Nanopuller-open data acquisition platform for AFM force spectroscopy experiments

Ultramicroscopy 164 (2016) 17–23

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Nanopuller-open data acquisition platform for AFM force spectroscopy experiments Konrad Pawlak 1, Janusz Strzelecki n,1 Institute of Physics, Faculty of Physics, Astronomy and Informatics, Nicolaus Copernicus University, Grudziadzka 5, 87-100 Torun, Poland

art ic l e i nf o

a b s t r a c t

Article history: Received 2 June 2015 Received in revised form 5 January 2016 Accepted 22 January 2016 Available online 10 March 2016

Atomic Force Microscope (AFM) is a widely used tool in force spectroscopy studies. Presently, this instrument is accessible from numerous vendors, albeit commercial solutions are expensive and almost always hardware and software closed. Approaches for open setups were published, as with modern low cost and readily available piezoelectric actuators, data acquisition interfaces and optoelectronic components building such force spectroscopy AFM is relatively easy. However, suitable software to control such laboratory made instrument was not released. Developing it in the lab requires significant time and effort. Our Nanopuller software described in this paper is intended to eliminate this obstacle. With only minimum adjustments this program can be used to control and acquire data with any suitable National Instruments universal digital/analog interface and piezoelectric actuator analog controller, giving significant freedom and flexibility in designing force spectroscopy experiment. Since the full code, written in a graphical LabVIEW environment is available, our Nanopuller can be easily customized. In this paper we describe the program and test its performance in controlling different setups. Successful and accurate force curve acquisition for standard samples (single molecules of I27O reference titin polyprotein and DNA as well as red blood cells) is shown. & 2016 Elsevier B.V. All rights reserved.

Keywords: Atomic force microscope Single molecule experiments Proteins DNA Force spectroscopy Nanoindentation

1. Introduction Our understanding of nanobiomechanics significantly increased during last 20 years [1]. In those studies, involving e.g. single molecule pulling [2], single bond breaking [3], single cell [4] or tissue [5,6] indentation, and detection of small movement in organisms [7], which are generally known as force spectroscopy, an Atomic Force Microscope (AFM) is a widely used tool [8–10]. Presently it is accessible from numerous vendors. Commercial solutions, while giving a standard basis for repeatable experiments are almost always source closed. Thus approaches for open, good quality force spectroscopy AFM were made [11–14]. Such setup can be easily assembled with universal piezoelectric actuators, optoelectronic components and flexible data acquisition interfaces which are low cost and readily available. However, suitable software to control such instrument was developed by each group separately and was not released. Developing such software from the beginning is time consuming and can be a real challenge for Life Science oriented investigators. Additionally, such custom solutions lack repeatability given by commercial setups and results from so many different platforms may be often difficult to adapt n

Corresponding author. 1 Both Authors contributed equally to this paper.

http://dx.doi.org/10.1016/j.ultramic.2016.01.008 0304-3991/& 2016 Elsevier B.V. All rights reserved.

and replicate by research community. Lack of a common platform to test custom made setups significantly hinders further evolution and wider application of force spectroscopy technique. Here we propose a solution by providing a Nanopuller - a software, which is intended to operate with any laboratory made AFM force spectroscopy setup steered with a National Instruments universal DAQ interface [11–14]. By providing the open software platform a true potential of using this interface for AFM force spectroscopy can be seen. The fact that NI-DAQ interface control/ acquisition is performed solely with analog voltage signals gives an enormous versatility, as virtually any piezoactuator controller module from many vendors available today can be used. Thus different setup geometries to perform various AFM force spectroscopy experiments are possible. At the same time the Nanopuller adjustment to different hardware configurations requires only minimum effort, the software can be used immediately after installation and no additional programming is required. It is however open sourced and ready to be modified and adjusted for specific experimental requirements. Since it is written in graphical LabVIEW environment such customization would be easy also for less computer science oriented researchers. The Nanopuller is particularly suited to perform force spectroscopy experiments manual mode, since it is greatly enhanced in Nanopuller by supporting a popular video game controller (e.g. DualShocks 2). In this paper we show architecture of Nanopuller software and

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its application on two different setup architectures and evaluate its performance on three standard biological samples with well researched mechanical properties: polyprotein construct, double stranded DNA and red blood cells.

2. Materials and methods 2.1. Construction of force spectroscopy AFM Construction of AFM force spectroscopy setup was already described [11–14], thus here only general considerations are specified with further details included in Nanopuller instruction manual. The main feature of force spectroscopy AFM suitable to be operated with Nanopuller software is utilization of National Instruments universal data acquisition card. It allows acquisition of data and instrumentation control with just few I/O channels for analog voltage signals (within 710 V range). Thus, an enormous variety of equipment can be used without any modification of primary Nanopuller code. Only initial minor adjustment (specifying a manufacturer given sensitivity of a piezoelectric closed loop piezoelectric actuator and DAQ channels used) is necessary when using the program for particular setup and equipment. A force spectroscopy AFM can be built in many configurations depending on experimental requirements. It can be a standalone instrument, suitable for pulling single molecules picked on uniform samples. Such setup, shown on Fig. 2a was built following design protocol published in [11] and was made with closed loop strain gauge Physik Instrumente P-841S.10 piezoactuator steered through E-509.S1 and E-505.00 controller modules by NI PCI-6251 interface. Alternatively, in applications, in which cantilever tip has to be placed in a particular spot on sample (like nanoindentation),

AFM must be capable to be combined with an optical/fluorescence microscope. Fig. 2b shows such configuration. It was built following guidelines published in [13], using PA 16/14 closed loop strain gauge actuator with 12V40 SG OEM analog controller from PiezoJena operated by NI-6221 interface. In both setups coarse tip vs. sample XY and Z movement was possible with mechanical translation stages and micrometer screws. The closed loop piezoelectric actuator allows cantilever vs. sample surface approach/retract movement. The NI-DAQ is generating a voltage ramp necessary to move piezoactuator forward and backward and is simultaneously acquiring the actual extension signal from the piezoactuator strain gauge position sensor. A force vs. distance curve is obtained by combining calibrated cantilever deflection signal with closed loop piezoelectric actuator extension signal (Fig. 1). The deflection is measured with either commercial or homemade AFM head. It is assumed, that this signal [(A þB)-(C þD)]/(A þB þ Cþ D) and a total laser spot brightness A þB þC þD are calculated using an external analog module [15] and both are acquired with specified NI-DAQ analog input channels. 2.2. AFM force spectroscopy test sample preparation I27O AFM reference polyprotein was obtained from Athena Environmental Sciences Company and diluted to 50 mg/ml concentration with PBS buffer (Sigma Aldrich P4417). A drop of such solution was introduced on gold evaporated glass substrate and incubated overnight. Sample was gently washed with PBS buffer before being secured in AFM for experimental session. MLCT-C AFM cantilevers from Bruker were used. For DNA pulling experiments we used the pUC18 plasmid (Fermentas, SD0051), linearized with the EcoRI enzyme

Fig. 1. General schematics of Atomic Force Microscope force spectroscopy experiment performed with Nanopuller: an open control and data acquisition software. A single polyprotein molecule unfolding experiment with corresponding hardware, a signal routing and software modules is presented. A force vs distance curve is obtained by simultaneous generation of voltage ramp on output channel (controlling extension/contraction of piezoactuator) and acquisition of feedback signal from extension sensor and cantilever bending by NI-DAQ interface.

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(Fermentas, ER0271). DNA was suspended in TE buffer (150 mM NaCl, 10 mM Tris (pH 8.0) and 1 mM EDTA) at a 50 mg/ml concentration. Before each experiment a drop of this solution was incubated overnight on a gold evaporated glass substrate. MLCT-C AFM cantilevers from Bruker were used. Red blood cells (RBC) acquired from healthy donors were suspended in 0.9% NaCl saline solution. A substrate for fixing erythrocytes was prepared by placing a drop of poly-L-lysine (Sigma Aldrich P8920) was left on glass bottom Petri dish to evaporate. RBC suspension was pipetted on the spot and incubated for one hour and vigorously washed with saline solution to remove any weakly bound cells. MLCT-D AFM cantilevers from Bruker were used. 2.3. Nanopuller program description A basic description necessary to understand program operation follows. A full explanation is present in operation manual, that can be downloaded from Nanopuller download page. Nanopuller software was created using National Instruments

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LabVIEW 2013 environment, and was compiled for Windows XP SP3 running under x86 or AMD64 processors. LabVIEW is a software development environment that contains numerous ready components, particularly suited for test, measurement, or control application. An intuitive, flowchart-like dataflow programming model naturally represents data-driven applications with timing and parallel execution. LabVIEW is also designed to work seamlessly with all National Instruments data acquisition devices. Thus it is perfectly suited to be employed in scientific experiment control. Source code of Nanopuller is using a state machine approach. It can be divided into four main parts (Fig. 2). The main loop, called idle state (Fig. 2a) is responsible for preparation of atomic force microscope setup for user action. This loop works in 20 millisecond intervals, refreshing DAQ card ports, both input and output. In the upper left corner of GUI (Supplementary Figure) real time previews for monitoring crucial values are placed. Those important parameters: cantilever bending, total sum of photodiode quadrants signals and current, piezoactuator elongation are thus monitored in real time. A cantilever bending signal, displayed both

Fig. 2. Force spectroscopy AFM setups used in Nanopuller testing. Setup shown on (a) is suitable for uniform samples, like molecular solutions, where optical visualization is not necessary. Setup (b), having an open optical path, can be placed on an inverted microscope, allowing placement of AFM tip on particular spot and targeting single red blood cells.

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as a numerical and a dynamically scaled scope, is used for coarse approaching with a micrometer screw, when a tip-surface contact event can be seen as a rapid voltage jump. It is also vital in centering the laser light on photodiode quadrants, as the value would approach zero. A total signal from photodiode quadrants allows for optimal laser spot positioning on cantilever. Piezo extension indicator gives information about total piezo extension, introduced both by signal from the DAQ interface and the controller offset knob. The main graph window of GUI shows an example acquired force curve for I27O protein unfolding. After the curve is obtained, quick measurements of forces and lengths between points of interest can be made with cursors (cursor sequence is not relevant). Cursors are also used when acquiring a sensitivity of a particular photodiode and cantilever configuration (commonly known as a “slope”) during calibration. Graph orientation can be transposed in advanced settings according to user preferences suitably for single molecule puling or indentation experiments. This module also contains all the necessary controls required for conducting typical force spectroscopy measurements in velocity clamp mode. Manual control of piezoactuator starting extension (Z-scanstart) and total extension during force spectroscopy scan (Z-scansize) is executed by large sliders. Execution of automatic experiment can also be triggered. Idle loop monitors safe values for experiment, and disables start triggers if unsafe configuration is detected. It is also responsible for slight correction of piezoactuator starting position in automatic mode. Apart from mouse and keyboard also a gamepad device can be enabled and used for ergonomic Nanopuller operation. To avoid any input collision or delay, the gamepad uses its own threads to acquire user input, and change corresponding values accordingly, during the standard 20 ms loop iteration. Second module of Nanopuller software is responsible for I/O configuration (Fig. 2b). It allows connecting signals with desired channels on National Instruments DAQ interface. Nanopuller is using three analog inputs (cantilever deflection, total photodiode sum, and piezoactuator extension sensor) and one analog output (voltage ramp for piezoactuator). Once I/O terminals are selected, the configuration can be stored as ASCII file, and loaded automatically during the next executions of the program. During the data acquisition input is synchronized with output clock. It is very important to run Nanopuller with system administrator privileges, since configuration files are stored in the installation location, which may be protected from unauthorized write operations. User can also configure game controller button assignment. Third (Fig. 2c) Nanopuller module is responsible for cantilever calibration with a thermal tune method [16–19]. In this window spectrum of cantilever thermal vibrations can be obtained. Clicking Start sampling triggers cantilever bending signal acquisition. The resulting spectrum range is limited by Sampling frequency slider, and it cannot exceed limitations posed by the NI-DAQ interface used. By increasing the number of repetitive averaged acquisitions we can get a smoother and more accurate spectrum. Next step is setting the interval of the resonance peak integral using built in cursors (from gray to black). By clicking Integrate button, a power peak value will be calculated and displayed. A procedure can be repeated if the spectrum acquired is not satisfactory by clicking Restart. Otherwise, the window is closed and we return to the main GUI. Afterwards cantilever sensitivity should be obtained by approaching surface, acquiring a force curve on a hard substrate and placing plot cursors on contact portion of obtained plot. Initially graph is scaled in Volts vs nanometers, and is altered to piconewtons vs. nanometers once the calibration procedure is done. This thermal tune Nanopuller module can also be used to revive older Atomic Force Microscopes, which are still suitable for high

quality research and only lack the ability to calibrate cantilevers, such as a still widely used Veeco Multimode III. In such case cantilever deflection can be fed to NI-DAQ by dedicated signal access equipment. Final Nanopuller part, responsible for starting force curve acquisition and saving data remains in idle mode until it is triggered with user actions (Fig. 2d). Start button enables the piezoactuator Z-scan and acquires a single force curve, with predicted time of this operation shown in controls menu. After the process is completed, the software enters idle state again, the recently acquired force curve is displayed on main graph window and controls are restarted. If a force curve is valuable, then a Save button can be clicked, which triggers saving with appropriate headers to the ASCII file (with or without cantilever bending deflection correction) as well as to LabVIEW datalog file or PUNIAS analysis software (Protein Unfolding and Nano-Indentation Analysis Software, http://punias. voila.net/) [20] compatible ASCII. The indicator prompts if current curve was saved. Save file formats can be selected and autosave mode can be enabled in the Advanced setting tab. Additionally right click with a mouse button on graph enables to capture of plot as image or raw data directly to clipboard. Output files containing force curves data can be stored in any directory chosen by the user, each file is labeled with system date, time and current save iteration. Compiled Nanopuller software, its source and support files as well as a comprehensive manual can be found on Sourceforge project site at: http://sourceforge.net/projects/nanopuller/ and on GitHub https://github.com/Czaper/Nanopuller. Since code of the software is published as open-source, under ISC license, the end user can compile it for its own setup, also using Linux and Mac versions of LabVIEW. A full version of LabVIEW is not necessary to use compiled Nanopuller software and only a prior installation of free LabVIEW Run-Time Engine for Windows is needed. All required DAQ drivers are included in Nanopuller installation package. (Fig. 3)

3. Results and discussion We used Nanopuller operated AFM setups for single molecule (I270 and DNA) pulling and erythrocyte indentation experiments. A commercial I27O reference protein was chosen for primary testing of Nanopuller operated setups as titin I27 module is well researched and widely employed for single molecule force spectroscopy experiments [21–24]. This polyprotein stretching was successfully performed in automatic as well as manual mode. Fig. 4a shows typical I27 unfolding force curve, with characteristic repetitive saw-tooth like pattern. Nanopuller software allowed us to perform pulling experiment at different speeds (100, 400, 1000 and 5000 nm/s). Thus, a dynamic force spectroscopy test of Nanopuller controlled setup could be performed. The inset graph shows fitting with an analytical expression in order to obtain unfolding rate ku ¼0.00032 s  1 and unfolding distance xu ¼0.24 nm constants, which are within agreement with protein manufacturers note (0.00033 s  1 and 0.24 nm respectively) [24]. This indicates that both piezoactuator control as well as force and extension measurement performed with Nanopuller operated setup is satisfactory. To test the Nanopuller further in applications, where automatic force curve acquisition is not suitable we performed test of single molecule DNA force spectroscopy (Fig. 4b). A careful single molecule picking and gradual unfolding after slight distancing from the substrate surface is necessary in order to obtain a significant number of single molecule force curves showing large deformations. As the program supports use of ergonomic game controllers

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Fig. 3. Block diagram of Nanopuller software. A code idea of Nanopuller is based on a simple state machine. Program activities can be divided on four main parts:. (1) Idle state, where channels are constantly updated, and program is awaiting for user interaction, such as data acquisition parameters (2) Input/Output configurator, where user can define I/O DAQ ports, hardware limitations, and assign buttons for gamepad input device. (3) Calibration module, dedicated for acquiring and integrating cantilever vibration spectra for thermal tune spring constant derivation. (4) Data acquisition module, working in either in manual or automatic mode.

such manual single molecule pulling is very easy compared to mouse and keyboard operation. Applying Nanopuller allowed us to stretch single double stranded DNA molecules with very high forces, up to the possible limits of covalent bonds. Fig. 4b shows examples of two such highly deformed double stranded DNA force curves. A characteristic overstretching plateau dependent on topological arrangement and rotational constraints of the stretched double helix can be observed. Based on average from 10 curves the overstretching force can be observed at 65.3 71.1 and 108.97 3.5 pN, which is in perfect agreement with previous studies [25–27] and corresponds to unconstrained and constrained double helix pulling. As overstretching transition appears precisely for those forces, DNA was already considered as a nanomechanical force standard [28]. Thus, those measurements indicate that Nanopuller operated setup can calibrate AFM cantilevers and measure forces with accuracy adequate for single molecule force spectroscopy. Manual operation of single molecule pulling allowed us to stretch DNA molecules far beyond typical pulling force range. This enabled us to discover a new, third mechanical transition in dsDNA, which can be seen as a small plateau at  1 nN on constrained DNA force curve, as we recently reported [29]. To evaluate Nanopuller operated setup in nanoindentation force spectroscopy we performed nanomechanical test of human erythrocyte stiffness. Red blood cells (RBC) were chosen as being

widely evaluated mechanically with diverse techniques [30]. In order to place cantilever tip precisely on selected red blood cell an inverted microscope based setup (Fig. 4c) was used. Nanopuller required only minor adjustment to adapt for this different AFM architecture and experiment type. Obtained 30 nanoindentation curves were fitted with Herz model (with spherical approximation of AFM tip) [31] in order to obtain Young modulus values. Results are shown in inset histogram. The calculated values are within range reported in previous research on RBC nanoindentation [4]. Thus we believe the Nanopuller operated setup can be easily adjusted for different AFM setups and successfully employed for deformation and force measurements also in nanoindentation experiments. We also successfully employed Nanopuller operated systems for evaluation of adhesive properties of caddisfly (Trichoptera) silk surface [32,33] and unfolding of native neural proteins [34–36]. This software platform can thus be considered a versatile tool in bionanomechanical research.

4. Conclusion We presented Nanopuller software allowing flexible arranging, control and data acquisition In AFM force spectroscopy

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Fig. 4. Examples of Nanopuller software experimental applications. A simple performance test of reference I27O polyprotein pulling is shown (a) with a pulling dynamics dependence displayed in the inset graph. Solid line corresponds to fitting an analytical solution to obtain I27 domains unfolding rate and unfolding distance constants. Manual force spectroscopy enables careful pulling of double stranded DNA, allowing examination of single molecule mechanics in very high force regime (b). Nanopuller controlled setup combined with optical microscope allows placing AFM tip in particular spot (c), enabling red blood cell indentation. Cell deformation curve is shown, with histogram of Young modulus obtained with Herz model fitting (gray solid line) in the inset plot.

experiment. We presented the program architecture and described the interface. The Nanopuller was tested by performing pulling experiments on single molecules of I27 titin polyprotein and DNA and erythrocyte nanoindentation. Two types of AFM instruments were used. Those tests show that Nanopuller operated setup can be

successfully used to perform accurate force spectroscopy measurements. We believe that Nanopuller software will bring standardized, high quality single molecule experiments to many laboratories and further popularize the technique in scientific community.

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Acknowledgment We thank Joanna Strzelecka from Medical Physics Department, Oncology Center, I. Romanowskiej 2, 85-796 Bydgoszcz, Poland for RBC experiment collaboration and experimental setup construction. We thank Szymon Topolewski for help with RBC elasticity measurements analysis. K. Pawlak and J. Strzelecki acknowledge the research Grant for young scientists 2055-F and 2058-F from Institute of Physics, Nicolaus Copernicus University. This work was supported by Polish National Science Center grant 2012/05/N/ST3/03178.

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Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ultramic.2016.01. 008.

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