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

In our laboratory, we develop new measurement technologies for nanoscale biology. Particularly we focus on development of high-speed atomic force microscopy to study dynamic biological processes at the nanometer scale. And for this, we've recently developed a new imaging mode, which we call Photothermal Off Resonance Tapping, or PORT.

In this mode, we are using a dedicated drive laser to actuate the cantilevers very controlled at a high speed, which lets us get the data and up to two orders of magnitude faster than with conventional off-resonance tapping. The gold standard in high speed AFM is resonant mode imaging. In this mode, the cantilever is actuated at this resonant frequency, which means that the cantilever position cannot be controlled, only its oscillation amplitude.

For normal speed AFM, off-resonance modes are increasingly common. However, the actuation frequency is limited here, which makes this imaging modes quite slow. By using a secondary laser to actuate the cantilever, we can achieve higher actuation frequencies, meaning higher imaging speeds.

This approach allows us to maintain the advantages associated with other off-resonance staffing modes, meaning we can optimize the balance between imaging sensitivity and speed. We're limited in the imaging speed by the highest portrait. If we wish to increase the portrait, we need to increase the laser power in order to maintain the same actuation amplitude.

This can lead to poor force control and damage to the sample. Enhancing the bandwidth of the cantilevers by reducing their size and by improving the laser absorption efficiency will enable us to attain higher port rates while ensuring adequate sample clearance. This advancement will facilitate quicker imaging rates that is crucial for examining the intricacies of assembly dynamics.

To begin, clean and prepare the cantilevers. Mount cantilevers on a holder compatible with the scanning electron microscopy or SEM system. Then heat the precursor gas to be used on the gas injection system to grow the new tip.

Once the vacuum reaches below 10 to the power of negative five millibar, purge the gas injection line 10 times for two seconds each to remove any residual air from the nozzle line. Use the SEM to locate the end of the cantilever. Tilt the holder to an angle to align the cantilever correctly for atomic force microscopy imaging.

Adjust the position and focus of the SEM for a clear view of the cantilever's tip where the carbon nano tip will be grown. Next, set the deposition parameters in the software to grow the new tip. Initiate the deposition process to grow the tip with a radiation of the electron beam on the cantilever tip while injecting the precursor gas and stop the gas injection once the deposition is complete.

Perform post growth SEM imaging to assess the quality and characteristics of the newly grown tip, including its radius and length. Remove the holder from the SEM chamber. To begin, prepare a high speed atomic force microscope.

Use tweezers to place an ultra short cantilever under the spring clip on the cantilever holder. Using a syringe, add 50 microliters of liquid through the left fluid access port. Then use three knobs on the AFM head to align the readout laser on the cantilever.

Observe the shadow of the cantilever on a white paper while maximizing the sum. Then center the laser spot on the photo diode using the two dedicated knobs. Next, check the excitation enabled box in excitation VI to switch on and align the drive laser, actuating and oscillating the cantilever.

Display the cantilever excitation and deflection signals on an oscilloscope. Use the shadow method and maximize the oscillation amplitude with drive laser adjustment knobs. To adjust the cantilever oscillation amplitude in port, add a DC voltage to the laser diode control circuit in the configuration box in excitation VI and input the peak-to-peak AC input for the laser diode control circuit.

To obtain interaction curves, set the Z controller VI to contact mode and click Start to approach the sample surface. Once the surface is reached, perform a force versus distance curve in ramp VI for cantilever deflection sensitivity calibration. Click withdraw to retract the Z piezo from the surface where the tip cannot reach.

After complete retraction, switch to port mode in the Z controller VI and turn on the excitation laser in excitation VI.Set the port mode to the desired frequency. Clear interaction curves were obtained by subtracting free oscillation from contact oscillation crucial for non-destructive bio-sample imaging. At 100 kilohertz port rate, good imaging quality was achieved.

However, as excitation frequency neared cantilever resonance, feedback control deteriorated, leading to obscured interaction curves and degraded image quality. To begin, prepare a 10 millimolar solution of magnesium acetate. Using a Hamilton syringe, inject 50 microliters of the solution into the fluidic channel of the cantilever holder, creating a drop of liquid that englobes the cantilever.

Set the scan size in the scan VI to 800 by 800 nanometers and the line rate to 100 hertz. To scan the surface, click the frame arrow to check for quality. After scanning, click withdraw in the Z controller VI to retract the cantilever from the surface.

Using a Hamilton syringe, remove the buffer solution from the cantilever holder. Then prepare a diluted DNA three point star solution and inject 50 microliters of this solution into the cantilever holder. Perform imaging with an 800 by 800 nanometer area at a default line rate of 100 hertz.

After the initial scan, adjust the imaging size and speed and scan VI to the specified values for further data acquisition Maintain the input for setpoint in the Z controller VI setpoint box at the lowest level necessary for accurate tracking throughout the imaging process. Repeat this for all required sample areas. Using this protocol, the realtime assembly of DNA three point star motifs into stable islands was observed by high speed port AFM.

Clear images were obtained at 100 and 200 hertz line rates for a 100 kilohertz port rate. However, higher imaging speeds without increasing port rates reduced image quality due to fast topography changes. Imaging results of DNA three point star for the lowest peak-to-peak AC input showed intact structures while the highest peak-to-peak AC input resulted in observable sample damage due to higher forces.

Similarly, the lowest DC offset input revealed intact structures while the highest DC offset input caused structural damage.