AFM Studies and Surface Assisted Self-Assembly of DNA Nanostructures

TECHNISCHE UNIVERSITÄT MÜNCHEN

Fortgeschrittenenpraktikum für Physiker Experiment Guide

Enzo Kopperger, Ali Aghebat Rafat, October 9, 2015 Contents

1 Introduction 2

2 Theory 4 2.1 Atomic Force Microscopy ...... 4 2.1.1 Basic Principle ...... 4 2.1.2 Imaging Modes ...... 6 2.1.3 Substrate ...... 7 2.2 DNA, our Biopolymer for todays experiment ...... 8 2.2.1 Strand Hybridization ...... 9 2.2.2 DNA Nanostructures - DNA as a Building Block ...... 10 2.3 Order ...... 12

3 Experiment 14 3.1 AFM Operation Instructions ...... 14 3.1.1 The Instrument ...... 14 3.1.2 Central Parameters ...... 15 3.1.3 Reoccurring Procedures ...... 16 3.2 General Preparation ...... 19 3.3 Folding of DNA Origami ...... 19 3.4 Imaging of M13mp18 Genome ...... 19 3.5 Imaging of DNA Rectangles ...... 21 3.6 Surface Assisted Ordering by Close Packing ...... 22

4 Questions 24

1 1 Introduction

Nanotechnology is widely understood as a field that deals with the creation and characterization of functional structures with features of 1-100 nm size. Popular everyday-life examples for the applications of nanotechnology range from colloidal particles in sunscreen over "lotus-effect" car polish to computer chips architectures with features of less than 10 nm width. While these examples are all of inorganic nature, bionanotechnology deals with understanding and creation of nanoscale systems as found in living organisms. While bacteria and cells are sized in the micrometer scale (E.Coli: length ≈ 2 µm), they are composed of much smaller functional units (organelles, genome, proteins ...). These basic components’ sizes are on the typical length scale of nanotechnology. For example, a typical size for ribosomes is about 20 nm, F1 ATPase measures roughly 10 nm. Chemical reaction networks allow cells to sense and react in complex ways on external stimuli. Inspired by the complexity and potential of naturally occurring systems bionanotechnology seeks to replicate form and function on a synthetic basis. While small sized structures pose all kinds of challenges to design and fabrication techniques also analysis and imaging become more difficult when coming closer to the length scale of single molecules. This makes the right choice of imaging methods all the more important. Imaging techniques can be divided into three major categories: optical microscopy, electron microscopy and scanning probe microscopy. Classic optical microscopy is limited to the Abbe diffraction limit, which states that two light λ emitting spots with distance d can not be resolved if d . 2 . This fundamental limitation to resolution can be overcome by super-resolution microscopy techniques like STORM, PALM and STEDM1. These methods also caught public attention with 2014’s Nobel Prize in Chemistry. Although those methods improved drastically over the last decades, spacial and temporal resolution is still insufficient for many applications. The fact that not the structure itself but strategically placed fluorescent dye molecules are imaged might also pose a problem for experiments. Typical parameters of optical super-resolution microscopy are a lateral resolution of 10 - 50nm and a time resolution of several seconds to minutes per frame. A more direct image can be constructed via electron microscopy. Here the sample is either scanned (SEM) or transilluminated (TEM) by a focused electron beam. As an electron beam’s wavelength is much shorter than that of visible light, it allows resolution reaching down to the single molecule level. Not only individually marked spots but the whole structure can be visualized2. A disadvantage of this technology is the high price of electron microscopes and the fact that imaging usually takes place in

1Stochastic Optical Reconstruction Microscopy / Photoactivated Localization Microscopy / Stimulated Emission Depletion Microscopy 2If you are interested electron microscopy, we can recommend the experiment "Strukturelle DNA Nanotechnologie" next door.

2 vacuum. Imaging under vacuum conditions requires the samples to be either dried or frozen; hence dead. In a biological context it is favorable to analyze structures as close to their native environment as possible. A Scanning Probe Microscope (SPM) relies on direct physical ("near field") proximity between the sample surface and a sharp tip. The first microscope of this category, the Scanning Tunneling Microscope, was presented 1981. Here, a voltage between tip and sample is applied while the tip moves over the surface. The electron tunneling current between tip and sample is strongly dependent on the tip-surface distance and, when measured, can be used to reconstruct an image of the analyzed surface. While there is a vast diversity of imaging techniques, which utilize different types of surface-probe interactions the atomic force microscope is most universally used. An image can be reconstructed by the tip "feeling" the surface while scanning along each pixel of the image. A more detailed view on tips "feeling" surfaces will be given in the next section.

3 2 Theory

2.1 Atomic Force Microscopy

2.1.1 Basic Principle

four quadrant diode laser diode

chip z-piezo chip

cantilever x,y-piezo tip (a) Schematic setup of an atomic force microscope. (b) Reﬂection of the laser beam on top of the cantilever.

Figure 2.1

Figure 2.2: Scanning electron micrographs of typical AFM probes. Left: Bruker SCANASYST-AIR. Cantilever length ≈ 115 µm. Right: Olympus AC-40TS. Cantilever length ≈ 40 µm. When new, the tip radius of both probes is about 2 nm. Photos: Bruker / Asylum Research Probe Stores [1, 2].

Figure 2.1a illustrates the basic principle of an atomic force microscope. While the tip (on the bottom of a cantilever) scans the surface it will react according to the interaction it "feels". These reactions however are very small movements. These movements can be ampliﬁed for measurement by exploiting the light pointer principle: A laser is focused onto the back of the cantilever and reﬂected to hit the center of a four quadrant photo diode (PD), which tracks the spot position. Small changes in the cantilever’s angle will translate into large changes in the laser spot position on the diode. For the instrument used in this experiment, rather than moving the tip itself along the specimen, the tip

4 2.1 Atomic Force Microscopy is fixed and relative movement between tip an sample is realized by placing the sample on a finely positionable stage. High precision positioning at high rates is reached with piezoelectric crystals. Piezoelectric material contracts or extends with high accuracy according to an externally applied voltage. This allows to move the sample in all three dimensions with (ideally) subatomic precision. Figure 2.1a gives an impression of typical probes that are used to scan the surface. The very small and pointy tip is positioned at the end of a cantilever. The cantilever itself is again connected to a so-called chip. The purpose of the chip is solely to be big enough to be handled by the operator so it can be fixed in the machine. The properties (size, shape, stiffness) of the cantilever determine how the surface-tip interaction translates into a cantilever movement and how much force and disturbance will be applied to the sample. The small and sharp tip under the cantilever is the part that is actually interacting with the surface. It is intuitive that a high resolution image is only possible with a very pointy tip. How exactly the tip shape is connected to the resulting image will be explained in the next section. A unit of tip and cantilever mounted to a chip is called probe. Depending on the application (e.g. scanning in air or liquid, soft or hard sample, flat or rough surface, desired scan area and speed) there is a large variety of probes available.

Atomic Force The interaction between tip and surface is a superposition of various interactions summed up as "atomic force", the interaction atoms are experienced when they are brought to very short distances. This potential is usually described as Lennard-Jones potential depending on the distance r with minimum potential ε at distance rm. r r V = ε[( m )12 − 2( m )6] LJ r r

The potential well with its minimum at r = rm is the result of a superposition of attractive van- der-Waals forces ∼ r−6 and a repulsion of the atom’s electron orbitals ∼ r−12. Depending on the imaging mode the tip is either kept in the attractive regime (green) or repulsive regime (red). In any case it is important to be conscious about which regime is currently used for imaging.

repulsive ~r-12 tip artifact surface Measured Surface Signal scan scan r direction direction Tip attractive

~ -r-6 Surface

(a) Schematic plot of Lennard- (b) Inﬂuence of the tip shape on the resulting image. The measured Jones potential. surface signal is superimposed with the tip geometry. Mathe- matically, this can be described as a convolution (dt. Faltung) of two functions describing surface and tip.

Figure 2.3

5 2 Theory

Tip Shape and Image As mentioned above, the tip shape has a strong influence on the recorded image. As can be seen in figure 2.2, real AFM probes do not have a perfectly sharp tip. Depending on the specific model they have a characteristic geometry and tip radius. The probes we use in today’s experiment are conic and have a tip radius of 2-10 nm. When the surface is scanned not only the outmost part, but the whole tip is interacting with the surface according to the Lennard-Jones potential. This can lead to imaging artifacts as illustrated in figure 2.3b.

2.1.2 Imaging Modes

DC Mode The most intuitive approach for imaging is to use the AFM tip like the needle of a turn table. This is also called Constant Height Mode . Here the tip is brought to the repulsive regime, and the steep ∼ r−12 incline causes the tip-surface distance to be largely constant even if the contact pressure varies. Observing the laser spot movements on the photo diode while scanning allows to reconstruct a hight map of the surface. The downside of this approach is, that cantilever calibration prior to each measurement is necessary to convert the laser spot movements into a z-length scale. Furthermore, the force which is applied to the sample varies with the hight of the surface features. A rough surface might bend the cantilever excessively, which results in sample or cantilever breakage. For this reason, usually the Constant Force Mode is the preferred method. Here the tip and sample are brought close enough to induce a cantilever deﬂection. While scanning, a feedback mechanism adjusts the z-piezo (which controls the sample elevation) with the aim to always keep the laser spot centered on the PD. This way, the tip-surface interaction is kept on a constant level. Recording the z-piezo motion allows to reconstruct a hight map of the surface. This approach allows scanning of rougher surfaces as the z-length scale is only limited by the range of the z-piezo. Quality of the feedback mechanism determines the maximum scan speed. As the tip is dragged along the surface, both experience lateral forces. While this is acceptable for solid state samples, it poses a great issue for soft surfaces as found in biological samples. Here the tip might push the surface features out of the way, which makes imaging impossible.

Tapping Mode The issue of lateral forces is overcome by imaging with an oscillating cantilever. In Tapping Mode AFM1 the cantilever is oscillated near its resonance frequency (see ﬁgure 2.4). Tip-surface interaction induces variations in the cantilever’s oscillation amplitude, which the feedback loop aims to minimize. Monitoring the z-piezo motion allows to reconstruct the sample topography.

Imaging Medium All of the mentioned imaging techniques can be performed in vacuum, air or any non-corrosive liquid, although hydrodynamic distortions of the cantilever make liquid imaging generally more challenging. For biological applications, however the possibility of imaging in native buﬀer conditions is a strong advantage of AFM over other imaging techniques. In todays experiment we will start with imaging in air before moving on to the slightly more delicate imaging in buﬀer.

1Depending on the manufacturing company the terms Intermittent Contact, AC Mode or Tapping Mode are used.

6 2.1 Atomic Force Microscopy

Figure 2.4: Cutout from tune panel of NanoScope Software: Typical frequency spectrum of a AFM cantilever in liquid Tapping Mode. For imaging the cantilever is excited on the slope 6% left of the resonance peak. This way slight changes in the resonance in one direction will lead to an amplitude increase, while changes in the other direction lead to an amplitude decrease.

2.1.3 Substrate

If the species of interest is not a nano-patterned surface itself, it must be fixed on a substrate surface in order to image. Common substrates are flat surfaces like polished glass slides, silicon wavers or cleaved mica . Mica is a mineral with a layer-like crystal structure. With the help of scotch tape the top layer can be easily removed to retain a clean, atomically flat and negatively charged surface. The negative surface charge makes it a convenient substrate for imaging of DNA. At first glance an obvious questions arises: how does having a negative surface charge help to adhere negatively charged DNA? Shouldn’t the charges repel each other? Experimentally, however, it can be shown that DNA adheres strongly to mica when the buffer contains mainly divalent cations (such as Mg2+ or Ni2+). As shown below, this can be explained by specific geometric arrangement of the counter ions. We assume an unscreened case of two negatively charged plates representing mica and DNA. On each plate discrete binding sites repeat periodically. The occupation parameters φi and φj describe the binding of ions to the corresponding binding sites. For an ion bound to the ith binding side on th mica the occupation parameter equals φi = 1, while if the i binding side on mica is empty φi = 0. The binding sites on DNA are described by φj in the same way. The resulting electrostatic force can be described by summing over all binding sites [3]:

2 e d X (1 − zφi)(1 − zφj) Fc(d) = (2.1) ε (x2 + d2)3/2 i,j i,j with ε representing the dielectric constant and e the electron charge. Here d is the distance between 2 2 1/2 th the counter ion layers of DNA and mica and (xi,j + d ) is the distance between j DNA site th and i mica site. The ion valence is denoted as zi = zj. The force between the two plates can only become negative (attractive) if zi,j > 1. The minimum (most attractive) force is realized by a staggered (dt. "gestaﬀelt") arrangement of ions as illustrated in ﬁgure 2.5. It can be shown, that by minimizing the free energy of the system the staggered arrangement is favored. This is intuitive, as

7 2 Theory this arrangement maximizes the distance between likely charges, while still allowing a screening of the charged surface. The addition of monovalent cations leads to a competition between the monovalent and divalent cations [4]. As can be seen in equation 2.1, swapping a divalent ion for a monovalent one is weakening the attractive force. We will use this eﬀect to tune the DNA-mica-interaction so it will be strong enough to keep DNA structures adhered, but weak enough to allow diﬀusive motion of structures on the surface.

j=1 j=2 j=3 j=4 j=5 j=6 DNA ------… ++ ++ ++ d … ++ ++ ++ mica - b - - - - - … i=1 i=2 i=3 i=4 i=5 i=6

Figure 2.5: Staggered arrangement of divalent ions leads to an attractive force, between two negatively charged surfaces. This eﬀect is also referred to as salt bridge. Figure adapted from [4].

2.2 DNA, our Biopolymer for todays experiment

In deoxyribonucleic acid, DNA, four bases occur: adenine, guanine, thymine, and cytosine. In ribonu- cleic acid, RNA, thymine is replaced by another member of the pyrimidine family, named uracil. One further important structural difference between DNA and RNA is a hydrogen instead of a hydroxyl at each furanose 2’ carbon, which is denoted as "deoxy" in the name deoxyribonucleic acid. The five nucleobases can be grouped into the aromatic compounds pyrimidines and purines. Pyrim- idines are six-membered carbon ring structures with nitrogen substituting carbon on position one and three. In a purine an additional five-membered ring is fused to a pyrimidine. In strands of nucleic acid, nucleosides are linked together by a phosphate group. The phosphate covalently links the 5’ carbon of one nucleoside’s furanose to the 3’ carbon of the succeeding one, creating a backbone of alternating sugar and phosphate. Thus, a DNA/RNA strand can be assigned a 5’ and a 3’ end. Since each phosphate is contributing with one negative charge, strands of nucleic acid are highly negatively charged polyelectrolytes. The so called primary structure of a strand of nucleic acid is defined by its nucleotide sequence. It cannot be changed without breaking any covalent bonds. Depending on the strand’s primary structure, the environmental conditions and whether there are complementary strands present, various secondary structures can be formed. Secondary structure denotes the geometric arrangement, which can be assumed by a macromolecule, without any change in its primary structure. The most common secondary structure of DNA was discovered by Rosalind Franklin2, James Watson and Francis Crick in 1953 [6, 7]. The B-form double helix is formed by two strands of complementary base sequence and antiparallel orientation of 3’ and 5’ ends. As further described below, the two strands are kept together by hydrogen bonds between the strands’ nucleobases. Base stacking interaction additionally 2 with help of Ray Gosling and in cooperation with Maurice Wilkins

8 2.2 DNA, our Biopolymer for todays experiment

Figure 2.6: Illustration of the B-DNA double helix. Adapted from [5].

stabilizes the structure. The double helix is right handed and holds a major groove of 2.2 nm and minor groove of 1.2 nm. It has been shown that other structures (A-form and Z-form, for example) can also be created, but B-DNA is by far most commonly occurring in living organisms. In ﬁgure 2.6 a cutout of a B-DNA strand is illustrated. Table 2.1 compares the geometric key properties of A-,B- and Z-DNA. A-DNA B-DNA Z-DNA Helix sense right handed right handed left handed Basepairs per turn 11 10.5 12 Rotation per basepair 32.7° 35.9° 30° Diameter 2.3 nm 2.0 nm 1.8 nm

Table 2.1: Geometric properties of A-,B- and Z-form DNA [8, 9, 10].

2.2.1 Strand Hybridization

Formation of the double helix structure, a process called strand hybridization, is promoted by two effects: Watson-Crick base pairing and base stacking interaction. James Watson and Francis Crick explained that the double helix structure occurs with the formation of hydrogen bonds between nucleobases. They also identified that the interactions are particularly strong for a pairing made up of adenine with thymine (uracil in RNA) and guanine with cytosine. The Watson-Crick base pairs for DNA are depicted in figure 2.7. Base stacking is a complex interaction generated by π electron systems in the aromatic ring structures, which are part of all nucleobases. The bases’ rings fit roughly on top of each other following the helix axis. Depending on the base sequence, however, they are still twisted and tilted against each other.

9 2 Theory

Figure 2.7: The two Watson-Crick base pairs for DNA. There are many other possibilities of base pairing, but since Watson-Crick pairings are the strongest, they play the major role in biology and DNA nanotechnology.

2.2.2 DNA Nanostructures - DNA as a Building Block

In 1982 the crystallographer Nadrian Seeman proposed the use of DNA to build 3D scaﬀolds, that could serve as organization grid for hard to crystalize proteins [12]. The key element to utilize the linear DNA polymer for the construction of two and three dimensional structures, is the four-way junction. The mobile version of the four-way junction is called Holliday junction, named after the molecular biologist Robin Holliday. It consists of two identical DNA duplexes. A Holiday junction occurs, when one strand has partly hybridized a complementary strand and swaps its partnering strand against an identical one with an adjacent duplex as shown in ﬁgure 2.8. As long as both participating duplexes are of identical base sequence, the junction can migrate via the dissociation of base pairs at two opposing double helices and duplex formation of those bases at the remaining two helices. Nonidentical strands enable the construction of an immobile four-way junction. DNA double helices

Figure 2.8: Schematic representation of a Holliday junction. Left: Each strand simplified as a straight line. Middle: Double helix illustration in open configuration. Right: Stacked configuration occurring under the presence of metal ions. Graphic adapted from Müller (2010) [13].

can be attached to each other with "sticky ends". This means one strand of a duplex is extended to remain unpaired. Structures which are designed to bind at this position carry the complementary domain as an unpaired extension, so the two extensions can hybridize and act as a link. On this basis variety of structures have been shown long before the DNA origami method was developed. It

10 2.2 DNA, our Biopolymer for todays experiment is important to note, that the DNA origami, as described below, is one technique among a many. Although it’s robustness, easy adaptation and catchy name helped it gaining high popularity among bionanotechnologists, other methods have their own advantages depending the individual structural needs.

DNA Origami

Figure 2.9: Design scheme of a DNA origami. The scaffold strand (black) is aligned parallel to assume a desired shape. Staple strands (colored) are inserted to confine the scaffold in its shape. Left: Helix representation. Right: Simplified representation with straight lines. Figure adapted from Rothemund 2006 [14]. .

The DNA origami technique was presented by Paul Rothemund in 2006 as a method for "Folding DNA to create nanoscale shapes and patterns" [14]. As illustrated by the word "origami", for this method, a long strand of circular virus DNA, called scaffold strand, is folded into a desired shape. This is achieved by a set of shorter oligonucleotides, called staple strands. Each of the typically about 200 staple strands is designed to be complementary to specific regions on the scaffold. When a staple hybridizes to more than one of those regions it is clinging them together. The only state in which all staple strands can hybridize completely is when the scaffold assumes the form given by the design. Assuming the correct buffer and ionic conditions, complete hybridization of all staple strands is energetically favorable, causing scaffold and staple strands to assemble themselves into the desired structure. Usually the helices are aligned in parallel, so each crossover of scaffold or staple strands from one double helix to an adjacent one forms a stacked shape four-way junction. Since 2006, the technology has evolved from 2D structures, established by Rothemund, to 3D structures as demonstrated Douglas et al. 2009 [15], which can even be bent and twisted following established design rules [16]. The structure that is imaged in today experiment is a regular rectangular origami structure ( see figure 2.10) with the approximate dimensions of 70 nm x 100 nm.

11 2 Theory

Figure 2.10: Some of the 2D origami structures by Rothemund to demonstrate the folding of arbitrary shapes. Top row: alignment of the scaﬀold strand. Middle row: Each line represents a double helix in the folded structures. The helices are distorted regularly due to the regular pattern of staple crossovers from one helix to another. Bottom row: AFM image of the same structure. Figure adapted from [14]. 2.3 Order

DNA is a convenient material for the self assembly of well defined structures, which can then again serve as a "molecular pegboard" for the precise geometric arrangement of smaller components [19, 20, 21]. The small size of structures themselves however, make it very difficult to orient them, with respect to each other and to the macroscopic world. In the last part of the experiment, we will demonstrate one approach to this problem called "surface assisted ordering". Apart from a purely subjective point of view, what is ordering of particles? One way to quantify orientational order can be borrowed from theory on liquid crystals. Here the orientational order parameter η is defined as:

3 1 η = h cos2 θ − i 0 < η < 1 (2.2) 2 2

θ describes the angle between each particle and the a "predominant direction". In a perfectly ordered system η equals 1, while an ensemble of perfectly random orientation gives a orientational order parameter of 0. With a small number of particles, this can be done by manual measurement of each particle’s orientation. If particle numbers get larger, image recognition software might become necessary. Poor image quality, possible imaging artifacts and the shape of the analyzed particles might make a reliable automatization quite challenging. Another way to study ordering on a software basis is to perform a discrete Fourier transform (DFT). Strongly simpliﬁed, a DFT describes a sequence of N values as a serious of sinusoids of given amplitude, frequency and phase. Doing so transforms the signal from spacial domain into frequency

12 2.3 Order

domain. Each point Xk in frequency domain can be calculated as:

N−1 X −i2π kn Xk = xn · e N , k ∈ Z (2.3) n=0

Figure 2.11 illustrates the transformation of a simple oscillating signal. For a two-dimensional dataset, like an image, the same principle can be extended to describe any Fk,l in the frequency domain:

N−1 N−1 X X −i2π( ki + lj ) Fk,l = fi,j · e N N , k, l ∈ Z (2.4) i=0 j=0

Computer software accomplishes this with an algorithm called fast Fourier Transform (FFT). This FFT image shows the periodicity and patterns in an image and their direction (see ﬁgure 2.12). FFT of an image is implemented as a standard feature into basic Image processing software (like imageJ). This algorithm is also used in a vast range of everyday applications. Scanner-Apps for smartphones, for example, use it to ﬁnd the text orientation a photographed document and correct the image orientation3. In our case the image’s FFT reveals the predominant orientations and frequency of repeating units in the image.

Figure 2.11: Left: The signal shown in blue is a superposition of a 20 Hz oscillation with amplitude 0.7 and a 100 Hz oscillation with amplitude 1. Right: Plot of the signal’s fast Fourier transform shows peaks the contributing frequencies. Ideally these two beaks should be inﬁnitely sharp and have amplitudes 0.7 and 1. The fact that the amplitudes are lower, and the peaks are surrounded by oscillations has to be seen as artifacts. Transformation of a longer dataset with a higher time resolution decreases these artifacts.

Figure 2.12: Left: 256x256 pixel image of 2 pixel wide vertical stripes in spacial domain and frequency domain. 1 The maximum frequency is given by 2 pixels. As the stripes repeat with a frequency of 4 pixel 1 they are visible as white spots at ± 4 pixel in the transformed image. Right: Superposition with 45◦ rotated stripe pattern. In the transformed image the new diagonal stripes become visible as additional frequencies on the rotated axis. Images adapted from [22]. 3JPEG image compression also relies on FFT.

13 3 Experiment

3.1 AFM Operation Instructions

3.1.1 The Instrument

Figure 3.1: Overview of the system setup. The Microscope setup consists of (from left to right): a computer, a light source, alignment optics, the AFM body, and a controller. For better damping of environment vibrations the AFM body is put onto the "damping bungee" during scanning.

Setup Components The experiment will be conducted at a Veeco Multimode V AFM. While our instrument is already in use since many years, it is still manufactured (with minor upgrades) and shipped in large numbers which makes it one of the most wide spread models in nanotechnology. Keep in mind that you are not working with a robust student/lab course instrument but with a serious state-of-the-art microscope. Make sure to prepare well and work in a focused and professional manner. The main components of the AFM setup are a computer with NanoScope software, controller and AFM body. While the user is usually working at the computer or the AFM body and the controller is stored securely under the table, the electronics in the controller are the actually complicated part of atomic force microscopy. The compact form factor of the body makes it very robust with respect to internal vibrations. As shown in ﬁgure 3.1 the AFM body is placed below a camera, which will make it easier to align the laser spot onto the tiny cantilever. When the system is ready for scanning it will be placed on the "damping bungee". The heavy granite plate hanging from the ceiling on rubber bands, is a simple an eﬀective way to isolate the instrument from vibrations within the building (e.g.

14 3.1 AFM Operation Instructions caused by the ventilation system or elevator). Figures 3.2a - 3.2d further explain the function of each component.

Startup When you arrive at the instrument the AFM should be left in STM Mode, the controller and PC should be running and the NanoScope software should be closed. - If not, tell your supervisor.

3.1.2 Central Parameters

The following will explain the most important imaging parameters. Refer to ﬁgure 3.4 in order to get familiar with the NanoScope user interface.

Drive Frequency For tapping mode imaging the cantilever is periodically excited by the chip holder’s shake piezo. Sweeping over a large range of frequencies allows to ﬁnd the cantilever’s resonance. As the resonance varies for each cantilever, a tune has to be performed prior to imaging in order to ﬁnd the correct Drive Frequency. Once it is found it will stay unchanged throughout the measurement. Section "Reoccurring Procedures" will explain how to tune correctly.

Drive Amplitude The shake piezo is actuated with an oscillating voltage. This parameter determines the amplitude of cantilever excitation. Keep in mind that, this is only the excitation amplitude and NOT the resulting cantilever amplitude (this is rather given by the RMS value, see below). De- pending on the quality of the cantilever’s resonance, the same Drive Amplitude can result in very diﬀerent cantilever oscillation amplitudes.

Amplitude Setpoint The distance between tip and sample that is maintained throughout the measurement. Because the tip-sample distance is controlled by a voltage on the z-piezo, this parameter carries the unit "Volts" instead of "Meters".

Integral Gain In constant height mode and Tapping Mode the feedback loop translates the sample in z direction with the aim to keep the tip-sample interaction constant. The Integral Gain determines how strong the feedback mechanism reacts to a change in cantilever behavior. With a low Integral Gain the feedback will be too slow to recognize small surface features. A high Integral Gain will cause ampliﬁcation of unwanted oscillations in the instrument, an thus lead to stripe like artifacts. If you see (or even hear) strong oscillations, immediately reduce the Integral Gain in order to avoid damage to the electronics!

Scan Size Area that is scanned in nanometers.

Scan Speed Number of lines scanned per second. Note that this parameter does NOT give the tip velocity. The actual tip velocity is derived from scan size and scan speed, but not of special interest here.

15 3 Experiment

RMS This value is displayed on the Base display instead of the horizontal difference (see figure 3.2b), as soon as the microscope is switched to Tapping Mode. It gives the oscillating tip’s Root- Mean-Square displacement from its rest position. As the cantilever deflection is measured as a voltage difference between upper half and lower half of the PD, this value is also given in the unit "Volts".

3.1.3 Reoccurring Procedures

Center Laser Spot Open the camera image with right click -> undock into the small window on the bottom left. Look at the camera image and use the two laser alignment knobs (Fig 3.2c) to place the laser spot on the cantilever.

Maximize Sum Signal Watch the sum signal on the Base display while ﬁne adjusting the laser spot position until you ﬁnd the maximum sum signal.

Zero PD Watch the vertical and horizontal diﬀerence of the PD on the base display. Use the PD adjustment knobs to minimize both values. Values below 0.3 count as "close enough to zero".

Pre-Approach Turn the focus wheel to focus the camera image onto the mica surface. The mica surface can be identiﬁed by cracks, running across with a high contrast. When the surface is found raise the focus plane by a 5◦ turn of the focus wheel. The following step is the most delicate step regarding tip breakage. Slowly (and carefully!) lower the tip with the step motor lever. Be even more careful when you see the tip entering the focus plane. Lower the tip until it is exactly in focus. This is as close as you can safely approach the surface without risking tip damage. Do not use the step motor to engage any further.

Tune Click the tune icon. Click autotune and manually change sweep width to 20 kHz, so the whole resonance peak is visible. Check whether the automatically found resonance frequency is on the left slope of the peak with roughly 30 kHz. If not, your supervisor will help you to do it manually. Close the tune window with exit.

Approach Click the engage button and wait. The software performs a very careful approach algorithm to bring the tip near to the surface without crushing into it. This might take up to 3 minutes. The approach was successful when the PC plays a short "beep" sound.

16 3.1 AFM Operation Instructions

(a) Closer look at the AFM Body. The body con- (b) Display on the base. The signal of the PD sists of (from top to bottom): head, scanner and summed up over all four quadrants is shown on base. For alignment the body is places on a X- the bottom. The aim of laser alignment will be Y translation stage under the camera. The light to maximize the sum signal. The top left shows source illuminates the sample from above in order the difference between the PD’s two top quad- to allow a clear camera image for alignment. rants and the two bottom quadrants, - also called vertical difference. The top right shows the difference between the PD’s two left quadrants and the two right quadrants, - also called horizontal difference. When the AFM is switched to Tapping Mode, this value is replaced by the RMS value which is an indicator for the cantilever oscillation amplitude.

(c) Closer look at the AFM head. The sample stage (d) Back view of the AFM head. Chip holder screw translation knobs allow to translate the sample rel- ﬁxes the chip holder inside the AFM head (hand ative to the tip in a very corse manner. The laser tight is enough!). The mirror lever is used for very adjustments knobs allow X-Y translation of the rough laser alignment and should not be changed. laser spot on the cantilever. Sample and Chip The PD adjustment knobs allow X-Y translation holder will be placed in the head. of the Photodiode. These will be used to minimize the vertical and horizontal diﬀerence (see 3.2b).

Figure 3.2 17 3 Experiment

Figure 3.3: Left: Chip holder for air imaging with mounted probe. Right: Chip holder for imaging in liquid. Through the glass the shake piezo is visible. When an oscillating voltage is applied to the electrical contacts, the whole holder will vibrate. Choosing the frequency close to the cantilevers resonance, allows strong cantilever vibration at comparatively low vibrations of the remaining instrument.

Figure 3.4: Screenshot of the NanoScope software user interface. The important parameters are highlighted in yellow. Do not change parameters that are not highlighted in this graphic.

18 3.2 General Preparation

3.2 General Preparation

Before you start the ﬁrst experiment ensure that:

• your supervisor introduced you to the chip holder for dry imaging and mounted a probe

• you have a sample of M13mp18 scaﬀold

• you have a metal puck with a mica disc on top

• the instrument is placed on the alignment stand below the camera unit

• the NanoScope software is closed

3.3 Folding of DNA Origami

Folding of origami structures is done as one-pot annealing process. The main components of the folding solution are

• M13mp18 virus genome. The 7249 nucleotide long circular strands of double stranded DNA will serve as the scaﬀold strand. It is provided dissolved in water at 100 nM concentration

• Staple Strands. When bought these strands are delivered separated in 96 well plates, at a concentration of 100 µM each. For more convenient use they are mixed in equal parts and stored in one tube. In our case 186 staple strands are stored in the "staple-stock" solution.

• 10-fold Folding Buﬀer. The folding buﬀer is provided as stock solution with 10-fold concentration.

Prepare a folding solution with a total volume of 100 µL, scaffold strand 50 nM and 3-fold excess of staple strands over scaffold strands. Use the 10-fold folding buffer (10xTAE + 125 mM Mg2+) to adjust the buffer conditions to 1-fold folding buffer. Mix the solution well, place the reaction tube into the thermocycler and run the program ”ori70H20”. This will run the folding ramp which will heat the solution to 65◦C and will decrease the temperature by 0.5◦C per minute until it reaches 45◦C.

3.4 Imaging of M13mp18 Genome

The genome of the M13mp18 bacteriophage is imaged in air, that means the sample must be dried before imaging. As DNA is a rather soft material, we use Tapping Mode for imaging, for it applies lower forces to the sample than contact mode.

• sample preparation – dilute the M13mp18 scaﬀold strands to 1 nM in imaging buﬀer – cleave mica surface with scotch tape

19 3 Experiment

– add 50 µL of dissolved scaﬀold strands on the cleaved mica and wait for 5 minutes – wash the sample using 1 mL water – gently dry the sample by use of the nitrogen nozzle – put sample puck on the AFM stage

• alignment – drive sample stage down as far as possible. – insert chip holder. Ensure it lies flat and fix it by tightening the chip holder screw. Moderately tight is enough, do not over tighten the screw! – drive sample closer to the tip until a surface-tip distance of ≈ 5mm is reached – first align the laser spot roughly and then maximize the sum signal and zero PD – Perform pre-approach. – If the sum signal decreased or horizontal/vertical difference rose above 0.3 realign the laser and/or PD.

• tune – change the imaging mode switch to Tapping Mode. Note that the display switches from showing the horizontal diﬀerence into showing Root-Mean-Square (RMS) cantilever amplitude. – ask your supervisor to place the instrument onto the damping-bungee – close the camera image in the NanoScope software and tune

• image – change the Drive Amplitude while looking at the RMS value until it shows a value of 0.12 mV. – perform approach. – change the Amplitude Setpoint to a 400 mV. This value is intentionally chosen much too high, to bring the tip to a safe distance while it is oscillating. Since the tip cannot feel the surface you just see a straight line as the hight signal. – from this safe distance slowly lower the Amplitude Setpoint until you see a change in the height signal. If you reach 50 mV without any signal change something is wrong. Please ask you supervisor to have a look. – when you notice the hight signal changes, but it is not yet a clear image, lower there Amplitude Setpoint further until a ﬂat surface is emerging. – once the surface is found optimize Amplitude Setpoint and Integral Gain on the basis of image quality. Optimize the parameters for an image size of 2 µm x 2 µm.

20 3.5 Imaging of DNA Rectangles

– when you are satisﬁed with the image quality, click frame down and record a complete image. – when done, bring the tip to a safe distance by clicking withdraw.

3.5 Imaging of DNA Rectangles

For liquid imaging the sample surface is prepared similar to dry imaging, but instead of drying the surface a large droplet of sample containing buﬀer is placed onto the mica surface.

• sample preparation – dissolve origami structures to 50µL of 3 nM concentration. – cleave mica surface with scotch tape – add 5 µL of dissolved origami structures, spread solution evenly and add 50 µL buﬀer on top – put sample puck on the AFM stage

• alignment – drive sample stage down as far as possible – insert liquid imaging chip holder and fasten it – drive sample closer to the tip until a surface-tip distance of ≈ 5mm is reached – roughly align laser spot on cantilever – usually you can barely see the laser spot on top of the cantilever once it is emerged in liquid. Try to ﬁnd the right spot position by maximizing the sum signal. To do so perform very small search movements with the spot around its current position – when the the sum signal is maximized, zero PD – perform pre-approach – if the sum signal decreased or horizontal/vertical diﬀerence rose above 0.3 realign the laser and/or PD as you did before

• tune – change the imaging mode switch to Tapping Mode. – ask your supervisor to place the instrument onto the damping-bungee – close the camera image in the NanoScope software and tune

• image – change the drive frequency while looking at the Base display until it shows a value of 0.15 mV.

21 3 Experiment

– perform approach. – change the Amplitude Setpoint to 400 mV. This value is intentionally chosen much too high, to bring the tip to a safe distance while it is oscillating. Since the tip cannot feel the surface you just see a straight line as the height signal. – from this safe distance slowly lower the Amplitude Setpoint until you see a change in the height signal. If you reach 50 mV without any signal change something is wrong. Please ask you supervisor to have a look. – when you notice the height signal changes, but it is not yet a clear image, lower the Amplitude Setpoint further until a ﬂat surface emerges. – when you are satisﬁed with the image quality, click frame down and record a complete image. – when you are done bring the tip to a safe distance by clicking tip withdraw.

3.6 Surface Assisted Ordering by Close Packing

We made sure that the M13mp18 genome has assembled into rectangular nanostructures. In the next step we will try have them orient themselves by close packing on the mica surface. First we will increase the concentration of structures until the surface is saturated. The second step is to weaken the DNA-mica interaction enough to allow the structures to diﬀuse on the surface.

• sample preparation – dissolve origami structures to 50µL of 8 nM concentration. – cleave mica surface with scotch tape – add 5 µL of dissolved origami structures, spread solution evenly and add 50 µL buﬀer on top – put sample puck on the AFM stage

• Packing – add 5 µL of 8 nM rectangle solution on the cleaved mica – if the sum signal decreased or horizontal/vertical diﬀerence rose above 0.3 realign the laser and/or PD as you did before – tune – approach

– ﬁnd imaging parameters for a good quality scan – when image is saved, click withdraw

• Better Packing

22 3.6 Surface Assisted Ordering by Close Packing

– prepare freshly cleaved mica – add 50 µL solution containing 40 nM rectangle origami in "mobility buﬀer" (1xTAE+ 150 mM NaCl and 12.5 mM Mg2+) on the cleaved mica – repeat the imaging as you just did for above

• Image Analysis – export representative images of all your measurements – compare the images of DNA rectangles you took at low density, high density and after addition of mobility buﬀer – use the NanoScope software or imageJ to perform a FFT of each image. Can you ﬁnd a predominant direction? Estimate the particle size in your AFM image judging from the pattern in the FFT image. – use an image processing tool (e.g. imageJ) and measure the particle orientations on each image. For each image, make a histogram plot, showing the particles’s angular distribution. How does this change when instead of the whole image you only analyze an area half the size of the original image?

23 4 Questions

1. The folding buﬀer, in which DNA origami structures are assembled, contains a buﬀer called TAE (Tris, Acetate, Ethylenediaminetetraacetic acid) and MgCl2. What do you think is the role of those two components?

2. Figure 4.1 shows an image with a common artifact. What do you think is wrong with this image, and what could you do to get rid of this artifact?

3. Figure 4.2 shows a diﬀerent sample. On the left you see an image recorded with sub-optimal imaging parameters. Based on your knowledge about AFM, what went wrong here? What parameters could you adjust to get the image on the right?

4. Modern high speed AFMs can reach speeds of over 100 frames per second in Tapping Mode. What are the limiting factors for scan speed? If we had a perfect AFM, what maximum scan speed would make sense?

Figure 4.1: Imaging Error 1

Figure 4.2: Imaging Error 2

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