JP7622973B2 - Scanning Ion Conductance Microscope - Google Patents

Description

The present invention relates to a scanning ion conductance microscope, and in particular to a probe control structure that enables faster scanning speeds.

A scanning ion conductance microscope (SICM), also called a scanning ion conductance microscope, is a microscope belonging to the scanning probe microscope (SPM).
SICM uses an electrode inserted inside a nanopipette as a probe and fills it with an electrolyte liquid. By applying a voltage between the electrode on the electrolyte side holding the sample and measuring the ionic current that occurs, the distance between the nanopipette and the sample can be controlled, and surface information can be obtained from the positional information obtained at that time.
This makes it possible to measure even soft biological samples such as cells, and because visualization is possible on a nanoscale, it is expected that the uptake of viruses, etc. may also be visualized continuously.
In this case, a technical problem with the conventional SICM is that the time resolution in continuous scanning is insufficient.

Japanese Patent Laid-Open No. 2003-233633 discloses a scanning probe microscope including a scanning probe having two piezoelectric actuators with different response times.
The publication discloses two piezoelectric actuators, one of which is for performing a repeated cycle (hopping motion) of the tip of the probe against the surface of the sample, and the other is described as being for avoiding collisions between the probe and the surface of the sample due to delayed response of the actuator.
When scanning a sample surface in the hopping mode, the hopping operation of moving the tip of the probe toward and away from the sample surface is a bottleneck in increasing the imaging speed.
Use of another piezoelectric actuator for retraction, as disclosed in the same publication, makes it possible to avoid collisions between the probe and the surface of the sample due to delayed response of the actuator; however, the unevenness of a cell is approximately 10 μm, and an actuator that performs repeated cycles of approach and retraction must have a long driving range to match the unevenness of the cell, and the resonance frequency related to the response speed is low, which is one of the factors that makes it difficult to increase the speed of scanning measurement.

US Patent No. 09354249 (US20130312143)

The present invention aims to provide a scanning ion conductance microscope with increased scanning speed.

The scanning ion conductance microscope according to the present invention comprises a sample stage for holding a sample, a probe holding means for holding a probe, an XY direction control means for controlling the relative horizontal position between the surface of the sample and the tip of the probe, and a Z direction control means for controlling the vertical distance between the surface of the sample and the tip of the probe, and the Z direction control means is characterized in that it comprises a retract mode actuator for controlling the retraction of the tip of the probe relative to the sample surface, a hopping mode actuator for performing a hopping operation of the tip of the probe relative to the sample surface, and a trace actuator for performing a tracing operation of the tip of the probe relative to the sample surface.

In the present invention, the actuator for controlling the position in the Z direction is divided into three as described above for the following reason.
As explained above, the actuator with the repetitive cycle described in the cited document 1 was not fast enough to image the surface of a cell or the like having an unevenness of about 10 μm.
Therefore, in the present invention, the repeating cycle actuator described in the above cited reference 1 is divided into a tracing actuator for tracing the entire sample surface such as a cell along the large uneven shape, and a hopping mode actuator for repeatedly approaching and retracting from the sample surface.
As a result, the driving range of the hopping mode actuator can be made shorter than that of the cited document 1, and high speed can be achieved.
Therefore, it is preferred that the range of movement of the escape mode actuator is smaller than the range of movement of the hopping mode actuator, and the range of movement of the trace actuator is larger than the range of movement of the hopping mode actuator.
It is also preferable that the retract mode actuator has a faster response than the hopping mode actuator.

This will now be described in more detail.
When the tip of the probe is brought close to the surface of a sample, the ionic current between the electrode provided inside the probe and the electrode on the sample side changes.
Based on this change in ion current, information on the height of the sample surface and various other pieces of sample surface information can be obtained.
In the hopping mode, as the tip of the probe approaches the sample surface, the ion current decreases, and when the current decreases to a predetermined threshold, the probe tip hops away from the sample surface.
For example, the range of movement of the tip of the probe during hopping is on the order of 0.001 to 5 μm.
To achieve this high speed approach and retraction, it is necessary to use actuators such as piezoelectric elements that move at high speed. However, because the unevenness of a cell is on the order of 10 μm, using a relatively long actuator that can capture the entire image of the cell would result in a corresponding decrease in time resolution.
Therefore, the present invention is characterized in that separate actuators are used for the hopping motion and for tracing the cell surface.
This makes it possible to perform high-speed hopping operations while simultaneously imaging cells with uneven surfaces.

In addition to speeding up the hopping operation, to reduce the waiting time that occurs with each step-like movement in the horizontal XY direction, for example, as shown on the right side of Figure 3, hopping is performed while moving at a constant speed in the X-axis direction, and the XYZ coordinates are recorded when the sample surface is reached. These coordinates are applied to XY coordinates divided into, for example, 1024 x 1024, and the gaps between each point are further interpolated by calculation to match the pixel size, eliminating the need to move in the XY direction in fixed steps after hopping and making it possible to eliminate the waiting time after moving in steps in the XY direction.

In the present invention, three actuators corresponding to the retraction mode, hopping mode, and trace mode are provided as a means for controlling the tip of the probe in the Z direction perpendicular to the sample surface, thereby enabling faster continuous measurement scanning.

1 shows a schematic diagram of an XYZ scanning mechanism of a SICM according to the present invention. (a) shows the mounting structure of the actuator and measuring instrument that can be detached from the microscope body, and (b) shows an image diagram of measuring a sample made of cells. 1 shows an example of a scanning method using the SICM according to the present invention.

An example of the structure of an XYZ axis direction control mechanism (scanning mechanism) of a scanning ion conductance microscope according to the present invention will be described below with reference to the drawings.
As shown in FIG. 1, an XY stage 22 is provided on the upper surface of a vibration isolation table 11, the movement of which can be controlled in the horizontal X and Y directions.
In this embodiment, a sample stage 23 for holding a sample is provided on the XY stage 22, and electrodes (not shown) are attached to the sample stage 23.
The X-axis and Y-axis directions are controlled by piezoelectric elements.
In the Z-axis direction, a stepping motor 13 capable of being controlled to move vertically via a support portion 12 standing on a vibration isolation table 11 is provided.
In this embodiment, the actuator for controlling the probe 24 in the Z-axis direction and the small measuring device 20 for measuring the ion current are structured to be removable from the stepping motor (microscope body), but the actuator mechanism may also be attached directly to the stepping motor.

The mounting fixture 15 has mounting portions 15a and 15b having a recessed shape (or holes) as shown in FIG. 2, and is detachable from the pillar-shaped fixing portions 14a and 14b on the stepping motor 13 side by using screw members 14c and 14d.
A trace actuator 16, a hopping mode actuator 17 and a retraction mode actuator 18 are attached in this order to the lower side of the fixture 15, and a probe holding means 19 is connected to the retraction mode actuator 18 at the bottom end.
Each of the three actuators is made of a piezoelectric element.
A probe 24 is detachably attached to the probe holding means by using a fixing screw 19a.
The probe 24 is made of, for example, a glass nano-pit, has an electrode inserted therein, and is filled with an electrolyte.

The present invention is characterized in that the actuator that controls the position in the Z-axis direction is divided into three for each function, and there are no limitations on the mounting method or structure of the measuring device 20 that detects and measures ion current, etc.
In this embodiment, a measuring instrument 20 for applying a voltage between the electrode on the probe 24 side and the electrode on the sample side and measuring the minute ionic current flowing between the two electrodes is provided integrally with this actuator via a measuring instrument holder 21 suspended from the mounting fixture 15.

An example of scanning measurement will be described with reference to FIG.
FIG. 2( b ) shows a schematic diagram of a cell as sample 1 .
The tip of the probe 24 is brought close to the surface of the sample 1 .
When the tip of the probe 24 is positioned near the sample surface, the hopping mode actuator 17 is activated and the probe 24 moves further down toward the sample surface while measuring the ion current value.
When the ion current decreases and falls to a predetermined threshold, the retraction mode actuator 18 is activated to quickly retract the tip of the probe 24 a short distance, and then the hopping mode actuator 17 is activated to perform hopping 2 as shown by the arrow in Figure 2(b).
During this hopping motion, the surface shape of the cell is traced 3 by relatively moving in the X-axis direction and/or the Y-axis direction.

In this way, the actuator in the Z-axis direction is divided into three parts in accordance with the three different functions described above, so that the scanning method shown on the right side of FIG. 3 can be performed at high speed.
In FIG. 3, the movement of the probe is indicated by arrows.
Conventionally, as shown on the left side of FIG. 3, movement in the X-axis direction was performed in steps, which required a certain waiting time to be inserted before performing the hopping operation. However, with the present invention, it is possible to perform measurements while moving continuously at a constant speed without inserting this waiting time.

REFERENCE SIGNS LIST 1 sample 2 hopping 3 tracing 11 vibration isolation table 12 support 13 stepping motor 14a fixed part 14b fixed part 15 attachment 16 trace actuator 17 hopping mode actuator 18 retraction mode actuator 19 probe holding means 20 measuring instrument 21 measuring instrument holding part 22 XY stage 23 sample stage 24 probe

Claims (1)

A sample stage for holding a sample;
a probe holding means for holding the probe,
an XY direction control means for controlling a relative horizontal position between the surface of the sample and the tip of the probe;
a Z-direction control means for controlling a vertical distance between the surface of the sample and the tip of the probe,
The Z-direction control means has three actuators for each function, a hopping mode actuator, a trace actuator, and an escape mode actuator,
the hopping mode actuator lowers the tip of the probe toward the sample surface while measuring the ion current value, and when the ion current decreases to a predetermined threshold, the retraction mode actuator is activated to quickly retract the tip of the probe by a small distance, and then the hopping mode actuator performs a hopping operation;
1. A scanning ion conductance microscope, wherein the trace actuator traces the surface shape of a sample during a hopping motion .

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