**3. Single cell analysis based on an E‐SEM nanorobotic manipulation system**

Single cells analysis needs to be investigated through the micro/nano scale techniques based on the local environmental measurements and controls. We developed the Environmental‐ SEM (E‐SEM) nanorobotic manipulation system to manipulate and control the local environ‐ ments for biological samples in nano scale (Figure 2). It realized that direct observation and manipulation of water‐containing biological samples under nanometer high resolution imaging.

**Figure 2.** Single Cell Nano-surgery System with various nano-tools

microscopes (SEMs) and transmission electron microscopes (TEMs). For example, M. F. Yu et. al presented the tensile strength of individual CNTs inside a SEM [18]. Howeverthe resolution of SEM, generally ~1 nm resolution, is approximately one order in magnitude lower than that of a TEM. High resolution and transmission image of TEMs are useful for measurement and evaluation of nano‐scale objects. Kizuka et.al proposed the manipulation holder inside high‐ resolution transmission electron microscope (HR‐TEM). The manipulator was specially

Micro-Nano Mechatronics — New Trends in Material, Measurement, Control, Manufacturing and Their Applications in

However, the specimen chamber and observation area of TEM are too narrow to contain manipulators with complex functions. Hence, special sample preparation techniques are also needed. We proposed a hybrid nanorobotic manipulation system which is integrated TEM and SEM nanorobotic manipulators as core system for the Nanolaboratory [20, 21]. The strategy is named as hybrid nanomanipulation so as to differentiate it from those with only an exchange‐ able specimen holder. The most important feature of the manipulatoris that it contains several passive DOFs, which makes it possible to perform relatively complex manipulations whereas to keep compact volume to be installed inside the narrow vacuum chamber of a TEM [22].

Recently single cells analysis has been much more attentions because of the progress of the micro/nano scale techniques on the local environmental measurements and controls [23]. Under conventional SEMs and TEMs, the sample chambers of these electron microscopes are set under the high vacuum (HV) to reduce the disturbance of electron beam for observation. To observe water‐containing samples, for example bio‐cells, the appropriate drying and dying treatments are needed before observations. Hence, direct observations of water‐containing

On the other hand, the environmental‐SEM (E‐SEM) can be realized the direct observation of water‐containing samples with nanometer high resolution by specially built secondly electron detector [24]. The evaporation of water is controlled by the sample temperature (~0 ‐ ~40 °C) and sample chamber pressure (10 ‐ 2600 Pa). The unique characteristic of the E‐SEM is the direct observation of the hydroscopic samples with non‐drying treatment. Hence, the nano‐ manipulation inside the E‐SEM is considered to be an effective tool for a water‐containing

**3. Single cell analysis based on an E‐SEM nanorobotic manipulation**

Single cells analysis needs to be investigated through the micro/nano scale techniques based on the local environmental measurements and controls. We developed the Environmental‐ SEM (E‐SEM) nanorobotic manipulation system to manipulate and control the local environ‐ ments for biological samples in nano scale (Figure 2). It realized that direct observation and manipulation of water‐containing biological samples under nanometer high resolution

samples are normally quite difficult through these electron microscopes.

sample with nanometer resolution [25‐28].

**system**

imaging.

designed with atomic level positioning resolution [19].

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356

In this chapter, the novel local stiffness evaluation, local cutting, and local extraction of biological organism are presented by micro‐nanoprobes based on the E‐SEM nanorobotic manipulation system for future cell diagnosis and surgery system.

### **3.1. Adhesion force measurement of single cell using nano‐putter**

Cell activities, such as embryogenesis, mitosis, morphogenesis, cell orientation, cell motility, and survival depend on attachment to neighboring cells and the extracellular matrix. Cellular attachment to extracellular matrices influences cell morphology, cell function, and signaling mechanisms that direct cellular proliferation and differentiation [29]. Cell‐surface interaction is important in the development of any material or device for biomedical applications, since the performance of a medical device in the body must be compatible with the surrounding tissue [30, 31]. Understanding of the cell adhesion process would benefit the development of suitable biomaterials or device for both tissue engineering and medical fields.

Cell adhesion processes are influenced by numerous parameters, such as the nature of the biomaterial and its surface characteristics (roughness, topography, chemical composition, surface wettability, surface charge and surface treatments) have been investigated. However, the effect of ambient humidity on cell adhesion has had less attention, especially at the single cell level. Understanding the adhesion force at various humidity conditions could help us to better understand the processes of cell‐directed integration during water evaporation. The understanding is also useful in controlling yeast infections at wet environment. Moreover, cell adhesion is influenced by the surface energy of substrate strongly, but the mechanism is still not clear[32]. The study of cell adhesion on substrates with different surface energy could help us to understand the adhesion mechanism better.

We presented a yeast cell adhesion force measurement performed using the nanorobotic manipulation system inside the ESEM [33, 34, 35]. Figure 3 (A) shows a typical force‐displace‐ ment curve during the single cell adhesion force measurement. Figure 3 (B) shows the initial position of the micro putter and the single cell. The micro putter was driven by the nanorobotic manipulation system. First, it was moved towards the cell until it contacted the cell (Figure 3 (C)). Then, a continuous movement was applied to the micro putter by the nanomanipulator. The micro putter beam deflected owing to the increasing pushing force. Figure 3 (D) shows the deflection of the micro putter during the adhesion force measurement. Finally, the cell was detached from its initial position under a certain force (Figure 3 (E)). The maximum force during this manipulation procedure was defined as the adhesion force.

Single yeast cell adhesion force measurement was performed at three humidity conditions, i.e. 100%, 70% and 40%. The mean adhesion force and the deviation are with 95% confidence at each humidity conditions. It demonstrates that the yeast cell adhesion forces range from 10 to 25 μN at various humidity conditions. The adhesion forces were 11.0 ± 5.1 μN, 17.4 ± 4.7 μN and 23.5 ± 6.1 μN at 100%, 70% and 40% relative humidity conditions respectively. It showed clearly that the cell adhesion was affected by the ambient humidity. The cell adhesion force is larger at low humidity than at high humidity. For example, the cell adhesion force was 23.5 μN at a humidity of 40%, which was 1.14 times largerthan the force 11.0 μN at humidity 100%.

#### **3.2. Single cell cutting using nano‐knife**

Cell cutting is an important step in cell analysis processes. For instance, it was widely used to prepare cell specimen slices for the observation of an inner structure [36]. Different to group cells analysis,research on individual cells could give accurate data ratherthan average results. Single cell analysis can help us to understand the biological processes more accurately. In‐situ single cell cutting technique could potentially benefit cell analysis, such as single cell operation and disease treatment.

Recently, a nano knife fabricated from a carbon nanotube (CNT) has been developed for the purpose of cell cutting [37]. The nano knife was designed by welding a CNT across two tungsten needles inside a scanning electron microscopy (SEM). This device can reduce the angle by which the sample is bent during cutting, due to the small diameter of the CNT. It can be seen clearly that the nano knife can leave a mark on the epon resin surface, which means it can cut very thin slices of cells. However, the bonding force between the CNT and tungsten probes by using electron beam induced deposition (EBID) method is quite small. There are still certain types of hard specimens such as bone, plants, and thick‐walled spores. The CNT based nano knife may not be able to deal with such samples, since a larger cutting force is required, especially when the sample size is large.

We presented a nano knife with a buffering beam was designed for an in‐situ single cell cutting purpose [38]. A schematic drawing of the single cell cutting using a nano knife is shown in Figure 4. The nano knife was immobilized to the nanomanipulator by using the electrical conductive tape inside the ESEM chamber. Underthe driving of the nanorobotic manipulation system, the nano knife can move towards and cut the single cell finally. The cutting force can be calculated based on the deformation of the nanokinfe's beam.

**Figure 3.** Single cell adhesion force measurement using micro putter inside ESEM.

Single Cell Nanosurgery System 359

**Figure 3.** Single cell adhesion force measurement using micro putter inside ESEM.

We presented a yeast cell adhesion force measurement performed using the nanorobotic manipulation system inside the ESEM [33, 34, 35]. Figure 3 (A) shows a typical force‐displace‐ ment curve during the single cell adhesion force measurement. Figure 3 (B) shows the initial position of the micro putter and the single cell. The micro putter was driven by the nanorobotic manipulation system. First, it was moved towards the cell until it contacted the cell (Figure 3 (C)). Then, a continuous movement was applied to the micro putter by the nanomanipulator. The micro putter beam deflected owing to the increasing pushing force. Figure 3 (D) shows the deflection of the micro putter during the adhesion force measurement. Finally, the cell was detached from its initial position under a certain force (Figure 3 (E)). The maximum force

Micro-Nano Mechatronics — New Trends in Material, Measurement, Control, Manufacturing and Their Applications in

Single yeast cell adhesion force measurement was performed at three humidity conditions, i.e. 100%, 70% and 40%. The mean adhesion force and the deviation are with 95% confidence at each humidity conditions. It demonstrates that the yeast cell adhesion forces range from 10 to 25 μN at various humidity conditions. The adhesion forces were 11.0 ± 5.1 μN, 17.4 ± 4.7 μN and 23.5 ± 6.1 μN at 100%, 70% and 40% relative humidity conditions respectively. It showed clearly that the cell adhesion was affected by the ambient humidity. The cell adhesion force is larger at low humidity than at high humidity. For example, the cell adhesion force was 23.5 μN at a humidity of 40%, which was 1.14 times largerthan the force 11.0 μN at humidity 100%.

Cell cutting is an important step in cell analysis processes. For instance, it was widely used to prepare cell specimen slices for the observation of an inner structure [36]. Different to group cells analysis,research on individual cells could give accurate data ratherthan average results. Single cell analysis can help us to understand the biological processes more accurately. In‐situ single cell cutting technique could potentially benefit cell analysis, such as single cell operation

Recently, a nano knife fabricated from a carbon nanotube (CNT) has been developed for the purpose of cell cutting [37]. The nano knife was designed by welding a CNT across two tungsten needles inside a scanning electron microscopy (SEM). This device can reduce the angle by which the sample is bent during cutting, due to the small diameter of the CNT. It can be seen clearly that the nano knife can leave a mark on the epon resin surface, which means it can cut very thin slices of cells. However, the bonding force between the CNT and tungsten probes by using electron beam induced deposition (EBID) method is quite small. There are still certain types of hard specimens such as bone, plants, and thick‐walled spores. The CNT based nano knife may not be able to deal with such samples, since a larger cutting force is

We presented a nano knife with a buffering beam was designed for an in‐situ single cell cutting purpose [38]. A schematic drawing of the single cell cutting using a nano knife is shown in Figure 4. The nano knife was immobilized to the nanomanipulator by using the electrical conductive tape inside the ESEM chamber. Underthe driving of the nanorobotic manipulation system, the nano knife can move towards and cut the single cell finally. The cutting force can

during this manipulation procedure was defined as the adhesion force.

**3.2. Single cell cutting using nano‐knife**

required, especially when the sample size is large.

be calculated based on the deformation of the nanokinfe's beam.

and disease treatment.

Biomedical Engineering

358

The in situ single cell cutting experiment was performed using these three nano knives. Figure 5(A) shows the initialposition ofthe nano knife anda single cell. Figure 5(B) shows the touching between the nano knife tip and the single cell. The deformation of the nano knife beam and the single cell during the cutting is shown in Figure 5(C). The deformation of the beam can be measured from the ESEM image directly using image analysis software. Therefore, the cutting force can be calculated based on Hooke's law. The separated single cell after cutting is shown in Figure 5(D). The sample slice angle can be measured from the ESEM image directly as well. Figure 5(E) shows the single cell cutting image using the 25o knife. Figure 5(F) shows the sample slice angle after cutting. The images of single cell cutting and sample slice angle after cutting using 45o knife are shown in Figure 5 (G) and Figure 5 (H).

**4. Conclusion**

one of the model organisms [40, 41]

**Acknowledgements**

**Author details**

**References**

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tive: University of California Press; 1986.

TRENDS in Pharmacological Sciences 2005;26 265‐273.

This chapter presents the single cell nanosurgery system based on nanomanipulation techni‐ ques.The micro‐nano tools have been proposed to investigate single cell analysis to manipulate and control the local environment in micro‐nano scale. The E‐SEM nanomanipulation system was constructed to realize the local stiffness evaluation, local cutting, and local extraction of biological organism in nano‐meter scale. The adhesion force measurement was presented by micro‐putter for single cells. The single cell cutting was also described using nano‐knife. As future direction, the multiple micro‐nanotools are used continuously depending on the purposes by exchanging machinery system (NTExS: Nanotool Exchanger System) [39]. We are investigating on the nanoinjection applications for the Caenorhabditis elegans (C. elegans) as

Single Cell Nanosurgery System 361

The authors are grateful to Prof. T. Inada N. Uozumi for providing with W303 yeast cells. This work was partially supported by a Grant‐in‐Aid for Scientific Research from the Ministry of

Education, Culture, Sports, Science and Technology of Japan.

Toshio Fukuda, Masahiro Nakajima, Yajing Shen and Masaru Kojima

Department of Micro‐Nano Systems Engineering, Nagoya University, Nagoya, Japan

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**Figure 4.** Schematic drawing of the single cell cutting experiment using nano knife inside ESEM.

**Figure 5.** Single cell cutting using nano knife inside ESEM.
