental method to verify the validity of the model. 5 times amplified model is more easily carried out PIV experiment for the rate of infusing is larger than the secreted rate of aqueous humor in real eye, while we are trying to validate under a real scale model. To verify, we focus on the fluid streamlines, and the flow field distribution. The measured fluid distributions are similar to the results obtained by finite element method, so it can be explained that this method is valuable, and it is feasible to continue to improve in the verification experiment. We plan to improve the simulation model and use the advanced experimental equipment to complete the measurement of the flow field in low speeds.The values of velocity and pressure of aqueous humor formulated in steady state makes no difference in horizontal position or vertical orientation. Therefore, under the rapid eye movement, the study of the mixing aqueous humor in the anterior chamber becomes more meaningful. Modarreszadeh  carried The relationship between the deformation of iris and the intraocular pressure is of the most interest in this study. Ritch  argued tOur study provides a means to verify the validity and reliability of the finite-element analysis model. Through PIV experiment, we have captured the flow field in anterior chamber in the simulation device and calculated the corresponding velocity distributions. The valid model of idealization geometry of human eye can be helpful to investigate the relationship among localization, the changes of iris elasticity modulus and high IOP, so as to make further exploration in the pathogenesis of glaucoma.


Consequently, for experiments where cells require irradiation and observation for extended time periods, it is important to provide a controlled environment. The highly customised nature of the microscopes used on VMB systems means that there are no commercially available environmentally controlled microscope systems for VMB systems. The Automated Microbeam Observation Environment for Biological Analysis (AMOEBA) is a highly flexible modular environmental control system used to create incubator conditions on the end of a VMB. The AMOEBA takes advantage of the recent “maker” movement to create an open source control system that can be easily configured by the user to fit their control needs even beyond VMB applications. When applied to the task of controlling cell medium temperature, CO2 concentration and relative humidity on VMBs it creates a stable environment that allows cells to multiply on the end of a VMB over a period of 36 h, providing a low-cost (costing less than $2700 to build), customisable alternative to commercial time-lapse microscopy systems. AMOEBA adds the potential of VMBs to explore the long-term effects of radiation on single cells opening up new research areas for VMBs.Vertical Microbeams (VMB) are used to irradiate individual cells with low MeV energy ions. The irradiation of cells using VMBs requires cells to be removed from an incubator; this can cause physiological changes to cells because of the lower CO Noticeatabolism . It was Vertical Microbeams(VMBs) such as at Columbia University’s Radiological Research Accelerator Facility (RARAF) or at Surrey University’s Ion Beam Centre comprise the disciplines of ion-beam physics, radiation biology and microscopy. This makes them unique tools that allow for the simultaneous observation and irradiation of cells using particle radiation ,8. Experhttps://www.python.org/) and the Arduino environment , computer-automated manufacturing techniques, for example, 3D printing, and widely available sensors to allow for quick development times the AMOEBA system provides a low cost homemade alternative to the high cost commercial systems. AMOEBA’s open source nature, relatively low cost (costing less than $2700) and flexibility also make AMOEBA a useful technology for the new Do-It-Yourself Biology (DIYBio) movement allowing experimental capabilities that cost $100,000 s to be carried out on equipment of that costs a fraction of that [The Automated Microbeam Observation Environment for Biological Analysis (AMOEBA) was developed to integrate environmental control into VMBs. The AMOEBA is a modular environmental control system which taps into the vast resources of the “maker” community reducing development costs and allowing easy reproduction of parts, ultimately creating a system that can be reproduced in other laboratories . By taki of that ,13. The The AMOEBA was designed with the flexibility to be applied to other applications without having to write any additional code. This meant that different sensors and actuators could be configured independently depending on the task at hand. This flexibility was achieved by designing the system around a single Controller Area Network (CAN) bus to which all the devices are connected, a schematic is shown in The AMOEBA has two main parts, the desktop software and a collection of different AMOEBA modules that connect together using CAT 5 cable. The server module connects the AMOEBA system to a computer using a USB  cable and each of the other modules has a different sensor or actuator attached.The structure of an AMOEBA module can be seen in The single ATMEGA 328P microcontroller used in each AMOEBA module was chosen as the standard AMOEBA microcontroller for many reasons. First, the microcontroller is capable of controlling devices that operate using many standard bus protocols including I2C, SPI , and serial communication. It is also capable of reading analogue signals and outputting pulse width modulated signals that can be easily converted to an analogue output. These microcontrollers are compatible with the Arduino programming environment, which is widely used by the “maker” community so numerous resources exist to interface with off-the-shelf sensors, significantly reducing development time. The Arduino software is designed to be used by those who do not have a background in electronics, so it hides a lot of the intricacies of developing embedded code from the user. This simplified setup will allow 3rd party users who wish to use the AMOEBA to more effectively implement their own sensors. The Arduino programming environment is also useful because it works across a range of ATMEGA microcontrollers; this means that the code designed for the ATEMGA 328P can easily be used on a more powerful ATMEGA microcontroller if a module requires it.A CAT5 cable is used for communication between the modules and to supply power. Of the eight wires available in a CAT5 cable three are used for ground, two are used for power, two are used for the CAN bus, and one is spare. If any module requires more than 0.5A then an external power supply can be connected to the device.There are four different types of AMOEBA modules that are connected to the bus: sensor modules, actuator modules, server modules and terminator modules. Sensor modules are modules that monitor system parameters, such as temperature. Actuator modules are modules that control a parameter, for example, a variable valve or a heater. The server module connects the AMOEBA system to a computer running the AMOEBA desktop client by a USB interface; it contains the main power supply and sets up the bus at the start of the experiment and can interface with up to 254 other modules. Finally, there is a sole terminator module used to terminate the AMOEBA bus. Each module on the AMOEBA system communicates using a standard protocol designed for the AMOEBA system, this means that any actuator module can process the data gathered by any sensor module. With this modular approach, the AMOEBA system can be quickly configured to suit any application.The AMOEBA system uses a CAN 2.0 bus that links all the modules together. The packets used are structured so that each module can communicate with any other module. This is achieved by using the first byte in each packet to designate the packet type. Each module also has its own unique address and a common shared address that can be used to send commands to other modules. The majority of the AMOEBA modules are either sensor or actuator modules, these are used to control or monitor various parameters. At the start of an experiment, each controller module on the bus is configured by the server module to monitor a certain sensor on the bus and the set point of that sensor. Once a start command is received the actuator module repeatedly polls the sensor module and adjusts its output accordingly. This continues until the actuator receives a stop command. We configured the AMOEBA system to work in a polled mode, it could easily be modified to operate in a triggered mode although this would not be required for environmental control since environmental control on cell culture does not require a fast response time.A computer running the AMOEBA desktop client connects to the server module by a USB cable. The AMOEBA software records the data gathered by the sensors and sets up the bus to control the environment. Once gathered, the data can be saved either into a specific AMOEBA XML file or to a character separated file that can be read using a customised script or any commercial spreadsheet software. For more complex experiments, the AMOEBA desktop client is also capable of running customised Python scripts specific to the experiment. 2 concentration and cell medium temperature. This was achieved by creating a controlled volume above the cells in which relative humidity and CO2 are controlled and by heating the cell dish to regulate cell medium temperature.Recreating incubator conditions on the end of a microbeam requires the control of 3 parameters: relative humidity, CO2 rich atmosphere can be seen in 2 within the shroud is read by a CO2 sensor ) and maintained (e.g. at 5%) by the variable valve module that controls the flow of CO2 from a gas cylinder. The humid air and CO2 mix within the environmental shroud to create a humid CO2 rich atmosphere.The processes used to create the humid, CO2 required to maintain 5% atmosphere is very low (0.05 l per hour); but if desired it would be possible to design and build a sealed chamber if desired.An AMOEBA environmental shroud  is used 3. To construct it uses a total volume of 157 cm3 of material to print with 100% infill. It is compatible with cell dishes with a diameter below 86 mm and could be easily be redesigned to cater for other plating systems if necessary without having to change any of the electronics.This work uses a shroud designed around the RARAF microbeam endstation to validate the AMOEBA ,10. The 2 monitoring, and mass flow analyser. The actuator modules are a variable valve, under dish heater, heated pipes and a solenoid valve module. There is also a sole server and terminator module. Collectively these modules cost $2380 to construct including the cost of the sensors.Since a variety of factors need to be controlled using the RARAF AMOEBA, it uses a variety of self-contained independent modules each serving their own purposes. The sensor modules are thermocouples, relative humidity and COthermocouple module is a bank of eight thermocouples, where each channel contains its own MAX31855KASA+  thermocouple preamplifier, which communicates with the microcontroller using an SPI bus with a maximum sensor refresh time of 100 ms. Although eight thermocouples are available the RARAF AMOEBA configuration uses four thermocouples: two are used to monitor the temperature of the compressed air, one is used to measure cell media temperature, and the fourth is used to measure the temperature of the water in the humidifier. The two thermocouples used to measure the temperature of the compressed air were adapted so that they can fit inside the airline by sealing it inside a Wye Push to Connect gas fitting using Permapoxy 5 min General Epoxy Resin . The Thermocouples module cost $120 to build and the thermocouples cost $90. It has a mass of 0.477 kg.The relative humidity and CO2monitoring is a single module with two HYT 271 relative humidity and temperature probes  and a Gas Sensing Solutions SprintIR 100% CO2 sensor. The HYT 271 communicate with the module’s microcontroller via an I2C bus with a 1 s sampling time. The CO2 sensor communicates with the AMOEBA microcontroller using UART  serial communication with a 50 ms sampling time. During most AMOEBA experiments only one relative humidity and the ambient temperature sensor is used since the relative humidity and ambient temperature is only measured inside of the AMOEBA chamber. The module cost $45 to build, each of the Relative Humidity and Temperature sensors costs $36 each and the CO2 sensor costs $155. The module has a mass of 0.266 kg.The variable valve module is used to control the flow rate of the CO2 gas. The module uses an FSV12  electronically controlled proportional valve that allows a maximum flow rate of 13LPM (216.7 cm3 s−1) of air. The behaviour of the valve is controlled using a Proportional-Integral-Derivative (PID) control algorithm on the module’s microcontroller that can be tuned by the user depending on the application [The under dish heater module is a MOSFET switch that controls the current going through an under dish heater, created by wrapping nichrome wire around the brass cap protecting the microbeam exit window. The under dish heater module cost $43 to construct. The mass of the module is 0.218 kg.In microbeam experiments, the dish containing cells is often in close proximity to a metallic section of beam pipe that can serve as a major heat sink. The heated pipe module heats air to create the humid environment necessary for the cells. Three 1 m sections of heated pipe were created by wrapping nichrome wire around 0.25-inch outer diameter PTFE (Polytetrafluoroethylene) pipe. The pipe is heated by passing an electrically controlled current through the wire that surrounds the pipe. Each section of heated pipe is attached to a heated pipe control board, which contains a MOSFET switch to control the current through the Nifethal 70 wire by pulse width modulation and an onboard MAX31855KASA+  thermocouple preamplifier to monitor the external temperature of the pipe. Each section of heated pipe cost $40 to build. Each heated pipe module has a mass of 160 g.The solenoid valve module for gas control consists of three solenoid valves controlled using the digital output pins on the microcontroller and they are driven using MOSFET switches. The valves are used to turn the CO2 and compressed airflow on at the start of an experiment and to turn them off at the end of the experiment. The three valves cost $200 and the electronics to control them cost $50. The mass of the module is 1.239 kg.The server module is used to interface between the AMOEBA software and the AMOEBA modules. The server module contains an Arduino Uno that was connected to additional CAN circuitry so it could interface with the AMOEBA modules. It also contained a 15 V 10A power supply that provides the power for all the AMOEBA modules. The cost of the server module was $125. The mass of the server module is 1.432 kg.The terminator module that contains a resistor that is used to terminate the bus. The terminator modules cost $15 to construct. The terminator has a mass of 0.1 kg.CAN buses need to be terminated to achieve their best operation; as a result, the AMOEBA system has a The AMOEBA system is also capable of interfacing with pre-existing lab equipment through the use of communication protocols such as GPIB, RS-232, USB or Ethernet. An example of this is the RS-232 AMOEBA module. It was created to interface with the JENCO 6230N handheld metre that connects to a range of ion sensors that can be used to measure factors such as dissolved oxygen or pH. This can be achieved due to the flexibility of the ATMEGA328P microprocessors used by each module and a large amount of freely available code already available online.mass flow analyser module is available to measure gas flow although it is not often used because the flow rates it can measure is too low. A Dwyer Instruments GFM-1108 Gas Mass Flow Metre  is used and it outputs a 0–5 V signal that is read by the module’s microcontroller and converted to a flow rate. The GFM-1108 is capable of measuring airflow up to 2 l per Minute (LPM) (33.3 cm3 s−1). It is also capable of measuring other gases using the supplied calibration factor. The mass flow analyser cost $785 and the control electronics cost $40 to produce. The module had a mass of 930 g.A 2 and 99% ± 1% relative humidity) environment. To test the stability of the AMOEBA system, an AMOEBA experiment was set up using a cell dish containing 2 ml of cell media and run for 6 h; the dish was removed from the environmental shroud every 2 h to measure the mass of the cell dish. The second step was to test its ability to sustain cells within the device. To test this, cells were observed in the AMOEBA system for 36 h, with an image being taken every 10 min.The application of the AMOEBA system for environmental control on the RARAF VMBs was tested in two steps. The first step was to test the ability of the AMOEBA system to create and sustain an incubator like   on the polypropylene sheet and cells were added on top of this.HeLa  cells were first grown in DMEM  supplemented with 10% Foetal Bovine Serum  and 1% of Pen/Strep  using T75 culture flasks in a 5% COprotocol . The mic−1 Cell-Tak solution and 10 μl of 0.2 M sodium bicarbonate solution  into to the well. The dish was then placed in a Series 8000 Direct-Heat CO2 Incubator (Thermo Fisher Scientific) at 37 °C and 5% CO2 for 30 min. Once removed from the incubator the dish was rinsed 3 times with 5 ml of sterile phosphate buffered saline solution. After this, the dishes can be stored for up to two weeks in a refrigerator.The dish wells coated with Cell-Tak were coated by putting 10 μl of 100 μg ml5cell/ml in a 1–3 μl volume droplet. Once placed into the cell dish the well was covered with a 22 mm by 22 mm autoclaved glass coverslip  and the cell dish was placed into the incubator for half an hour. Upon removal from the incubator, the coverslip was taken off and 5 ml of DMEM was added to the cell dish, before returning it to the incubator at 37 °C and 5% CO2. The dish was left in the incubator for a minimum of 3 h before being placed onto the microbeam endstation for observation.Cells were plated by placing a droplet containing 100–300 cells into the Cell-Tak coated well of the dish at a density of 102 with the stain for 30 min and then the medium was replaced with fresh DMEM supplemented with 10% Foetal Bovine Serum and 1% Pen/Strep prior to observation.To observe the cells using the RARAF endstation, cells were stained using Hoechst 33342  at a concentration of 100 nM in DMEM . The celA detailed description of the microscope system used on Microbeam II, including details on the hardware and software systems used to control the endstation and camera can be found in Bigelow et al. . It cons2 5% ± 0.5% and relative humidity 99.5% ± 0.5%. The spikes seen every two hours in the relative humidity, cell dish temperature and ambient CO2 are caused by the removal of the cell dish from the system so that it can be weighed and would not occur if the system was left for normal operation.2 within the chamber initially peaked at 8%, settling to 5% within 145 s after inserting the dish. When the dish was removed from the chamber to weigh it, ambient CO2 spiked to a maximum of 14.2% after the chamber was closed. The mean settling time of the system after being disturbed was 371 s (6.19 min), the signal oscillated within the acceptable boundaries at an approximate frequency of 8mHz. Over the 6 h period, the mean CO2 value was 4.994% and the RMS Error of the CO2 signal was 0.370%, including the spikes caused by removing the cell dish. In the times between the spikes, the means and RMS error values can be seen in When first inserted into the AMOEBA the cell dish temperature reached 37 °C within 6 min, reaching a maximal overshoot temperature of 40 °C, with the dish temperature above 38 °C for 121 s. The same occurred when the dish was replaced after being weighed. The dish temperature spiked to a maximum of 41 °C, these temperatures returned back to the acceptable limits within 130 s. The mean dish temperature over the 6 h period was 36.840 °C and the RMS Error was 2.754 °C including the spikes experienced when the dish was removed the temperature oscillated within the acceptable boundaries at a frequency of 10.2 mHz. The CO2. Once the environment was established during the 6 h period, the relative humidity did not drop below 99% other than the times the system was disturbed to measure the mass of the cell dish.During the undisturbed periods of operation when the system exceeded the bounds the AMOEBA successfully restored the parameter to the acceptable limits within a maximum of 1.2 min for cell dish temperature and 36 s for ambient CO−1 and the maximum mass loss was 0.067 g h−1. The mean mass loss was 6 mg h−1 with a variance of 0.002 g h−1. The control experiments shown in −1 with a variance of 0.0002 g h−1. As a result the AMOEBA system has improved the loss of medium due to evaporation by a factor of 94.5 times. If the mean evaporation rate was sustained over a 12 h period the dish would lose 72 mg and over a 36 h period the dish would lose 216 mg.Two other similar experiments were carried out using the AMOEBA; the results of these experiments can be seen in A dish of Hoechst 33342 stained HeLa cells was examined under the microscope for 36 h; during the 36-h experiment, the AMOEBA regulated the environment at incubator conditions and an image was taken every 10 min. Cell division was successfully observed with two examples are highlighted in The AMOEBA was designed to be a modular environmental control system that would be flexible enough to be applied to many situations beyond the area of environmental control. The single bus design allows modules to be easily added to and removed from the AMOEBA system. Its flexibility is also attested to by two facts: firstly that the AMOEBA system has worked on two beamlines, the Permanent Magnet Microbeam and Microbeam 2, at the RARAF facility, secondly, the AMOEBA system is in use in two VMB laboratories each with their own unique environmental setups(RARAF at Columbia University and at the Surrey Ion Beam Centre at Surrey University) .The AMOEBA system is portable and can be moved between systems with little effort. The AMOEBA system can be adapted to almost any microscope since it would only require a new environmental shroud rather than require the whole system to be redesigned. If another parameter needs to be controlled, such as oxygen, the sensor would simply require a new module to be developed before adding it to the system.Another testimony of the power and flexibility of the AMOEBA system is its ability to use standard communication protocols, this is shown by the RS-232 module that is able to interface with a Jenco 6230N . This feature of the AMOEBA system means that items of lab equipment with standard interface protocols such as USB, RS232 or GPIB, can be used as part of the AMOEBA control system.2 levels stayed within the desired bounds of 5% ± 0.5% over the whole 6 h period; when it did briefly stray from outside this range it returned within 2 min.In this work, we demonstrated that the AMOEBA is capable of creating an incubator-like environment for extended live cell experiments. During the 6 h test, the temperature of the cell media stayed within the desired temperature bounds (37 °C ±1 °C) once it had reached its desired temperature. The four occasions when the temperature did exceed these bounds the temperature of the cell dish returned to desired range within 2 min. Likewise, the CO2 values briefly overshot their desired set point, however, these parameters settled within minutes. Of these two values the temperature is the most critical for cell health, but the temperature spike is too short to damage the cells [When starting the system the temperature and the COhe cells . This ov2 initially dropped to below 1% due to the chamber being opened, the relative humidity also dropped for the same reason but because there was still a net flow of humid air and CO2 into the chamber, and the chamber was not opened for long enough for more CO2 or humid air to diffuse out of the chamber, as a result the relative humidity and CO2 did not drop to ambient levels. The temperature measured by the thermocouple measuring cell medium temperature dropped when the cell dish was being weighed because when the cell dish was removed to be weighed the dish and the thermocouple measured the temperature of the air in the chamber. When the dish was replaced the CO2 experienced a spike because the variable valve had opened more to compensate for the drop in CO2, resulting in an increased flow of CO2 in the shroud causing a spike when the chamber was closed.When the cell dish was removed, the COWe used the AMOEBA to observe cells for a 36 h period including instances of cell division. The unique culture conditions required to irradiate cells VMB, likely reduced and delayed cell division during this time. This delay is likely to have been caused by the polypropylene substrate, this can be deduced because Hoechst 33342 has previously been demonstrated to not be toxic to HeLa cells , and theThe AMOEBA system provides a flexible low-cost alternative to commercial environmental control systems. Unlike commercial systems, its modular design means it can be rearranged and within minutes be ready for almost any task that is required of it. The AMOEBA system opens up the potential of VMBs to irradiate and observe cells for extended periods of time. This system will enable microbeams to carry out experiments that were not possible previously, paving the way for microbeams to be able to assess cell motility, explore the effects of radiation over several cell divisions and to observe mutagenic effects of particle radiation without having to remove the cells from the microbeam.Supplemental