I have prepared DNA nanostructures having calculated dimension of 20 nm X 4 nm (monomeric unit) and polymeric 2D array structure. But I am facing problems during imaging of these structures in AFM. DNA structure concentration is 1μM. Is any treatment required on mica surface prior to imaging? What amount of sample should be loaded on mica surface? How to dry the mica surface? What should be the optimum parameters to be set in AFM?
What are the specific problems you are facing during imaging? Are you imaging in air or in liquid? Mica is the most common substrate for DNA nanostructure imaging, but there are definitely problems that can occur. Here are a few important factors to take into account:
1. You should cleave the top-most mica layer with tape before deposition, this ensures you have a smooth, clean mica layer to work with.
2. You should have an adequate Mg2+ concentration to promote the adsorption of DNA nanostructures to mica. This is outlined in many publications for both plasmid DNA and DNA origami/nanostructures and is ~12 mM Mg2+.
3. The coverage of DNA nanostructures on mica is mostly dependent on solution concentration. If you are having trouble finding your nanostructures, you may want to try increasing or decreasing your concentration.
4. I have found the most important step is rinsing and drying the samples. I use about 200 uL of water to rinse excess salts off the surface and then dry with a steady stream of purified nitrogen gas for 2-3 minutes. If I do not do this, my nanostructures tend to aggregate and my sample is ruined.
5. You should use tapping mode AFM for imaging in air. The fragile nanostructures will be destroyed if you use contact mode.
I hope this helps with your specific issues.
You might search for journal articles by Y. Lyubchenko. They have developed well-respected protocols to image DNA with AFM.
Thank you, Prof. J. J. Weimer, for your suggestion. I went through selected publications of Dr. Y. Lyubchenko. His papers certainly helped me in solving my problems regarding AFM imaging. However, I have still few questions in my mind about imaging DNA nanostructures using AFM. While imaging larger DNA like plasmid or λ- DNA, the possibility of damaging DNA sample during sample preparation for AFM imaging might not be there. But doing such thing with self-assembled DNA nanostructures may disturb the structural integrity of the structure. Many researchers are successfully doing wonderful imaging of DNA nanostructures under AFM. So can I get the precise protocol for imaging DNA nanostructures, please?
What are the specific problems you are facing during imaging? Are you imaging in air or in liquid? Mica is the most common substrate for DNA nanostructure imaging, but there are definitely problems that can occur. Here are a few important factors to take into account:
1. You should cleave the top-most mica layer with tape before deposition, this ensures you have a smooth, clean mica layer to work with.
2. You should have an adequate Mg2+ concentration to promote the adsorption of DNA nanostructures to mica. This is outlined in many publications for both plasmid DNA and DNA origami/nanostructures and is ~12 mM Mg2+.
3. The coverage of DNA nanostructures on mica is mostly dependent on solution concentration. If you are having trouble finding your nanostructures, you may want to try increasing or decreasing your concentration.
4. I have found the most important step is rinsing and drying the samples. I use about 200 uL of water to rinse excess salts off the surface and then dry with a steady stream of purified nitrogen gas for 2-3 minutes. If I do not do this, my nanostructures tend to aggregate and my sample is ruined.
5. You should use tapping mode AFM for imaging in air. The fragile nanostructures will be destroyed if you use contact mode.
I hope this helps with your specific issues.
I agree with Michelle for the use of a divalent cation such as Mg, the concentration is a concern though. Less is more in this case and the rinsing and drying procedure is critical. I usually put a pipette tip on the hose attached to the N tank to blow the DNA structures dry on the mica surface. This process also tends to straighten them out and separate aggregations making for wonderful AFM imaging. I have also had good luck getting DNA to dry onto mica using the blow-drying technique without treating the mica in any way or using Mg.
Good luck.
Thanks, Michelle and Glenn for your constructive suggestions. I will definitely focus on the points you mentioned.
You can also try APTES vapour deposition technique. In this method, you basically silanize freshly cleaved mica surface and this helps you to moderate the electrostatics better. You could leave your mica in a desiccator containing APTES (freshly distilled) and DIPEA (also fresh) and leave this for 2 hrs under vacuum. This creates a hydrophobic surface (which you may want to check with contact angle measurement). DNA immobilisation can be carried out thereafter on such surfaces. You can also store these surfaces for up to a month or so under Argon atmosphere.
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