There is an avalanche-like increase of reports, where molecules of nucleic acids (DNA and RNA) appear as an object of nanotechnology research and/or as material for nano-sized devices. In many cases scanning probe microscopy (SPM) is the most powerful and informative research tool. Examinations as well as precise manipulations can be performed this way. What is important when SPM is applied to molecular level experiments? Three facets are illustrated further.
- Appropriate probes
- Substrates and deposition protocols
- AFM long-term stability
The Importance of Probe Radius on Resolution
Tiny features of the relief can not be detected if the probe tip radius is too large. When imaged with conventional probe, the width of the DNA molecule is 10-20 nm usually, while real strand diameter is about 2 nm. Here are shown short poly(dG)–poly(dC) DNA fragments deposited on modified HOPG (see Figure 1.). Small unwound single-strand fragments can be seen (bold arrow on the scan) and even helical pitch of the DNA molecule can be resolved (thin arrows) with a sharp enough tip (like DLC probe tip shown on the inlet). See comprehensive discussion on sub-molecular imaging in “Highresolution atomic force microscopy of duplex and triplex DNA molecules” Klinov D. et al. Nanotechnology (2007), V18, N22, p.225102.
Figure 1. Short poly(dG)–poly(dC) DNA fragments deposited on modified HOPG
Depositing DNA onto Substrates
Pretreatment Substrates to Promote DNA Deposition
Figure 2. DNA molecules (plasmid) were deposited onto mica surface from solution containing Mg2+.
DNA molecules (plasmid) were deposited onto mica surface from solution containing Mg2+. Pre-treatment of mica by pure water increases density of surface negative charge (because of cations loss). Binding of DNA in this case is fast and strong, circular plasmid becomes compacted (A). Freshly cleaved surface has lower density of the negative charge, thus DNA binding is slower and lateral diffusion occurs during deposition (B).
Binding DNA to Nica
Figure 3. Plasmid DNA deposited onto mica surface pretreated with APTES
Plasmid DNA deposited onto mica surface pretreated with APTES. The attachment occurs relatively fast.
Binding DNA to HOPG
Figure 4. Plasmid DNA deposited onto HOPG surface pretreated with organic modifier
Plasmid DNA deposited onto HOPG surface pretreated with organic modifier (CH2)n(NCRH2CO)m–NH2. Attachment occurs relatively slow.
AFM Stability for Precise and Long Term Imaging
Temperature Drift and AFM Measurements
Figure 5. Low temperature drifts
Temperature drifts are serious obstruction for long-term experiments on small fields. Typical drifts values are 10-15 nm per hour in best commercial AFM devices. Due to this effect objects with sizes of tens of nanometers can be lost during long observations. On the left images it is shown how carbon nanotube (that is similar to DNA in terms of its dimensions) can be moved by AFM probe. Right pare of scans show this object in long-term experiment. Displacement for 7 hours is small enough and the same particles remain in the field of view. Sample courtesy of Dr. H.B.Chan, Department of Physics, University of Florida, USA.
Closed Loop Correction for Ensuring Correct Probe Repositioning
Figure 6. Atomic lattice of mica imaged with CL sensors
Closed loop (CL) sensors are essential for correct probe repositioning and manipulations. Because CL sensors put some electronic noise they usually are not available below 100 nm. NTEGRA Therma allows CL correction even below 10 nm. Here is atomic lattice of mica imaged with CL sensors.