High-Speed Atomic Force Microscopy Imaging of DNA Three-Point-Star Motif Self Assembly Using Photothermal Off-Resonance Tapping

Summary

Here, we show the imaging protocol for observing biomolecular interactions with photothermal off-resonance tapping (PORT), where we optimized imaging parameters, identified system limits, and investigated potential improvements in imaging three-point-star DNA motif assembly.

Abstract

High-speed atomic force microscopy (HS-AFM) is a popular molecular imaging technique for visualizing single-molecule biological processes in real-time due to its ability to image under physiological conditions in liquid environments. The photothermal off-resonance tapping (PORT) mode uses a drive laser to oscillate the cantilever in a controlled manner. This direct cantilever actuation is effective in the MHz range. Combined with operating the feedback loop on the time domain force curve rather than the resonant amplitude, PORT enables high-speed imaging at up to ten frames per second with direct control over tip-sample forces. PORT has been shown to enable imaging of delicate assembly dynamics and precise monitoring of patterns formed by biomolecules. Thus far, the technique has been used for a variety of dynamic in vitro studies, including the DNA 3-point-star motif assembly patterns shown in this work. Through a series of experiments, this protocol systematically identifies the optimal imaging parameter settings and ultimate limits of the HS-PORT AFM imaging system and how they affect biomolecular assembly processes. Additionally, it investigates potential undesired thermal effects induced by the drive laser on the sample and surrounding liquid, particularly when the scanning is limited to small areas. These findings provide valuable insights that will drive the advancement of PORT mode’s application in studying complex biological systems.

Introduction

High-speed atomic force microscopy (HS-AFM) is a rapidly growing imaging technique^1,2,3,4. It operates at speeds that allow researchers to visualize biomolecular interactions in real time^5,6,7,8,9. Photothermal off-resonance tapping (PORT) is an off-resonance imaging mode similar to peak force tapping^10,11, pulsed force mode^12,13, or jumping mode¹⁴. However, rather than vertically oscillating the scanner, PORT vertically oscillates only the cantilever through an excitation laser focused on the cantilever (usually close to the clamping point). The cantilever deforms due to the bimorph effect: a power-modulated excitation laser periodically heats the coated cantilever, which bends due to the different thermal expansion coefficients of the cantilever and the coating materials¹⁵. Cantilever and sample heating can be minimized by using a drive laser that is periodically switched off and back on during each oscillation cycle, rather than using a full sinusoidal drive⁵.

DNA has been used to form biologically relevant, structurally interesting, and biochemically useful motifs for a number of years^{16,17,18,19,20}. In addition, DNA structures have been proven ideally suited to characterize AFM imaging quality²¹ and to assess the tip-effect of high speed AFM²². Blunt-end DNA three-point-stars (3PS) became practical as a programmable model system for investigating the supramolecular organization of similarly structured molecules in otherwise complex biological systems¹⁹. Previously, the self-assembly of lattices formed by blunt-ended trimeric DNA monomers was tracked via HS-AFM²³. Eventually, these organize into large networks with hexagonal order. Here, the self-assembly of DNA 3-point stars¹⁹ is imaged with the PORT technique at scanning speeds fast enough to track the self-assembly and its correction mechanisms²⁴ while assuring minimal disruption of the process or sample damage. As with any HS-AFM mode, there is a trade-off between achievable imaging quality, imaging speed, and the unwanted disturbance of the sample. By choosing the right compromise, one can better understand the self-organization patterns of supramolecular assemblies. This protocol will, therefore, use a similar setup with DNA 3PS as a model system to optimize the parameters specific to PORT. This will allow operation at fast imaging speeds at large enough scan sizes while minimizing sample damage.

Protocol

1. Sample and buffers NOTE: The DNA tile used in this study is the 3-point-star motif developed at the Mao laboratory at Purdue University19,25. All oligonucleotides used in this study were purchased from Integrated DNA Technologies, Inc. Gather the necessary materials and reagents. Mix the single-stranded DNAs (ssDNAs) at a 1:3:3 molar ratio (S1 0.6 µM, 1.8 µM for S2, and 1.8 µM for S3) in t…

Representative Results

In this investigation, the dynamic assembly process of DNA 3-point-star motifs into stable islands was successfully observed utilizing the capabilities of the HS-PORT AFM. This technique allowed us to capture the assembly of these structures in real-time. In Figure 2A,B, we get a clear image scanning at 100 Hz and 200 Hz line rates, respectively, for 100 kHz PORT rate (800 nm by 800 nm scan size). This corresponds to 3.9 and 1.95 oscillation cycles per pixel, respectively. H…

Discussion

When imaging delicate biological samples, off-resonance tapping imaging modes in AFM are particularly useful since they can directly control the tip-sample interaction forces¹⁰. Among them, the PORT mode stands out due to the higher oscillation rates it can reach, which enables higher scan rates. As PORT directly and only actuates the cantilever with a laser, it allows excitation at much higher frequencies than conventional off-resonance tapping modes, particularly when using ultrashort cantilever…

Materials

AC10DS	Olympus	BL-AC10FS-A2	Discontinued
Biometra Compact XS/S	Biometra GmbH	846-025-199	Electrophoresis  unit
Biometra TRIO	Biometra GmbH	207072X	thermocycler for annealing
Custom AFM setup	Laboratory for Bio-Nano Instrumentation, Interfaculty Bioengineering Institute, School of Engineering, Ecole Polytechnique Fédérale Lausanne		Obtainable through Laboratory for Bio-Nano Instrumentation
EDTA	ITW Reagents	A5097	In annealing buffer
Laser Power Meter	Thorlabs	PM100D	Digital Handheld Optical Power and Energy Meter Console
Lively 3AP Power Supply, MP-310	Major Science	MP-310	Electrophoresis Power Supply
MgAc2	ABCR GmbH	AB544692	In annealing buffer
TBE	Thermo Scientific	327330010	Running buffer for electrophoresis
TRIS	Bio-Rad	1610719	In annealing buffer