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 mo....

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 University^19,25. All oligonucleotides used in this study were purchased from Integrated DNA Technologies, Inc. Gather the necessary materials and reagents.

1. 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 the annealing buffer (5 mM TRIS, 1 mM EDTA, and 10 mM MgAc₂). The final concentration of the DNA motif must be 0.6 µM (49 ng/µL wi....

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

Name	Company	Catalog Number	Comments
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