Biomod/2011/TeamJapan/Sendai/Results/Atomic Force Microscope: Difference between revisions

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We used a high-speed  [http://en.wikipedia.org/wiki/Atomic_force_microscopy.html AFM]([http://www.ribm.co.jp/equipment/index.html RIBM], Nano Live Vision：NLV) for observation.
We used a high-speed  [http://en.wikipedia.org/wiki/Atomic_force_microscopy.html AFM]([http://www.ribm.co.jp/equipment/index.html RIBM], Nano Live Vision：NLV) for observation.
This high-speed AFM enables us to capture moving molecules in solution clearly without blurring.<br/>
This high-speed AFM enables us to capture moving molecules in solution clearly without blurring.<br/>
So we can confirm not just the molecular robot, but also observe <big>'''at video-rate'''</big> the molecular robot moving over the DNA origami field by using this.<br/>
So we can confirm not just the molecular robot, but also observe the molecular robot moving over the DNA origami field at video-rate.<br/>
In the following flowchart it is drawn the different project stages that were verified by the AFM.<br/>
The following figure below shows our observation flowchart.<br/>
[[Image:2d 3d field field_robot .png|900px|center|thumb|Project stages verified by AFM]]<br/>
[[Image:flowchart 1102.png|900px|center|thumb|Project stages verified by AFM]]<br/>


== Observation of 2D and 3D structures ==
== Observation of 2D and 3D structures ==
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First,we checked 2D nanostructures.The shape of 3D nanostructures resemble that of aggregation.On the other hand,the shape of 2D nanostructures are rectangle and differ from aggregation clearly.  
First, we checked 2D nanostructures. The shape of 3D nanostructures resemble that of aggregation. On the other hand,the shape of 2D nanostructures are rectangle and differ from aggregation clearly.
That is why 2D nanostructures are easier to observe than 3D ones.Therefore, before doing the annealing for getting the triangular prism we verified the 2D structure. The size of 2D structure in AFM is 67 nm × 23 nm(Figure 1).<br/>As compared to our design(60 nm × 20 nm),the maximum error is 15percent.From electoric rebounding between double helix, the size of 2D nanostrucutre fall within the error range. We can affirm 2D nanostructures since our original design (Figure 2) depicts similar dimensions (60 nm × 20 nm)
That is why 2D nanostructures are easier to observe than 3D ones. Therefore, before doing the annealing for getting the triangular prism, we verified the 2D structure. The size of 2D structure in AFM is 67 nm × 23 nm(Figure 1).<br/>As compared to our design(60 nm × 20 nm), the maximum error is 15percent.From electoric rebounding between double helix, the size of 2D nanostrucutre fall within the error range. We can affirm 2D nanostructures since our original design (Figure 2) depicts similar dimensions (60 nm × 20 nm).


There are surplus scafold(M13) from the middle of 2D nanostructure(Figure 3).
We observed leftover scaffold strand from the middle of 2D nanostructure(Figure 3).  
We experimented to cut surplus M13 by [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] because we were afraid that surplus M13 inhibit folding and moving of structure.
We used M13mp18 for the sdaffold, though our robot only uses 1100 bases. That was the reason why the surplus M13 inhibit folding and moving of structure.<br/>
After we cut surplus M13,we also observed 2D structure made from cut M13(Figure 1).
So, by [http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Design#Procedure_for_cutting_DNA restriction enzyme], we cut the surplus M13 in order to avoid unintended folding and interference when the robot moves on the Field during the race. After we cut the surplus M13,we also observed 2D structures made from cut M13(Figure 1).  
It proved that our experiment by PCR succeeded and we got 2D nanostructure using cut M13.  
It proved that our experiment by restriction enzyme succeeded and we got 2D nanostructures using cut M13.




*Sample conditions:<br/>
*Sample conditions:<br/>
・Concentration M13:staples (1:10)<br/>
・Concentration M13:staples (1:10)<br/>
・Mg<sub>2</sub><sup>+</sup> concentration 12.5 mM<br/>
・Mg<sup>2+</sup> concentration 12.5 mM<br/>
・Annealing time: 3 hours from 90°C to 25°C<br/>
・Annealing time: 3 hours from 90°C to 25°C<br/>
・Buffer: Tris-HCl<br/>
・Buffer: Tris-HCl<br/>
・Only cut M13<br/>
・cut M13<br/>


We confirmed the size of 2D structures in this experiment.<br/>
Next experiment, we reffered to the size of 2D structures and checked 3D structures.<br/>




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===Triangular prism 3D structure ===
===Triangular prism 3D structure ===


[[Image:Robotfold.png|250px|right|thumb|Figure 3. design of 3D structure]]
[[Image:Robotfold.png|250px|right|thumb|Figure 4. design of 3D structure]]
[[Image:tri3D.jpg|450px|right|thumb|Figure 4. AFM 3D strucuture]]
[[Image:tri3D.jpg|450px|right|thumb|Figure 5. AFM 3D strucuture]]






Figure 3 is our design of 3D structure and Figure 4 shows the 3D structure of triangular prism in AFM.  
Figure 4 is our design of 3D structure and Figure 5 shows the 3D structure of triangular prism in AFM.  
3D structure consists of the three DNA rectangular faces. (Figure 3).<br/>
3D structure consists of the three DNA rectangular faces. (Figure 4).<br/>


As compared to the figure of 2D structures, there are some differences.
As compared to the figure of 2D structures, there are some differences.
We can see the closed structure in figure 4.The closed structure was not observed in Figure 2.
We can see the closed structure in figure 5.The closed structure was not observed in Figure 1.
This structure was formed by self-assembly using three edging staples. There are two sticky ends in both ends of the 2D structures.<br/>
This structure was formed by self-assembly using three edging staples. There are two sticky ends in both ends of the 2D structures.<br/>


We found that the shape of the structure seemed to be like triangle because three DNA rectangular faces were opened up and adsorbed on mica (Figure 4).   
We found that the shape of the structure seemed to be like triangle because three DNA rectangular faces were opened up and adsorbed on mica (Figure 5).   
The reason of this phenomenon is that the 3D structure on mica was distorted and M13 linked to DNA rectangular faces were cut by AFM tip (Figure 5).  
The reason of this phenomenon is that the 3D structure on mica was distorted and M13 linked to DNA rectangular faces were cut by AFM tip (Figure 6).  
It seems that the 3D structures were not stable in the presence of the external force by AFM.<br/>
It seems that the 3D structures were not stable in the presence of the external force by AFM.<br/>


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・M13:staples= 1:10(ratio of concentration)<br/>
・M13:staples= 1:10(ratio of concentration)<br/>
・Mg<SUP>2+</SUP> concentration 12.5 mM<br/>
・Mg<SUP>2+</SUP> concentration 12.5 mM<br/>
・anealing time:3 hours<br/>
・anealing time:3 hours from 90°C to 25°C<br/>
・Buffer:Tris-HCl<br/>
・Buffer:Tris-HCl<br/>
・cutting M13<br/>
・cut M13<br/>






[[Image:tubure nagare3.png|450px|right|thumb|Figure 5. Model of 3D structure adsorbed on mica]]<br/>
[[Image:tubure nagare3.png|450px|right|thumb|Figure 6. Model of 3D structure adsorbed on mica]]<br/>




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<big>
<big>
<p style="text-align">
<p style="text-align">
*[http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Result_Atomic_Force_Microscope/detail_Observation_2D_and_3D_structure '''Detail:Older design of 2D and 3D structures''']
*[http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Result_Atomic_Force_Microscope/detail_Observation_2D_and_3D_structure '''Older design of 2D and 3D robot''']
</p>
</p>
</big>
</big>
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<html><div style="clear:both;"></div></html>


== Observation of the Field　==
== Observation of the Field ==


[[Image:field_endo.png|380px|right|thumb|Figure 6. design of AFM Field]]
[[Image:field_endo.png|380px|right|thumb|Figure 7. design of AFM Field]]
[[Image:field_wiki.jpg|380px|right|thumb|Figure 7. AFM Field]]
[[Image:field_wiki.jpg|380px|right|thumb|Figure 8. AFM Field]]




Figure 6 is design of the Field. Figure 7 is AFM image of the Field.<br/>
Figure 7 is design of the Field. Figure 8 is AFM image of the Field.<br/>
According to this image, redundant M13 was also observed.<br/>
According to this image, redundant M13 was also observed.<br/>
The size of Field in AFM picture is similar to that of design.<br/>
The size of the Field in AFM image is similar to that of design.<br/>
Therefore we succeeded in observing the Field.<br/>
Therefore, we succeeded in observing the Field.<br/>




*Condition<br/>
*Condition<br/>
・M13:staples= 1:10(ratio of concentration)<br/>
・M13:staples= 1:10(ratio of concentration)<br/>
・Mg2+ concentration 12.5 mM<br/>
・Mg<sup>2+</sup> concentration 12.5 mM<br/>
・anealing time:3 hours<br/>
・anealing time:3 hours from 90°C to 25°C<br/>
・Buffer:Tris-HCl<br/>
・Buffer:Tris-HCl<br/>






We got clear pictures of Field.<br/>
 
Before this experiment,the 2D and 3D structure were also observed using the high-speed AFM.<br/>
In other words,we accomplished observing <big>'''all the elements necessary for our race'''</big> by using high-speed AFM.<br/>
Next,we move on to combine the 3D structure with Field.<br/>
{{-}}
{{-}}
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== Observation structure on the Field ==
== Observation of structure on the Field ==
 


===Triangular prism 2D structure on the Field ===


===Triangular prism 2D structure on the Field　===
[[Image:field_2d_tobumae.jpg|380px|right|thumb|Figure 9. 2D structure on the Field]]
[[Image:field_2d_tobu.jpg|380px|right|thumb|Figure 10. No structure on the Field]]


[[Image:field_2d_tobumae.jpg|380px|right|thumb|Figure 8. 2D structure on the field]]
[[Image:field_2d_tobu.jpg|380px|right|thumb|Figure 9. No structure on the field]]


In the previous section, we concluded that the Field,2D and 3D structure were also observed by using the high-speed AFM.<br/>
In other words,we accomplished observing <big>'''all the elements necessary for our race'''</big> by using high-speed AFM.<br/>
So we move on to combine the 3D structure with the Field.<br/>


　
 
Luckily we obtained the image of the 2D structure attached to the field using high-speed AFM.<br/>
 
 
Luckily we obtained the image of the 2D structure attached to the Field using high-speed AFM.<br/>
In this sample, there ought to be only robots (2D structures which couldn't be folded and 3D structures ) but several fields were observed in this sample.<br/>
In this sample, there ought to be only robots (2D structures which couldn't be folded and 3D structures ) but several fields were observed in this sample.<br/>
We supposed that the leftover fields on cantilever adsorbed on mica.<br/>
We suppose that the previous solution with origami fields remained on cantilever, then that solution was mixed with new one and adsorbed on mica.<br/>
We scanned this field with 2D structure about 10 times but the structure didn't go away. Next, we raised the scanning speed then the structure was gone.<br/>
We scanned this field with 2D structure about 10 times but the structure didn't go away. Next, we raised the scanning speed then the structure was gone.<br/>
The structure was detached from the field because cantilever scratched the structure.
The structure was detached from the Field because cantilever scratched the structure.


It seems that robots can be attached to the field.<br/>
It seems that robots can be attached to the Field.<br/>




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・M13:staples= 1:10(ratio of concentration)<br/>
・M13:staples= 1:10(ratio of concentration)<br/>
・Mg<SUP>2+</SUP> concentration 12.5 mM<br/>
・Mg<SUP>2+</SUP> concentration 12.5 mM<br/>
・Annealing time:3 hours<br/>
・Annealing time:3 hours from 90°C to 25°C<br/>
・Buffer:Tris-HCl<br/>
・Buffer:Tris-HCl<br/>


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== Condition for the robots to attach the fields ==
== Condition for the robots to attach the Fields ==




In order to optimize the condition for reproducible observation, we need to optimize
We confirmed the 2D structure attached to the Field using high-speed AFM. However this observation is lacking in reproducibility.<br/>
In order to obtain reproducible observation, we need to optimize the condition for the robots to attach the Fields.<br/>
So we moved on to combine the robots with the Fields on various conditions.<br/>




<FONT SIZE=5 COLOR=#FF0000>'''Please click for more information!!'''</FONT><br/>
 
<FONT SIZE=4 COLOR=#FF0000>'''Please click for more information!!'''</FONT><br/>


<big>
<big>
<p style="text-align">
<p style="text-align">
*[http://openwetware.org/index.php?title=Biomod/2011/TeamJapan/Sendai/Result_Atomic_Force_Microscope/detail_Observation_struture%2Bfield '''Detail:Observation structure on the Field''']
*[http://openwetware.org/index.php?title=Biomod/2011/TeamJapan/Sendai/Result_Atomic_Force_Microscope/detail_Observation_struture%2Bfield '''Detail:Observation of the robot on the the Field''']
</p>
</p>
</big>
</big>
<br/>
<br/>
<FONT SIZE=4>'''Flow chart of experiments to bind the robots and fields'''</FONT><br/>
<FONT SIZE=4>'''Flow chart of experiments to bind the robots and the Fields'''</FONT><br/>
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<table border="0" align="center" vertical-align: middle;>
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==　Observation diary　==
== Conclusion ==
 
We confirmed the robots and the Fields by using high-speed AFM.<br/>
In addition, luckily we observed the 2D robots attached onto the fields.<br/>
However, we have not obtained the reproducible observation of the robot on the field yet despite of our vigorous seek on the experimental condition .<br/>
But from  the results of AFM we convince that the robots successfully combine  with the fields, and we also have [http://openwetware.org/index.php?title=Biomod/2011/TeamJapan/Sendai/Result_Atomic_Force_Microscope/detail_Observation_struture%2Bfield some solutions] to solve this reproducibility<br/>
We believe that the robots will move on the fields once we succeeded the reproducible attachment on the field.; the simulation result clearly shows the realistic possibility of the successful movement after the attachment.<br/>
 
== Observation diary ==


We wrote the observation diary for AFM.
We wrote the observation diary for AFM.

Latest revision as of 06:06, 2 November 2011

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High-speed AFM

We used a high-speed AFM(RIBM, Nano Live Vision：NLV) for observation. This high-speed AFM enables us to capture moving molecules in solution clearly without blurring.
So we can confirm not just the molecular robot, but also observe the molecular robot moving over the DNA origami field at video-rate.
The following figure below shows our observation flowchart.

Project stages verified by AFM

Observation of 2D and 3D structures

Triangular prism 2D structure

Figure 1. AFM 2D structures
Figure 2. caDNAno design: 2D view of origami unfolded body
Figure 3. 2D structure using all of M13


First, we checked 2D nanostructures. The shape of 3D nanostructures resemble that of aggregation. On the other hand,the shape of 2D nanostructures are rectangle and differ from aggregation clearly. That is why 2D nanostructures are easier to observe than 3D ones. Therefore, before doing the annealing for getting the triangular prism, we verified the 2D structure. The size of 2D structure in AFM is 67 nm × 23 nm(Figure 1).
As compared to our design(60 nm × 20 nm), the maximum error is 15percent.From electoric rebounding between double helix, the size of 2D nanostrucutre fall within the error range. We can affirm 2D nanostructures since our original design (Figure 2) depicts similar dimensions (60 nm × 20 nm).

We observed leftover scaffold strand from the middle of 2D nanostructure(Figure 3). We used M13mp18 for the sdaffold, though our robot only uses 1100 bases. That was the reason why the surplus M13 inhibit folding and moving of structure.
So, by restriction enzyme, we cut the surplus M13 in order to avoid unintended folding and interference when the robot moves on the Field during the race. After we cut the surplus M13,we also observed 2D structures made from cut M13(Figure 1). It proved that our experiment by restriction enzyme succeeded and we got 2D nanostructures using cut M13.


  • Sample conditions:

・Concentration M13:staples (1:10)
・Mg2+ concentration 12.5 mM
・Annealing time: 3 hours from 90°C to 25°C
・Buffer: Tris-HCl
・cut M13



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Triangular prism 3D structure

Figure 4. design of 3D structure
Figure 5. AFM 3D strucuture


Figure 4 is our design of 3D structure and Figure 5 shows the 3D structure of triangular prism in AFM. 3D structure consists of the three DNA rectangular faces. (Figure 4).

As compared to the figure of 2D structures, there are some differences. We can see the closed structure in figure 5.The closed structure was not observed in Figure 1. This structure was formed by self-assembly using three edging staples. There are two sticky ends in both ends of the 2D structures.

We found that the shape of the structure seemed to be like triangle because three DNA rectangular faces were opened up and adsorbed on mica (Figure 5). The reason of this phenomenon is that the 3D structure on mica was distorted and M13 linked to DNA rectangular faces were cut by AFM tip (Figure 6). It seems that the 3D structures were not stable in the presence of the external force by AFM.

The length of the side is almost equal to the design. We think that 3D structures are not distorted in the solution if there is no scratch by AFM.
Therefore, we conducted that the 3D structure is successfully self-assembled.


  • Condition

・M13:staples= 1:10(ratio of concentration)
・Mg2+ concentration 12.5 mM
・anealing time:3 hours from 90°C to 25°C
・Buffer:Tris-HCl
・cut M13


Figure 6. Model of 3D structure adsorbed on mica





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Observation of the Field

Figure 7. design of AFM Field
Figure 8. AFM Field


Figure 7 is design of the Field. Figure 8 is AFM image of the Field.
According to this image, redundant M13 was also observed.
The size of the Field in AFM image is similar to that of design.
Therefore, we succeeded in observing the Field.


  • Condition

・M13:staples= 1:10(ratio of concentration)
・Mg2+ concentration 12.5 mM
・anealing time:3 hours from 90°C to 25°C
・Buffer:Tris-HCl




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Observation of structure on the Field

Triangular prism 2D structure on the Field

Figure 9. 2D structure on the Field
Figure 10. No structure on the Field


In the previous section, we concluded that the Field,2D and 3D structure were also observed by using the high-speed AFM.
In other words,we accomplished observing all the elements necessary for our race by using high-speed AFM.
So we move on to combine the 3D structure with the Field.



Luckily we obtained the image of the 2D structure attached to the Field using high-speed AFM.
In this sample, there ought to be only robots (2D structures which couldn't be folded and 3D structures ) but several fields were observed in this sample.
We suppose that the previous solution with origami fields remained on cantilever, then that solution was mixed with new one and adsorbed on mica.
We scanned this field with 2D structure about 10 times but the structure didn't go away. Next, we raised the scanning speed then the structure was gone.
The structure was detached from the Field because cantilever scratched the structure.

It seems that robots can be attached to the Field.


  • Condition

・M13:staples= 1:10(ratio of concentration)
・Mg2+ concentration 12.5 mM
・Annealing time:3 hours from 90°C to 25°C
・Buffer:Tris-HCl

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Condition for the robots to attach the Fields

We confirmed the 2D structure attached to the Field using high-speed AFM. However this observation is lacking in reproducibility.
In order to obtain reproducible observation, we need to optimize the condition for the robots to attach the Fields.
So we moved on to combine the robots with the Fields on various conditions.


Please click for more information!!


Flow chart of experiments to bind the robots and the Fields
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Conclusion

We confirmed the robots and the Fields by using high-speed AFM.
In addition, luckily we observed the 2D robots attached onto the fields.
However, we have not obtained the reproducible observation of the robot on the field yet despite of our vigorous seek on the experimental condition .
But from the results of AFM we convince that the robots successfully combine with the fields, and we also have some solutions to solve this reproducibility
We believe that the robots will move on the fields once we succeeded the reproducible attachment on the field.; the simulation result clearly shows the realistic possibility of the successful movement after the attachment.

Observation diary

We wrote the observation diary for AFM. If you want to check it, please click Observation diary.

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