JP2004156959A - Scanning probe microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a scanning probe microscope for submergence that does not require the positioning of a cantilever precisely and can stably excite the cantilever in liquid. <P>SOLUTION: The scanning probe microscope 100 comprises a liquid stage 110 for retaining liquid 102, the cantilever 124 having a probe 122 at a free end, a holder 126 for retaining the cantilever 124 in the liquid 102, a sample stand 132 for retaining the biological sample 104 in the liquid 102, an XYZ scanner 134 for scanning the biological sample 104 in XY directions and a Z direction to the cantilever 124, an excitation signal generation section 158 for generating an excitation signal having a frequency near the mechanical resonance frequency in the cantilever 124 for exciting the cantilever 124 at the mechanical resonance frequency, and a transducer 170 for generating ultrasonic waves according to the excitation signal and uniformly irradiating the cantilever and the periphery with the ultrasonic waves. The biological sample can be more accurately measured by this sort of scanning probe microscope. <P>COPYRIGHT: (C)2004,JPO

Description

【０１１１】
ここで、Ｈ_ｃは角振動数ω_ｃにおける振動子１７２への入力から変位検出センサー１４０の出力までの入出力特性（ゲイン）、Ａ_ｋ０はカンチレバー１２４と生体試料１０４が非接触状態時（初期状態）の変位検出センサー１４０の出力信号の振幅Ａ_ｋである。
【０１１２】
Ｈ_ｃは、振動子１７２が電気信号を受けて超音波を発生させるときの変換効率、超音波伝搬部１７４における超音波の伝搬効率、超音波伝搬部１７４と液体１０２の境界における超音波の透過率、液体１０２における超音波の伝搬効率、カンチレバー１２４と生体試料１０４が非接触状態のときの超音波によるカンチレバー１２４の励振効率（χ_０）、カンチレバー１２４の振動が変位検出センサー１４０で電気信号に変換されるときの変換効率（検出感度）を含んでいる。
【０１１３】
カンチレバー１２４と生体試料１０４が接触しているときの安定状態では次の関係式が成り立つ。
【０１１４】
【数２】

【０１１５】
ここで、χはカンチレバー１２４と生体試料１０４が接触状態のときの超音波によるカンチレバー１２４の励振効率である。（χ／χ_０）はカンチレバー１２４と生体試料１０４の接触状態を示しており、これを超音波によるカンチレバー１２４の励振効率比α（０≦α≦１）とする。励振効率比αは式（２）より次式で表されれる。
【０１１６】
【数３】

【０１１７】
従って、カンチレバー１２４を振動させるための信号（振動子１７２へ供給する信号）Ｓは、式（１）と式（３）より次式で表すことができる。
【０１１８】
【数４】

【０１１９】
ＫＨ_ｃは、変位検出センサー１４０と位相シフト回路５８２とゲイン調整回路５８６と加算回路５８８と振動子１７２と超音波伝搬部１７４とカンチレバー１２４とで構成される正帰還の帰還ゲインであり、０≦ＫＨ_ｃ＜１である。なお、ＫＨ_ｃが１以上になると発振が起きる。
【０１２０】
Ｈ_ｃとＡ_ｋ０は定数として扱えるので、規格化のためにＨ_ｃ＝１、Ａ_ｋ０＝１とおくと、式（４）のＳは、Ｋとαの関数として、次のように表せる。
【０１２１】
【数５】

【０１２２】
Ｓ（Ｋ，α）をＫのいくつかの代表値に対してプロットしたものを図１４に示す。図１４は、αをパラメーターとするＳ（０，α）とＳ（０．１，α）とＳ（０．５，α）とＳ（０．８，α）とＳ（０．９９，α）とを示している。
【０１２３】
図１４より、生体試料１０４とカンチレバー１２４の接触状態α、言い換えれば、カンチレバー１２４の励振効率比αに応じて、カンチレバー１２４を振動させるための信号（振動子１７２へ供給する信号）Ｓをコントロールできることがわかる。すなわち、カンチレバー１２４と生体試料１０４が接触していない状態ではＳを大きくし、カンチレバー１２４と生体試料１０４が接触している状態ではＳを減らすことができることがわかる。その効果は、Ｋを１に近づけていくほど、より大きくなることがわかる。
【０１２４】
なお、Ｋ＝０とすると、第一実施形態〜第三実施形態で示したように、励振信号生成部１５８から出力される励振信号をそのまま振動子１７２に供給することと同様になる。
【０１２５】
上述によると、Ｋ≒１（正確にはＫＨ_ｃ≒１）とするのが望ましいように思える。しかし、Ｋ（正確にはＫＨ_ｃ）を１に近づけるほど正帰還による発振が生じやすくなり動作が不安定になる。このため、Ｋ（正確にはＫＨ_ｃ）は実際には０．５〜０．８の値に設定されるとよい。
【０１２６】
次に、生体試料１０４とカンチレバー１２４の接触状態αに応じてカンチレバー１２４を振動させるための信号（振動子１７２へ供給する信号）Ｓをコントロールすることによって、生体試料１０４へ与えるダメージの低減にどれだけの効果があるかを評価する。
【０１２７】
生体試料１０４に与えるダメージＤは、超音波によるカンチレバー１２４の励振効率の損失分χ_０（１−α）とカンチレバー１２４を振動させるための信号（振動子１７２へ供給する信号）Ｓを掛け合わせたものとして考えることができる。すなわち、ダメージＤは、Ｋとαの関数として、次のように表せる。
【０１２８】
【数６】

χ_０は定数と見なせるので、規格化のためにχ_０＝１とおくと、式（６）のＤは次のように表せる。
【０１２９】
【数７】

【０１３０】
Ｄ（Ｋ，α）をＫのいくつかの代表値に対してプロットしたものを図１５に示す。図１５は、αをパラメーターとするＤ（０，α）とＤ（０．１，α）とＤ（０．５，α）とＤ（０．８，α）とＤ（０．９９，α）とを示している。
【０１３１】
図１５より、生体試料１０４とカンチレバー１２４の接触状態αに応じて、カンチレバー１２４を振動させるための信号（振動子１７２へ供給する信号）Ｓをコントロールすることによって、生体試料１０４に与えるダメージを急激に低減できることがわかる。その効果は、Ｋを１に近づけていくほど、より大きくなることかわかる。
【０１３２】
従って、生体試料１０４の種類や測定する情報などを考慮して、ゲイン調整回路５８６のゲインＫを適切に設定することにより、測定時に生体試料１０４に与えるダメージを適切に低減することができる。
【０１３３】
本実施形態の走査型プローブ顕微鏡５００は、第一実施形態の走査型プローブ顕微鏡１００と同様の利点を有している。これに加えて、本実施形態の走査型プローブ顕微鏡５００は、生体試料１０４とカンチレバー１２４の接触状態に基づいて、言い換えれば、変位検出センサー１４０の出力信号に基づいて、振動子１７２に供給する励振信号を調整する励振コントロール回路５８０を有しているため、測定時に生体試料１０４に与えるダメージを大幅に低減することができる。
【０１３４】
第六実施形態
第六実施形態の走査型プローブ顕微鏡を図１６に示す。図１６において、図１の部材と同一の参照符号で示された部材は同等の部材を示しており、それらの詳しい説明は記載の重複を避けて省略する。
【０１３５】
走査型プローブ顕微鏡は、変位検出センサー１４０からの変位信号に基づいて調整された励振信号を出力する励振コントロール回路６８０を有している。励振コントロール回路６８０は、カンチレバー１２４をその機械的共振周波数で自発的に振動させる（自励発振させる）とともに、測定時の試料に与えるダメージを低減する回路である。
【０１３６】
つまり、本実施形態の励振コントロール回路６８０は、第四実施形態の自励発振回路４８０と第五実施形態の励振コントロール回路５８０の機能を兼ね備えている。励振コントロール回路６８０は、言い換えれば、部分的に励振信号生成部を構成している。従って、励振コントロール回路６８０はトランスデューサー１７０と共にカンチレバー励振手段を構成している。
【０１３７】
これに伴い、本実施形態の走査型プローブ顕微鏡６００のコントローラー４５０は、励振信号生成部を有していない。つまり、コントローラー４５０は、第一実施形態のコントローラー１５０から励振信号生成部１５８を省いた構成となっている。
【０１３８】
図１７に示されるように、励振コントロール回路６８０は、変位検出センサーからの変位信号の位相を９０度進める位相シフト回路６８２と、位相シフト回路６８２の出力信号のレベルを一定に保つオートゲインコントロール回路６８４と、位相シフト回路６８２の出力信号に所定のゲインをかけるゲイン調整回路６８６と、ゲイン調整回路６８６の出力信号にオートゲインコントロール回路６８４の出力信号を加算する加算回路６８８とを有している。
【０１３９】
変位検出センサー１４０と位相シフト回路６８２とオートゲインコントロール回路６８４と加算回路６８８とトランスデューサー１７０とカンチレバー１２４はカンチレバー１２４の機械的共振周波数において正帰還を構成する。さらに、正帰還の帰還ゲインはオートゲインコントロール回路６８４によって１以上に調整される。この正帰還は、図１１に示した自励発振回路と等価な回路を構成している。このため、カンチレバー１２４はその機械的共振周波数で発振される。その振動振幅は、オートゲインコントロール回路６８４により所定のレベルに保たれる。
【０１４０】
変位検出センサー１４０と位相シフト回路６８２とゲイン調整回路６８６と加算回路６８８とトランスデューサー１７０とカンチレバー１２４はカンチレバー１２４の機械的共振周波数において正帰還を構成する。さらに、正帰還の帰還ゲインはゲイン調整回路６８６によって１未満に調整される。このため、この正帰還は、図１３に示した励振コントロール回路５８０と等価な回路を構成している。このため、生体試料１０４の種類や測定する情報などを考慮して、ゲイン調整回路６８６のゲインＫを適切に設定することにより、測定時に生体試料１０４に与えるダメージを適切に低減することができる。
【０１４１】
本実施形態の走査型プローブ顕微鏡６００は、第一実施形態の走査型プローブ顕微鏡１００と同様の利点を有している。これに加えて、本実施形態の走査型プローブ顕微鏡６００は、カンチレバー１２４を自励発振させる機能を有する励振コントロール回路６８０を有しているため、励振信号生成部を別途に設けることなく、カンチレバー１２４をその機械的共振周波数で振動させることができる。これは走査型プローブ顕微鏡の操作性の向上（操作手順の簡略化や操作時間の短縮）にとって有益である。
【０１４２】
また、本実施形態の走査型プローブ顕微鏡６００は、生体試料１０４とカンチレバー１２４の接触状態に基づいて、言い換えれば、変位検出センサー１４０の出力信号に基づいて、振動子１７２に供給する励振信号を調整する機能を有する励振コントロール回路６８０を有しているため、測定時に生体試料１０４に与えるダメージを大幅に低減することができる。
【０１４３】
【発明の効果】
本発明によれば、高い精度でのカンチレバーの位置決めを必要とせず、液体中においてカンチレバーを安定に励振できる液中用走査型プローブ顕微鏡が提供される。これにより、生体試料をより正確に測定することができる。
【図面の簡単な説明】
【図１】本発明の第一実施形態の走査型プローブ顕微鏡を示している。
【図２】図１のステージを上方から見た図である。
【図３】図１と図２に示されたトランスデューサーの上面図（Ａ）と側面図（Ｂ）と正面図（出力端側端面図）（Ｃ）とを示している。
【図４】図３に示されたトランスデューサーに代えて適用可能な別のトランスデューサーの上面図（Ａ）と側面図（Ｂ）と正面図（出力端側端面図）（Ｃ）とを示している。
【図５】図３に示されたトランスデューサーに代えて適用可能な別のトランスデューサーの上面図（Ａ）と側面図（Ｂ）と正面図（出力端側端面図）（Ｃ）とを示している。
【図６】図３に示されたトランスデューサーに代えて適用可能な別のトランスデューサーの上面図（Ａ）と側面図（Ｂ）と正面図（出力端側端面図）（Ｃ）とを示している。
【図７】図１に示された変位検出センサーの出力特性を示している。
【図８】本発明の第二実施形態の走査型プローブ顕微鏡を示している。
【図９】本発明の第三実施形態の走査型プローブ顕微鏡を示している。
【図１０】本発明の第四実施形態の走査型プローブ顕微鏡を示している。
【図１１】図１０に示された走査型プローブ顕微鏡の部分的に示す図であり、図１０に示された自励発振回路の構成を示している。
【図１２】本発明の第五実施形態の走査型プローブ顕微鏡を示している。
【図１３】図１２に示された走査型プローブ顕微鏡の部分的に示す図であり、図１２に示された励振コントロール回路の構成を示している。
【図１４】式（５）で表されたＳ（Ｋ，α）をＫのいくつかの代表値に対してプロットしたグラフを示している。
【図１５】式（７）で表されたＤ（Ｋ，α）をＫのいくつかの代表値に対してプロットしたグラフを示している。
【図１６】本発明の第六実施形態の走査型プローブ顕微鏡を示している。
【図１７】図１６に示された走査型プローブ顕微鏡の部分的に示す図であり、図１６に示された励振コントロール回路の構成を示している。
【図１８】従来の生体用原子間力顕微鏡の一例の構成を示している。
【図１９】あるカンチレバーの大気中での振動特性を示している。
【図２０】大気中において図１９に示された振動特性を持つカンチレバーの液中での振動特性を示している。
【図２１】液体中において図２０に示された振動特性を持つカンチレバーに対する、図１８に示された光てこセンサーの出力特性を示している。
【符号の説明】
１００ 走査型プローブ顕微鏡
１０２ 液体
１０４ 生体試料
１１０ 液体ステージ
１２２ 探針
１２４ カンチレバー
１２６ ホルダー
１３２ 試料台
１３４ ＸＹＺスキャナー
１４０ 変位検出センサー
１４８ 振幅検出回路
１５２ Ｘ走査信号生成部
１５４ Ｙ走査信号生成部
１５６ Ｚ制御回路
１５８ 励振信号生成部
１６０ ホストコンピューター
１６２ Ｘ駆動回路
１６４ Ｙ駆動回路
１６６ Ｚ駆動回路
１７０ トランスデューサー
１７２ 振動子
１７４ 超音波伝搬部
１７６ 出力端[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a scanning probe microscope. In particular, it relates to an atomic force microscope capable of observing a biological sample. The atomic force microscope can perform manipulation of a biological sample, observation and processing of a sample other than the biological sample, and the like.
[0002]
[Prior art]
A scanning probe microscope (SPM) is a general term for a device that mechanically scans a mechanical probe to obtain information on a sample surface, and includes a scanning tunneling microscope (STM), an atomic force microscope (AFM), and a scanning type microscope. It includes a magnetic force microscope (MFM), a scanning capacitance microscope (SCaM), a scanning near-field optical microscope (SNOM), a scanning thermal microscope (SThM), and the like.
[0003]
The scanning probe microscope can perform raster scanning of the mechanical probe and the sample relatively in the X and Y directions, obtain surface information of a desired sample region through the mechanical probe, and display the information on a monitor TV by mapping. it can.
[0004]
Atomic force microscopes, among other scanning probe microscopes, have attracted attention as having the potential to observe the movement of living biological samples in liquids at higher resolution than optical microscopes.
[0005]
Until now, the only device that can observe the movement of a biological sample is an optical microscope, but the optical microscope cannot observe the sample at a resolution equal to or less than the wavelength of light because of the diffraction limit.
[0006]
An electron microscope can realize high resolution on the order of nanometers, but cannot observe a living biological sample in a liquid because an object to be measured cannot be arranged in the liquid.
[0007]
In contrast, the AFM can be expected to have a high resolution on the order of nanometers, and can observe even a sample in a liquid. Moreover, it is one of the reasons that attention has been paid to the fact that it can be easily combined with an optical microscope.
[0008]
Such a biological AFM often employs a method (AC mode) of detecting an interaction acting between sample probes from the vibration characteristics of a cantilever. This is because there is an advantage that the force acting between the sample and the probe can be kept weak as compared with the normal mode.
[0009]
For example, FIG. 18 is a block diagram illustrating a configuration of an example of a conventional biological AFM.
[0010]
In FIG. 18, the stage 10 has been subjected to a water-repellent treatment, and can hold the liquid 14 by surface tension. The sample 7 held by the XYZ scanner 6 is arranged in the liquid 14, and can be scanned in the XYZ directions by the X drive circuit 3, the Y drive circuit 4, and the Z drive circuit 5 based on a signal output from the controller 2. It has become.
[0011]
In the liquid 14, a cantilever 8 having a probe at a free end is disposed so as to face the sample 7. This cantilever 8 is held by a holder 9 provided on a stage 10. A piezoelectric element 11 is provided on the holder 9 and can mechanically vibrate the cantilever 8 at a predetermined amplitude and frequency in response to an excitation signal from the controller 2.
[0012]
An optical lever sensor 16 for detecting the displacement of the cantilever 8 is arranged below the stage 10. The optical lever sensor 16 has a configuration similar to that shown in, for example, Japanese Patent Application Laid-Open No. 2002-82037, and outputs the laser beam 13 converged through the objective lens 15 and the transmission glass 12 provided on the stage 10. The free end of the cantilever 8 is irradiated to detect the displacement of the cantilever 8. The optical lever sensor 16 outputs a displacement signal indicating the detected displacement of the cantilever 8 to the amplitude detection circuit 17.
[0013]
The amplitude detection circuit 17 calculates the amplitude value of the displacement signal output from the optical lever sensor 16 and outputs an amplitude signal indicating the calculated amplitude value of the cantilever 8 to the Z control circuit 18 in the controller 2. The Z control circuit 18 controls the displacement of the XYZ scanner 6 in the Z direction through the Z drive circuit 5 so as to keep the amplitude signal output from the amplitude detection circuit 17 constant.
[0014]
In this AFM, the piezoelectric element 11 receives an excitation signal from the controller 2 and excites the cantilever 8 with a mechanical resonance frequency of the cantilever 8 and a predetermined amplitude. Further, the displacement of the cantilever 8 is detected by the optical lever sensor 16, and the XYZ scanner 6 is moved by the Z control circuit 18 and the Z drive circuit 5 in the Z direction (normal line of the sample 7) so that the vibration amplitude of the cantilever 8 becomes constant. Direction) to control the position of the sample 7 held by the XYZ scanner 6 in the Z direction.
[0015]
In parallel with this, the XYZ scanner 6 is driven by the X drive circuit 3 and the Y drive circuit 4 in the XY plane direction, and the sample 7 is two-dimensionally scanned with respect to the cantilever 8. The host computer 1 acquires drive signals of the X drive circuit 3 and the Y drive circuit 4, that is, applied voltage signals for displacing the XYZ scanner 6 in the X direction and the Y direction as position signals on the sample surface, and performs Z control. An output signal of the circuit 18, that is, an applied voltage signal for displacing the XYZ scanner 6 in the Z direction is acquired as a concavo-convex signal on the sample surface, and an image is formed and displayed based on the acquired position signal and concavo-convex signal.
[0016]
In the AFM in the AC mode described above, since the vibration of the cantilever is greatly attenuated by the liquid, the excitation efficiency of the cantilever is very poor. For this reason, there is a problem that the vibration characteristics of the holder that supports the cantilever appear as noise.
[0017]
FIG. 19 shows the vibration characteristics of a certain cantilever in the atmosphere, and FIG. 20 shows the vibration characteristics of the same cantilever in a liquid. As shown in FIG. 19, even in a cantilever exhibiting high excitation efficiency (Q value) at a mechanical resonance frequency in the atmosphere, the mechanical resonance frequency decreases in a liquid as shown in FIG. At the same time, the excitation efficiency (Q value) is extremely reduced.
[0018]
Therefore, the excitation efficiency of the cantilever with respect to the holder is reduced, and the vibration characteristics of the holder supporting the cantilever appear as noise in the displacement signal of the cantilever detected by the optical lever sensor. FIG. 21 shows the output characteristics of the optical lever sensor with respect to the cantilever having the vibration characteristics shown in FIG. It is no longer possible to determine the mechanical resonance frequency of the cantilever from the output characteristics of such an optical lever sensor. This is because mechanical vibration is transmitted to the cantilever via the holder.
[0019]
JP-A-7-174767 discloses a method of exciting a cantilever by irradiating a free end of the cantilever with a focused ultrasonic wave, instead of exciting the cantilever by transmitting mechanical vibration to the cantilever via a holder. Is proposed.
[0020]
[Patent Document 1]
JP-A-2002-82037
[0021]
[Patent Document 2]
JP-A-7-174767
[0022]
[Problems to be solved by the invention]
However, JP-A-7-174767 has the following problem.
[0023]
The size of the cantilever is as large as about 100 μm in length and about 30 μm in width, and as small as about 10 μm in length and about 2 μm in width. In order to accurately irradiate the free end of such a small cantilever with ultrasonic waves, it is necessary to converge the ultrasonic waves to several tens of μm or less and to position the cantilever with respect to the ultrasonic waves with high accuracy. Therefore, it is necessary to provide a position adjusting mechanism for controlling the position of the cantilever with high accuracy. However, providing such a position adjusting mechanism not only increases the size and complexity of the apparatus, but also increases the time required for measurement preparation such as cantilever replacement.
[0024]
In addition, an ultrasonic wave converged to several tens μm or less has an intensity distribution having a steep peak. Therefore, the excitation efficiency of the cantilever changes due to the relative drift of the focal length of the cantilever and the ultrasonic wave, that is, the drift of the position adjustment mechanism and the transducer. Therefore, in order to stably excite the cantilever, it is necessary to minimize vibration noise and thermal drift of the position adjustment mechanism and the transducer.
[0025]
The present invention has been made in view of such a situation, and has as its object the use in a liquid that can stably excite the cantilever in a liquid without requiring high-precision positioning of the cantilever. An object of the present invention is to provide a scanning probe microscope.
[0026]
[Means for Solving the Problems]
The present invention is a submerged scanning probe microscope for observing a biological sample in a liquid with a probe, and has an X axis and a Y axis that are both parallel to a horizontal plane and orthogonal to each other, and a Z axis that is orthogonal to the horizontal plane. The scanning probe microscope includes a liquid stage for holding a liquid, a cantilever having a probe at a free end, a holder for holding the cantilever in the liquid held on the liquid stage, and a liquid held for the liquid stage. A sample stage for holding the sample therein, a scanner for moving the sample with respect to the cantilever at least along the Z axis (Z scanning), a cantilever exciting means for exciting the cantilever at a frequency near its mechanical resonance frequency, A displacement detection sensor that optically detects the displacement of the free end of the cantilever and outputs a displacement signal reflecting the displacement, and a displacement detection sensor An amplitude detection circuit that calculates an oscillation amplitude of the cantilever based on a displacement signal from the sensor and outputs an amplitude signal reflecting the oscillation amplitude, and a cantilever and a sample along the Z axis based on the amplitude signal from the amplitude detection circuit. A Z control circuit that determines a target value of the interval and outputs a Z control signal corresponding to the target value; a Z drive circuit that controls displacement of the scanner along the Z axis according to the Z control signal; Along with calculating the information on the surface of the sample by calculation, it has a calculation display unit for displaying the obtained information on the surface of the sample, and the cantilever excitation unit generates an excitation signal according to the excitation signal generation unit and the excitation signal. It has a transducer that generates ultrasonic waves and irradiates the cantilever and its surroundings almost uniformly.
[0027]
The scanner should be able to move the sample along the X and Y axes (XY scan) relative to the cantilever in addition to the Z scan, and correspondingly, the scanning probe microscope should An X-scan signal generator for generating an X-scan signal for displacing along the X-axis, a Y-scan signal generator for generating a Y-scan signal for displacing the scanner along the Y-axis, and an X-axis of the scanner according to the X-scan signal It is preferable to further include an X drive circuit that controls displacement along the Y axis and a Y drive circuit that controls displacement along the Y axis of the scanner according to the Y scanning signal. Information on the surface of the sample is obtained by calculation based on the X scan signal and the Y scan signal.
[0028]
The transducer has a vibrator that generates ultrasonic waves according to an input excitation signal, and an ultrasonic wave propagation unit that propagates the ultrasonic waves generated by the vibrator. The ultrasonic wave propagation unit has an output end for emitting ultrasonic waves to the outside, and at least the output end of the ultrasonic wave propagation unit is located in the liquid held on the liquid stage.
[0029]
The ultrasonic wave propagation section preferably has a horn for amplifying the ultrasonic wave. Further, the ultrasonic wave propagation section is preferably made of a polymer material.
[0030]
The ultrasonic wave propagation unit is fixed to the liquid stage via, for example, a vibration isolating rubber.
[0031]
The transducer may be held by the scanner, and the ultrasonic wave propagation part of the transducer may also serve as the sample stage.
[0032]
The scanner may include a Z scanner for moving (Z scanning) the sample along the Z axis with respect to the cantilever, and the Z scanner may also serve as a transducer of the transducer. In this case, the scanning probe microscope further includes an adder for adding the Z control signal and the excitation signal, and the output signal of the adder is input to the Z drive circuit.
[0033]
The excitation signal generation unit may be a self-excited oscillation circuit that generates an excitation signal based on a displacement signal from the displacement detection sensor. The self-excited oscillation circuit includes, for example, a phase shift circuit that advances the phase of the displacement signal from the displacement detection sensor by 90 degrees, and an auto gain control circuit that keeps the level of the output signal of the phase shift circuit constant.
[0034]
The cantilever excitation unit may further include an excitation control circuit that adjusts an excitation signal from the excitation signal generation unit. The excitation control circuit includes, for example, a phase shift circuit that advances the phase of the displacement signal from the displacement detection sensor by 90 degrees, a gain adjustment circuit that applies a predetermined gain to the output signal of the phase shift circuit, and an excitation signal that is applied to the output signal of the gain adjustment circuit. And an addition circuit for adding the excitation signal from the signal generation unit.
[0035]
The excitation signal generator may output an excitation signal adjusted based on the displacement signal from the displacement detection sensor. Such an excitation signal generation unit includes, for example, a phase shift circuit that advances the phase of the displacement signal from the displacement detection sensor by 90 degrees, an auto gain control circuit that keeps the level of the output signal of the phase shift circuit constant, and a phase shift circuit. A gain adjustment circuit that applies a predetermined gain to the output signal of the first control circuit, and an addition circuit that adds the output signal of the automatic gain control circuit to the output signal of the gain adjustment circuit.
[0036]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[0037]
First embodiment
The present invention is directed to a scanning probe microscope for observing a biological sample in a liquid with a probe, and a scanning probe microscope of the first embodiment is shown in FIG. In the following description, two axes or two directions that are both parallel to the horizontal plane and that are orthogonal to each other will be referred to as the X axis and Y axis or the X and Y directions, respectively, and the axis or direction that is orthogonal to the horizontal plane will be the Z axis or the Z direction. Shall be called.
[0038]
The scanning probe microscope 100 includes a liquid stage 110 for holding a liquid 102 such as water, a cantilever 124 having a probe 122 at a free end, and a holder 126 for holding the cantilever 124 in the liquid 102 held on the liquid stage 110. Then, the sample stage 132 holding the biological sample 104 in the liquid 102 held on the liquid stage 110, the biological sample 104 is moved along the X axis and the Y axis with respect to the cantilever 124 (XY scanning), and Z And an XYZ scanner 134 for moving (Z-scanning) along the axis.
[0039]
The liquid stage 110 has a transmission glass 112 that transmits light and a frame 114 that holds the transmission glass 112. The liquid stage 110 has a water-repellent surface, and can hold the liquid 102 by surface tension. Here, to hold the liquid 102 means to keep the liquid 102 at one place.
[0040]
The liquid 102 may be held on a water-repellent slide glass. In this case, the slide glass holding the liquid 102 is placed on the liquid stage 110. Further, the liquid 102 may be held in a liquid cell such as a petri dish.
[0041]
The scanning probe microscope 100 further includes a displacement detection sensor 140 that optically detects the displacement of the free end of the cantilever 124 and outputs a displacement signal reflecting the displacement, and a cantilever based on the displacement signal from the displacement detection sensor 140. And an amplitude detection circuit 148 for calculating an amplitude of the vibration and outputting an amplitude signal reflecting the vibration amplitude.
[0042]
The displacement detection sensor 140 is composed of, for example, an optical lever sensor. Details of the optical lever sensor are disclosed in, for example, JP-A-2002-82037. However, the displacement detection sensor 140 is not limited to the optical lever sensor, and may be constituted by another sensor.
[0043]
The optical lever sensor 140 includes a light source that emits a light beam, an objective lens 142 that converges the light beam from the light source and irradiates the free end of the cantilever 124, and a photodetector that detects a light beam reflected from the cantilever 124. Contains. The objective lens 142 is disposed below the transmission glass 112.
[0044]
The light beam 146 emitted from the light source is converged by the objective lens 142, passes through the transmission glass 112, and irradiates the free end of the cantilever 124. The light beam reflected by the cantilever 124 enters the objective lens 142 through the transmission glass 112, and is guided to the photodetector. The photodetector has a light receiving surface, and the reflected light beam from the cantilever 124 forms a light spot on the light receiving surface. The light spot moves on the light receiving surface of the photodetector according to the displacement of the free end of the cantilever 124. The photodetector outputs a signal reflecting the position of the light spot formed on the light receiving surface, that is, a signal reflecting the displacement of the free end of the cantilever 124.
[0045]
The scanning probe microscope 100 has a controller 150. The controller 150 moves the XYZ scanner along the X-axis to generate an X-scan signal, and the XYZ scanner along the Y-axis. A Y-scan signal generation unit 154 for generating a Y-scan signal to be displaced; and a target value of a distance between the cantilever and the sample along the Z-axis is determined based on the amplitude signal from the amplitude detection circuit, and Z control corresponding to the target value is obtained. And a Z control circuit 156 for outputting a signal.
[0046]
The Z control circuit 156 may be of either analog control or digital control (software control).
[0047]
The scanning probe microscope 100 further includes an X drive circuit 162 that controls the displacement of the XYZ scanner along the X axis according to the X scan signal, and a Y drive circuit 164 that controls the displacement of the XYZ scanner along the Y axis according to the Y scan signal. And a Z drive circuit 166 for controlling the displacement of the XYZ scanner along the Z axis according to the Z control signal.
[0048]
Further, the scanning probe microscope 100 calculates the information on the surface of the biological sample 104 based on the X-scanning signal, the Y-scanning signal, and the Z control signal, and displays the obtained information on the surface of the biological sample 104. A host computer 160 functioning as a display unit.
[0049]
The scanning probe microscope 100 further has cantilever excitation means for exciting the cantilever at a frequency near its mechanical resonance frequency. The cantilever excitation means includes an excitation signal generation unit 158 that generates an excitation signal having a frequency near the mechanical resonance frequency of the cantilever 124, generates an ultrasonic wave according to the excitation signal, and transmits the ultrasonic wave almost uniformly to the cantilever and its surroundings. And a transducer 170 for irradiating the light.
[0050]
The excitation signal generator 158 is provided, for example, inside the controller 150, but is not limited to this. In other words, the controller 150 partially constitutes the excitation signal generator 158. That is, the controller 150 and the transducer 170 constitute cantilever excitation means.
[0051]
The transducer 170 includes a vibrator 172 that generates an ultrasonic wave according to an input excitation signal, and an ultrasonic wave propagation unit 174 that propagates the ultrasonic wave generated by the vibrator 172. Is fixed to the liquid stage 110 via the vibration damping rubber 178.
[0052]
The vibrator 172 includes, but is not limited to, a piezoelectric element, for example. The vibrator 172 and the ultrasonic wave propagation unit 174 are mechanically coupled, and the ultrasonic wave propagation unit 174 has an output end 176 for emitting ultrasonic waves to the outside. As shown in FIGS. 1 and 2, at least the output end 176 of the ultrasonic wave propagation unit 174 is located in the liquid 102 held on the liquid stage 110. The output end 176 is preferably located as close to the cantilever 124 as possible.
[0053]
The transducer 170 is arranged such that the irradiation surface of the output end 176 of the ultrasonic wave propagation unit 174 is orthogonal to the X axis, and emits ultrasonic waves in the X direction. Since the cantilever 124 is held at an angle of about 5 to 20 degrees with respect to the liquid stage 110 by the holder 126, the ultrasonic wave is applied to the surface of the cantilever 124 at an angle of about 5 to 20 degrees.
[0054]
Of course, the irradiation direction of the ultrasonic wave is not limited to this. In order to efficiently excite the cantilever 124, the ultrasonic wave may be irradiated at a small incident angle with respect to the surface of the cantilever 124, in other words, in a state close to vertical. For this reason, the ultrasonic wave may be irradiated from the lower left of the paper surface so as to impinge on the surface of the cantilever 124 at a small incident angle. The ultrasonic waves may be emitted in parallel with the YZ plane, for example, from above the paper surface.
[0055]
The output end 176 of the ultrasonic wave propagation unit 174 has a size sufficiently larger than the size of the cantilever 124. Therefore, the ultrasonic wave emitted from the output end 176 of the ultrasonic wave propagation unit 174 has a sufficiently large wavefront as compared with the size of the cantilever. For this reason, the ultrasonic wave is applied almost uniformly to the cantilever and its surroundings.
[0056]
Specifically, the large cantilever 124 has a length of about 100 μm and a width of about 30 μm, and the small cantilever 124 has a length of about 10 μm and a width of about 2 μm. On the other hand, the output end 176 of the ultrasonic wave propagation unit 174 has, for example, a size of about 1 mm in height (dimension in the Z direction) and about 1 mm in width (dimension in the Y direction).
[0057]
Ultrasound is not emitted vertically to the cantilever 124 but can be considered substantially uniform. The resonance frequency of the cantilever 124 is generally 100 kHz to 1 MHz, corresponding to the magnitude described above. Assuming that the speed of the ultrasonic wave in the liquid 102 is 1500 m / s, the wavelength of the ultrasonic wave is 15 mm to 1.5 mm. Therefore, the ultrasonic wave applied to the cantilever 124 can be considered substantially uniform not only for a small one but also for a large one.
[0058]
The material of the ultrasonic wave propagation section 174 may have a relatively small acoustic impedance in order to reduce reflection at the boundary surface with water. For this reason, the ultrasonic wave propagation section 174 is made of, for example, a polymer material such as Teflon, silicone, or acrylic resin. The polymer material is easy to mold and efficiently transmits ultrasonic waves.
[0059]
The vibration isolation rubber 178 is for preventing the ultrasonic waves generated by the vibrator 172 from being transmitted to the liquid stage 110 and the holder 126, and is preferably made of a material having low rigidity. In particular, the vibration isolation rubber 178 may be a material whose resonance frequency is much lower than the resonance frequency of the cantilever 124, for example, silicone rubber. The vibration isolation rubber 178 may be integrally formed with the ultrasonic wave propagation unit 174.
[0060]
The ultrasonic wave propagation unit 174 preferably has a horn that amplifies the ultrasonic wave propagating inside the ultrasonic wave propagation unit 174. For example, as shown in FIG. 3, the ultrasonic wave propagation unit 174 has a truncated quadrangular pyramid horn extending in the X direction to amplify the ultrasonic wave. The ultrasonic wave propagation unit 174 has a rectangular input end (an end face to which the vibrator 172 is joined) and an output end 176. For example, the input end has a height (dimension in the Z direction) of about 3 mm and a width (dimension in the Y direction) of about 3 to 20 mm, and the output end 176 has a height of about 1 mm and a width of about 1 mm.
[0061]
The horn has a cross-sectional area perpendicular to the X-axis that decreases as approaching the output end 176. Further, the cross section is reduced at a higher rate in the Y direction than in the Z direction. Thus, the horn amplifies higher in the Y direction than in the Z direction. This is because the degree of freedom in arranging the cantilever 124 in the Y direction is higher than in the Z direction (because there is a dimensional allowance in the Y direction than in the Z direction).
[0062]
As described above, in the transducer 170, the output end 176 of the ultrasonic wave propagation unit 174 is set to a size of 1 mm × 1 mm. This is a specific example of a preferable size in which the ultrasonic intensity around the cantilever 124 becomes substantially uniform and a horn effect (amplification factor) as large as possible can be obtained. Therefore, the output end 176 of the ultrasonic wave propagation unit 174 is not limited to this. If there is room in the space, it may be larger in order to obtain a more uniform ultrasonic wave. Also, if sufficiently uniform, it may be smaller in order to obtain stronger ultrasonic waves.
[0063]
Although the ultrasonic wave propagation section 174 of the transducer 170 has a horn having a truncated pyramid shape, the shape of the horn is not limited to this. Another transducer with a differently shaped horn, which can be applied in place of the transducer 170, is shown in FIG.
[0064]
As shown in FIG. 4, the transducer 170A includes a vibrator 172A such as a piezoelectric element that generates ultrasonic waves, and an ultrasonic wave propagation unit 174A that propagates the ultrasonic waves generated by the vibrator 172A. The sound wave propagating unit 174A has a horn shaped like a truncated elliptical cone extending in the X direction. The ultrasonic wave propagation unit 174A has a rectangular input terminal elongated in the Y direction and a circular output terminal 176A. The horn has a cross-sectional area orthogonal to the X-axis which decreases as approaching the output end 176A. Further, the cross section is reduced at a higher rate in the Y direction than in the Z direction. Thus, the horn amplifies higher in the Y direction than in the Z direction.
[0065]
Each of the ultrasonic wave propagating section 174 and the ultrasonic wave propagating section 174A is a conical horn whose cross-sectional area is reduced at a constant rate, but is an exponential type or catenoidal horn having a higher amplification factor. You may.
[0066]
Still another transducer that can be used in place of the transducer 170 is shown in FIG. As shown in FIG. 5, the transducer 170B has a vibrator 172B such as a piezoelectric element that generates ultrasonic waves, and an ultrasonic wave propagation unit 174B that propagates the ultrasonic waves generated by the vibrator 172B. The sound wave propagation section 174B has a horn having an amplifying function only in the Y direction. The horn has a cross-sectional area perpendicular to the X-axis that decreases as approaching the output end 176B. Further, in the cross section, the dimension in the Z direction is constant, and only the dimension in the Y direction decreases at a constant rate. Therefore, the horn does not amplify the ultrasonic wave in the Z direction, but only in the Y direction.
[0067]
Still another transducer that can be used in place of the transducer 170 is shown in FIG. As shown in FIG. 6, the transducer 170C has a vibrator 172C such as a piezoelectric element that generates ultrasonic waves, and an ultrasonic wave propagation unit 174C that propagates the ultrasonic waves generated by the vibrator 172C. The sound wave propagation section 174C does not have a horn for amplifying the ultrasonic wave. Therefore, the intensity of the ultrasonic wave emitted from the output end 176C of the transducer 170C for the same excitation signal is lower than that of the other transducers. For this reason, a piezoelectric element having a large output is preferably applied to the vibrator 172C.
[0068]
In the scanning probe microscope 100 of the present embodiment, an excitation signal is output from the excitation signal generator 158 in the controller 150. The excitation signal has a predetermined amplitude, and has the same frequency as the mechanical resonance frequency of the cantilever 124. The excitation signal is input to the transducer 172 of the transducer 170. The vibrator 172 receives the input of the excitation signal and generates ultrasonic waves having a predetermined amplitude having the same frequency as the mechanical resonance frequency of the cantilever 124. The ultrasonic wave propagates through the ultrasonic wave propagating section 174 and the liquid 102 and is uniformly applied to the cantilever 124 and its surroundings. As a result, the cantilever 124 vibrates (is excited) at a predetermined amplitude at the mechanical resonance frequency.
[0069]
In this state, the displacement detection sensor 140 detects the displacement of the free end of the cantilever 124, and the amplitude detection circuit 148 outputs an amplitude signal indicating the amplitude of the vibration of the free end of the cantilever 124. The Z control circuit 156 outputs a Z control signal for keeping the vibration amplitude of the cantilever 124 at a predetermined value based on the amplitude signal of the cantilever 124 input from the amplitude detection circuit 148. The Z drive circuit 166 drives the XYZ scanner 134 according to the Z control signal supplied from the Z control circuit 156, and moves the biological sample 104 held by the XYZ scanner 134 in the Z direction.
[0070]
That is, the Z control circuit 156 and the Z drive circuit 166 cooperate to control the position of the biological sample 104 in the Z direction with respect to the cantilever 124 by the XYZ scanner 134. The Z control circuit 156 and the Z drive circuit 166 cause the biological sample 104 to approach the cantilever 124 until the biological sample 104 and the cantilever 124 (more precisely, the probe 122) come into contact with each other. The position of the biological sample 104 in the Z direction is controlled so that the vibration amplitude of the cantilever 124 is kept constant.
[0071]
In parallel with such Z control, the X drive circuit 162 and the Y drive circuit 164 respectively control the XYZ scanner according to the X scan signal and the Y scan signal supplied from the X scan signal generation unit 152 and the Y scan signal generation unit 154, respectively. By driving the 134 in the X direction and the Y direction, the biological sample 104 is two-dimensionally scanned, that is, XY scanned with respect to the cantilever 124.
[0072]
During the XY scanning and the Z control, the host computer 160 obtains and displays information on the surface of the biological sample 104, for example, unevenness information, based on the X scanning signal, the Y scanning signal, and the Z control signal. To form and display an image of the surface of the sample.
[0073]
In the scanning probe microscope 100 of the present embodiment, since the cantilever 124 is excited by ultrasonic waves, vibration of members other than the cantilever 124, for example, vibration of the holder 126 and the like is relatively reduced. As a result, the output characteristics of the displacement detection sensor 140 are free from noise as shown in FIG.
[0074]
Further, since the cantilever 124 is excited by ultrasonic waves having a wavefront sufficiently larger than the size of the cantilever 124, the positioning accuracy of the transducer 170 required in the scanning probe microscope 100 of the present embodiment can be low. Further, since the intensity of the ultrasonic wave around the cantilever 124 is substantially uniform, it is hardly affected by vibration noise and thermal drift of the transducer 170. As a result, the scanning probe microscope 100 of the present embodiment can more stably excite the cantilever 124, so that a more accurate measurement result can be obtained.
[0075]
Second embodiment
FIG. 8 shows a scanning probe microscope according to the second embodiment of the present invention. 8, members denoted by the same reference numerals as those in FIG. 1 indicate the same members, and a detailed description thereof will be omitted to avoid duplication of description.
[0076]
The scanning probe microscope 200 of the present embodiment has a transducer 280 that constitutes a cantilever excitation unit that excites the cantilever 124 at a frequency near its mechanical resonance frequency, together with an excitation signal generation unit 158 in the controller 150. . The transducer 280 generates an ultrasonic wave according to the excitation signal from the excitation signal generation unit 158, and irradiates the ultrasonic wave almost uniformly to the cantilever 124 and its surroundings.
[0077]
The transducer 280 has a vibrator 282 such as a piezoelectric element that generates an ultrasonic wave in accordance with an excitation signal, and an ultrasonic wave propagation unit 284 that propagates the ultrasonic wave generated by the vibrator 282. The transducer 280 is held by the XYZ scanner 134, and the ultrasonic wave propagation unit 284 also serves as a sample stage. In other words, the sample stage 284 is connected to the XYZ scanner 134 via the transducer 282 of the transducer 280.
[0078]
The ultrasonic wave propagation unit 284 may be variously modified similarly to the ultrasonic wave propagation unit 174 of the first embodiment.
[0079]
The excitation signal generator 158 outputs an excitation signal having a frequency near the resonance frequency of the cantilever 124 and having a predetermined amplitude. The vibrator 282 generates ultrasonic waves of a predetermined amplitude having the same frequency as the mechanical resonance frequency of the cantilever 124 according to the input excitation signal. The ultrasonic wave propagates through the ultrasonic wave propagation section 284, the biological sample 104, and the liquid 102, and is uniformly applied to the cantilever 124 and its surroundings, and excites the cantilever 124 at a predetermined amplitude at its mechanical resonance frequency.
[0080]
The scanning probe microscope 200 of the present embodiment has the same advantages as the scanning probe microscope 100 of the first embodiment. In addition, since the surface of the cantilever 124 is irradiated with ultrasonic waves at a small incident angle, that is, almost perpendicular, the scanning probe microscope 200 of the present embodiment is different from the scanning probe microscope of the first embodiment. The cantilever 124 can be excited more efficiently than 100. Further, since the vibrator 282 for generating the ultrasonic wave is arranged away from the liquid stage 110 and the holder 126, the vibration damping rubber used in the first embodiment is not required. Therefore, the scanning probe microscope 200 has a simpler configuration than the scanning probe microscope 100 of the first embodiment.
[0081]
Third embodiment
FIG. 9 shows a scanning probe microscope according to a third embodiment of the present invention. In FIG. 9, members denoted by the same reference numerals as those in FIG. 1 indicate the same members, and a detailed description thereof will be omitted to avoid duplication of description.
[0082]
In the scanning probe microscope 300 of the present embodiment, the XYZ scanner 334 moves the biological sample 104 along the X axis and the Y axis with respect to the cantilever 124 (XY scanning). And a Z scanner 338 that moves (scans) the biological sample 104 along the Z axis.
[0083]
At the lower end of the Z scanner 338, a sample table 332 holding the biological sample 104 is fixed. The Z scanner 338 also functions as a transducer of the transducer, and the sample stage 332 also functions as an ultrasonic wave propagation unit of the transducer. That is, the Z scanner 338 and the sample stage 332 constitute a transducer. The sample stage 332, that is, the ultrasonic wave propagation unit may be variously deformed similarly to the ultrasonic wave propagation unit 174 of the first embodiment.
[0084]
In addition, the scanning probe microscope 300 includes an adder 382 that adds the Z control signal output from the Z control circuit 156 and the excitation signal output from the excitation signal generation unit 158. The output signal of the adder 382 is input to the Z drive circuit 166.
[0085]
Eventually, the excitation signal generator 158, the adder 382, the Z drive circuit 166, the Z scanner 338, and the sample table 332 cooperate to constitute cantilever excitation means for exciting the cantilever 124 at a frequency near its mechanical resonance frequency. ing.
[0086]
The excitation signal generator 158 outputs an excitation signal having a frequency near the resonance frequency of the cantilever 124 and having a predetermined amplitude. The excitation signal is superimposed on the Z control signal by the adder 382 and input to the Z drive circuit 166. Z drive circuit 166 causes Z scanner 338 to vibrate in the Z direction according to the excitation signal component. Accordingly, the Z scanner 338 generates an ultrasonic wave having a predetermined amplitude having the same frequency as the mechanical resonance frequency of the cantilever 124. The ultrasonic wave propagates through the sample table 332, the biological sample 104, and the liquid 102, and is uniformly applied to the cantilever 124 and its surroundings, and excites the cantilever 124 at a predetermined amplitude at its mechanical resonance frequency.
[0087]
The scanning probe microscope 300 of the present embodiment has the same advantages as the scanning probe microscope 100 of the first embodiment. In addition, since the surface of the cantilever 124 is irradiated with ultrasonic waves at a small incident angle, that is, near vertical, the scanning probe microscope 300 of the present embodiment has the same structure as the second embodiment, The cantilever 124 can be excited more efficiently than the scanning probe microscope 100 of one embodiment. Further, since the Z scanner 338 also serves as a transducer for generating ultrasonic waves, the scanning probe microscope 300 of the present embodiment can have a simpler configuration than the scanning probe microscope 200 of the second embodiment.
[0088]
Fourth embodiment
FIG. 10 shows a scanning probe microscope according to a fourth embodiment of the present invention. 10, members denoted by the same reference numerals as the members in FIG. 1 indicate the same members, and a detailed description thereof will be omitted to avoid duplication of description.
[0089]
As shown in FIG. 10, the scanning probe microscope 400 of the present embodiment includes a self-excited oscillation circuit 480 that generates an excitation signal based on a displacement signal from the displacement detection sensor 140. The self-excited oscillation circuit 480 is a circuit that causes the cantilever 124 to self-oscillate (self-excited oscillation) at its mechanical resonance frequency. Therefore, the self-excited oscillation circuit 480 constitutes an excitation signal generator. That is, the self-excited oscillation circuit 480 and the transducer 170 constitute a cantilever excitation unit.
[0090]
Accordingly, the controller 450 of the scanning probe microscope 400 according to the present embodiment does not have an excitation signal generation unit. That is, the controller 450 has a configuration in which the excitation signal generator 158 is omitted from the controller 150 of the first embodiment.
[0091]
As shown in FIG. 11, the self-excited oscillation circuit 480 includes a phase shift circuit 482 that advances the phase of the displacement signal from the displacement detection sensor 140 by 90 degrees, and an auto gain control that keeps the level of the output signal of the phase shift circuit constant. And a circuit 484.
[0092]
The displacement detection sensor 140, the phase shift circuit 482, the auto gain control circuit 484, the transducer 170, and the cantilever 124 form a positive feedback at the mechanical resonance frequency of the cantilever 124. Further, the feedback gain of the positive feedback is adjusted to 1 or more by the auto gain control circuit 484. Therefore, the cantilever 124 is oscillated at its mechanical resonance frequency. The vibration amplitude is maintained at a predetermined level by the auto gain control circuit 484.
[0093]
The scanning probe microscope 400 of the present embodiment excites the cantilever 124 using ultrasonic waves, similarly to the scanning probe microscope 100 of the first embodiment. Therefore, vibrations of members other than the cantilever 124, for example, vibrations of the holder 126 and the like are relatively reduced. As a result, the output characteristic of the displacement detection sensor 140 does not include noise as shown in FIG. Therefore, it is possible to configure a self-excited oscillation circuit of the cantilever.
[0094]
In a conventional scanning probe microscope in which a cantilever is vibrated by using a vibrator fixed to a holder, the output characteristic of the optical lever sensor includes a lot of noise as shown in FIG. It was impossible to construct a self-excited oscillation circuit.
[0095]
The scanning probe microscope 400 of the present embodiment has the same advantages as the scanning probe microscope 100 of the first embodiment. In addition, since the scanning probe microscope 400 of the present embodiment has the self-excited oscillation circuit 480 for self-oscillating the cantilever 124, the cantilever 124 can be provided without separately providing an excitation signal generation unit. It can be vibrated at the mechanical resonance frequency. This is useful for improving the operability of the scanning probe microscope (simplification of operation procedures and reduction of operation time).
[0096]
Fifth embodiment
Since a scanning probe microscope measures by actually bringing a mechanical probe into contact with an object to be measured, when the object to be measured is a soft biological sample, the biological sample is considerably damaged. However, the smaller the damage to the biological sample, the better.
[0097]
The present embodiment is directed to reducing the damage that a scanning probe microscope gives to a sample during measurement. Before describing the scanning probe microscope of the present embodiment, a phenomenon in which the scanning probe microscope damages a sample will be described first.
[0098]
When the cantilever comes into contact with the sample, the vibration amplitude of the cantilever decreases, that is, the excitation efficiency decreases. This phenomenon is considered as follows.
[0099]
The injection energy for vibrating the cantilever is E ₀ (Constant), the vibration energy of the cantilever is E _k , The leakage energy (loss energy) to the sample due to contact between the cantilever and the sample _s And the other losses are negligible, then E ₀ = E _k + E _s And the excitation efficiency E _k / E ₀ Is E _k / E ₀ = 1-E _s / E ₀ It is expressed as
[0100]
E when the cantilever is not in contact with the sample _s = 0, the excitation efficiency is E _k / E ₀ = 1. When the cantilever and the sample come into close contact, the leakage energy (loss energy) E _s , The excitation efficiency becomes E _k / E ₀ <1. When the cantilever and the sample come closer and come into full contact (they are always in contact), the excitation efficiency becomes E _k / E ₀ = 0. At this time, E _s / E ₀ = 1 and the injection energy E for oscillating the cantilever ₀ Are all leak energy E _s And injected into the sample.
[0101]
From the above, the leakage energy E _s Can be considered as a cause of damaging the sample. Therefore, to reduce damage to the sample, E ₀ Can be reduced (E _s / E ₀ ≤ 1). However, simply E ₀ Is small, the vibration energy E of the cantilever _k Is also small and not practical.
[0102]
Therefore, E depends on the contact state between the cantilever and the sample. ₀ It is good to change the size of. That is, when the cantilever and the sample are not in contact with each other, E ₀ Is increased, and when the contact between the cantilever and the sample is strong, E ₀ Can reduce the damage to the sample during measurement.
[0103]
FIG. 12 shows a scanning probe microscope according to a fifth embodiment of the present invention in which damage to a sample is reduced based on such considerations. 12, members denoted by the same reference numerals as the members in FIG. 1 indicate the same members, and detailed descriptions thereof will be omitted to avoid duplication of description.
[0104]
As shown in FIG. 12, the scanning probe microscope 500 includes an excitation control circuit 580 that adjusts an excitation signal from the excitation signal generator 158. The excitation control circuit 580 is a circuit that reduces damage to the sample during measurement. The excitation control circuit 580, together with the excitation signal generator 158 and the transducer 170, constitutes a cantilever excitation unit.
[0105]
As shown in FIG. 13, the excitation control circuit 580 includes a phase shift circuit 582 that advances the phase of the displacement signal from the displacement detection sensor 140 by 90 degrees, and a gain adjustment circuit that applies a predetermined gain to the output signal of the phase shift circuit 582. 586, and an addition circuit 588 for adding the excitation signal from the excitation signal generation unit 158 to the output signal of the gain adjustment circuit 586. Excitation control circuit 580 outputs the output signal of addition circuit 588 as an excitation signal.
[0106]
The displacement detection sensor 140, the phase shift circuit 582, the gain adjustment circuit 586, the addition circuit 588, the transducer 170, and the cantilever 124 form a positive feedback at the mechanical resonance frequency of the cantilever 124. Further, the feedback gain of the positive feedback is adjusted to less than 1 by the gain adjustment circuit 586.
[0107]
The excitation control circuit 580 can adjust an excitation signal for exciting the cantilever 124 according to the contact state between the biological sample 104 and (the probe 122 of) the cantilever 124. Hereinafter, this principle will be described.
[0108]
In FIG. 13, the angular frequency of the cantilever 124 at the mechanical resonance frequency is ω _c The excitation signal output from the excitation signal generator 158 in the controller 150 is ₀ sinω _c Let it be t. When the cantilever 124 is vibrating at the resonance frequency, the displacement of the cantilever 124 has a phase delayed by 90 degrees with respect to the excitation signal, so that the signal output from the displacement detection sensor 140 is A _k sin (ω _c t−π / 2). The output of the phase shift circuit 582 is A _k sinω _c t, the output of the gain adjustment circuit 586 is KA _k sinω _c t, the output of the adder circuit 588 is (A ₀ + KA _k ) Sinω _c t. Where A _k Is the amplitude of the output signal of the displacement detection sensor 140, and K is the gain of the gain adjustment circuit 586.
[0109]
In a stable state when the cantilever 124 is not in contact with the biological sample 104, the following relational expression holds.
[0110]
(Equation 1)

[0111]
Where H _c Is the angular frequency ω _c , Input / output characteristics (gain) from the input to the vibrator 172 to the output of the displacement detection sensor 140, _k0 Is the amplitude A of the output signal of the displacement detection sensor 140 when the cantilever 124 and the biological sample 104 are not in contact with each other (initial state). _k It is.
[0112]
H _c Are the conversion efficiency when the vibrator 172 receives an electric signal to generate an ultrasonic wave, the ultrasonic wave propagation efficiency in the ultrasonic wave propagation unit 174, the transmittance of the ultrasonic wave at the boundary between the ultrasonic wave propagation unit 174 and the liquid 102, The propagation efficiency of the ultrasonic wave in the liquid 102, the excitation efficiency of the cantilever 124 by the ultrasonic wave when the cantilever 124 is not in contact with the biological sample 104 (χ ₀ ), The conversion efficiency (detection sensitivity) when the vibration of the cantilever 124 is converted into an electric signal by the displacement detection sensor 140.
[0113]
In a stable state when the cantilever 124 and the biological sample 104 are in contact with each other, the following relational expression holds.
[0114]
(Equation 2)

[0115]
Here, χ is the excitation efficiency of the cantilever 124 by ultrasonic waves when the cantilever 124 and the biological sample 104 are in contact with each other. (Χ / χ ₀ ) Indicates a contact state between the cantilever 124 and the biological sample 104, which is defined as an excitation efficiency ratio α (0 ≦ α ≦ 1) of the cantilever 124 by ultrasonic waves. The excitation efficiency ratio α is expressed by the following equation from equation (2).
[0116]
[Equation 3]

[0117]
Therefore, a signal S for vibrating the cantilever 124 (signal supplied to the vibrator 172) can be expressed by the following equation from the equations (1) and (3).
[0118]
(Equation 4)

[0119]
KH _c Is a positive feedback feedback gain composed of the displacement detection sensor 140, the phase shift circuit 582, the gain adjustment circuit 586, the addition circuit 588, the vibrator 172, the ultrasonic wave propagation unit 174, and the cantilever 124, and 0 ≦ KH _c <1. In addition, KH _c Oscillation occurs when is greater than one.
[0120]
H _c And A _k0 Can be treated as a constant. _c = 1, A _k0 If = 1, S in equation (4) can be expressed as a function of K and α as follows:
[0121]
(Equation 5)

[0122]
FIG. 14 shows S (K, α) plotted against some representative values of K. FIG. 14 shows S (0, α), S (0.1, α), S (0.5, α), S (0.8, α) and S (0.99, α) with α as a parameter. ).
[0123]
From FIG. 14, it is possible to control the signal (signal supplied to the vibrator 172) S for vibrating the cantilever 124 according to the contact state α between the biological sample 104 and the cantilever 124, in other words, according to the excitation efficiency ratio α of the cantilever 124. I understand. That is, it can be seen that S can be increased when the cantilever 124 is not in contact with the biological sample 104, and S can be reduced when the cantilever 124 is in contact with the biological sample 104. It can be seen that the effect increases as K approaches 1.
[0124]
If K = 0, it is the same as supplying the excitation signal output from the excitation signal generator 158 to the vibrator 172 as it is, as described in the first to third embodiments.
[0125]
According to the above, K ≒ 1 (more precisely, KH _c It seems that 望 ま し い 1) is desirable. However, K (exactly KH _c ) Is closer to 1, oscillation due to positive feedback is more likely to occur, and the operation becomes unstable. Therefore, K (exactly KH _c ) Is actually set to a value of 0.5 to 0.8.
[0126]
Next, by controlling a signal (signal supplied to the vibrator 172) S for vibrating the cantilever 124 in accordance with the contact state α between the biological sample 104 and the cantilever 124, the damage to the biological sample 104 can be reduced. Evaluate whether there is any effect.
[0127]
The damage D to the biological sample 104 is the loss of the excitation efficiency of the cantilever 124 due to the ultrasonic waves. ₀ It can be considered that (1-α) is multiplied by a signal (signal supplied to the vibrator 172) S for vibrating the cantilever 124. That is, the damage D can be expressed as a function of K and α as follows.
[0128]
(Equation 6)

χ ₀ Can be regarded as a constant. ₀ Assuming that = 1, D in equation (6) can be expressed as follows.
[0129]
(Equation 7)

[0130]
FIG. 15 shows D (K, α) plotted against some representative values of K. FIG. 15 shows D (0, α), D (0.1, α), D (0.5, α), D (0.8, α) and D (0.99, α) with α as a parameter. ).
[0131]
From FIG. 15, by controlling a signal (signal supplied to the vibrator 172) S for vibrating the cantilever 124 according to the contact state α between the biological sample 104 and the cantilever 124, damage to the biological sample 104 is sharply reduced. It can be seen that it can be reduced. It can be seen that the effect increases as K approaches 1.
[0132]
Therefore, by appropriately setting the gain K of the gain adjustment circuit 586 in consideration of the type of the biological sample 104 and information to be measured, it is possible to appropriately reduce damage to the biological sample 104 during measurement.
[0133]
The scanning probe microscope 500 of the present embodiment has the same advantages as the scanning probe microscope 100 of the first embodiment. In addition to this, the scanning probe microscope 500 of the present embodiment is configured to drive the excitation 172 supplied to the vibrator 172 based on the contact state between the biological sample 104 and the cantilever 124, in other words, based on the output signal of the displacement detection sensor 140. Since the excitation control circuit 580 for adjusting the signal is provided, damage to the biological sample 104 during measurement can be significantly reduced.
[0134]
Sixth embodiment
FIG. 16 shows a scanning probe microscope of the sixth embodiment. In FIG. 16, members denoted by the same reference numerals as those in FIG. 1 indicate the same members, and detailed descriptions thereof will be omitted to avoid duplication of description.
[0135]
The scanning probe microscope has an excitation control circuit 680 that outputs an excitation signal adjusted based on a displacement signal from the displacement detection sensor 140. The excitation control circuit 680 is a circuit that causes the cantilever 124 to spontaneously vibrate (self-excited oscillation) at its mechanical resonance frequency and to reduce damage to the sample during measurement.
[0136]
That is, the excitation control circuit 680 of the present embodiment has the functions of the self-excited oscillation circuit 480 of the fourth embodiment and the excitation control circuit 580 of the fifth embodiment. In other words, the excitation control circuit 680 partially constitutes an excitation signal generation unit. Therefore, the excitation control circuit 680 and the transducer 170 constitute a cantilever excitation unit.
[0137]
Accordingly, the controller 450 of the scanning probe microscope 600 of the present embodiment does not have an excitation signal generation unit. That is, the controller 450 has a configuration in which the excitation signal generator 158 is omitted from the controller 150 of the first embodiment.
[0138]
As shown in FIG. 17, the excitation control circuit 680 includes a phase shift circuit 682 that advances the phase of the displacement signal from the displacement detection sensor by 90 degrees, and an auto gain control circuit that keeps the level of the output signal of the phase shift circuit 682 constant. 684, a gain adjustment circuit 686 that applies a predetermined gain to the output signal of the phase shift circuit 682, and an addition circuit 688 that adds the output signal of the auto gain control circuit 684 to the output signal of the gain adjustment circuit 686. .
[0139]
The displacement detection sensor 140, the phase shift circuit 682, the auto gain control circuit 684, the addition circuit 688, the transducer 170, and the cantilever 124 form a positive feedback at the mechanical resonance frequency of the cantilever 124. Further, the feedback gain of the positive feedback is adjusted to 1 or more by the auto gain control circuit 684. This positive feedback constitutes a circuit equivalent to the self-excited oscillation circuit shown in FIG. Therefore, the cantilever 124 is oscillated at its mechanical resonance frequency. The vibration amplitude is maintained at a predetermined level by the auto gain control circuit 684.
[0140]
The displacement detection sensor 140, the phase shift circuit 682, the gain adjustment circuit 686, the addition circuit 688, the transducer 170, and the cantilever 124 form a positive feedback at the mechanical resonance frequency of the cantilever 124. Further, the feedback gain of the positive feedback is adjusted to less than 1 by the gain adjustment circuit 686. Therefore, the positive feedback forms a circuit equivalent to the excitation control circuit 580 shown in FIG. Therefore, by appropriately setting the gain K of the gain adjustment circuit 686 in consideration of the type of the biological sample 104 and information to be measured, it is possible to appropriately reduce damage to the biological sample 104 during measurement.
[0141]
The scanning probe microscope 600 of the present embodiment has the same advantages as the scanning probe microscope 100 of the first embodiment. In addition, since the scanning probe microscope 600 of this embodiment has the excitation control circuit 680 having a function of causing the cantilever 124 to self-oscillate, the cantilever 124 is not provided separately. At its mechanical resonance frequency. This is useful for improving the operability of the scanning probe microscope (simplification of operation procedures and reduction of operation time).
[0142]
Further, the scanning probe microscope 600 of the present embodiment adjusts the excitation signal supplied to the vibrator 172 based on the contact state between the biological sample 104 and the cantilever 124, in other words, based on the output signal of the displacement detection sensor 140. Since the excitation control circuit 680 has a function of performing the measurement, damage to the biological sample 104 during measurement can be significantly reduced.
[0143]
【The invention's effect】
According to the present invention, there is provided a submerged scanning probe microscope capable of stably exciting a cantilever in a liquid without requiring high-precision positioning of the cantilever. Thereby, the biological sample can be measured more accurately.
[Brief description of the drawings]
FIG. 1 shows a scanning probe microscope according to a first embodiment of the present invention.
FIG. 2 is a view of the stage of FIG. 1 as viewed from above.
FIG. 3 shows a top view (A), a side view (B), and a front view (output end side end view) (C) of the transducer shown in FIGS. 1 and 2;
FIG. 4 shows a top view (A), a side view (B), and a front view (output end side end view) (C) of another transducer that can be used in place of the transducer shown in FIG. ing.
5 shows a top view (A), a side view (B), and a front view (output end side end view) (C) of another transducer that can be used in place of the transducer shown in FIG. 3; ing.
6 shows a top view (A), a side view (B), and a front view (output end side end view) (C) of another transducer that can be used in place of the transducer shown in FIG. ing.
FIG. 7 shows output characteristics of the displacement detection sensor shown in FIG.
FIG. 8 shows a scanning probe microscope according to a second embodiment of the present invention.
FIG. 9 shows a scanning probe microscope according to a third embodiment of the present invention.
FIG. 10 shows a scanning probe microscope according to a fourth embodiment of the present invention.
11 is a diagram partially showing the scanning probe microscope shown in FIG. 10 and shows a configuration of the self-excited oscillation circuit shown in FIG. 10;
FIG. 12 shows a scanning probe microscope according to a fifth embodiment of the present invention.
13 is a diagram partially showing the scanning probe microscope shown in FIG. 12, and shows a configuration of the excitation control circuit shown in FIG. 12;
FIG. 14 shows a graph in which S (K, α) represented by equation (5) is plotted against some representative values of K.
FIG. 15 shows a graph in which D (K, α) represented by equation (7) is plotted against some representative values of K.
FIG. 16 shows a scanning probe microscope according to a sixth embodiment of the present invention.
17 is a diagram partially showing the scanning probe microscope shown in FIG. 16, and shows a configuration of the excitation control circuit shown in FIG. 16;
FIG. 18 shows a configuration of an example of a conventional atomic force microscope for a living body.
FIG. 19 shows vibration characteristics of a certain cantilever in the atmosphere.
20 shows the vibration characteristics of the cantilever having the vibration characteristics shown in FIG. 19 in the liquid in the air.
21 shows the output characteristics of the optical lever sensor shown in FIG. 18 for a cantilever having the vibration characteristics shown in FIG. 20 in a liquid.
[Explanation of symbols]
100 scanning probe microscope
102 liquid
104 biological sample
110 liquid stage
122 probe
124 cantilever
126 holder
132 Sample stand
134 XYZ scanner
140 Displacement detection sensor
148 Amplitude detection circuit
152 X scan signal generator
154 Y scan signal generator
156 Z control circuit
158 Excitation signal generator
160 host computer
162 X drive circuit
164 Y drive circuit
166 Z drive circuit
170 transducer
172 vibrator
174 Ultrasonic wave propagation part
176 output terminal

Claims (13)

A submerged scanning probe microscope for observing a biological sample in a liquid with a probe, having an X axis and a Y axis that are both parallel to a horizontal plane and orthogonal to each other, and a Z axis that is orthogonal to the horizontal plane. Scanning probe microscope
A liquid stage for holding liquid,
A cantilever having a probe at its free end,
A holder for holding the cantilever in the liquid held on the liquid stage,
A sample stage for holding the sample in the liquid held on the liquid stage,
A scanner for moving (Z-scanning) the sample at least along the Z-axis with respect to the cantilever;
Cantilever exciting means for exciting the cantilever at a frequency near its mechanical resonance frequency,
A displacement detection sensor that optically detects the displacement of the free end of the cantilever and outputs a displacement signal reflecting the displacement;
An amplitude detection circuit that calculates a vibration amplitude of the cantilever based on a displacement signal from the displacement detection sensor, and outputs an amplitude signal reflecting the vibration amplitude;
A Z control circuit for obtaining a target value of the distance between the cantilever and the sample along the Z axis based on the amplitude signal from the amplitude detection circuit and outputting a Z control signal corresponding to the target value;
A Z drive circuit that controls displacement of the scanner along the Z axis according to the Z control signal;
A calculation display unit for calculating the information on the surface of the sample based on the Z control signal and displaying the obtained information on the surface of the sample;
The cantilever excitation unit has an excitation signal generation unit that generates an excitation signal, and a transducer that generates an ultrasonic wave according to the excitation signal and irradiates the ultrasonic wave to the cantilever and its surroundings almost uniformly. Probe microscope. The scanner can move (XY scan) the sample along the X axis and the Y axis with respect to the cantilever in addition to the Z scan, and the scanning probe microscope displaces the scanner along the X axis. An X scan signal generator for generating a scan signal; a Y scan signal generator for generating a Y scan signal for displacing the scanner along the Y axis; and an X for controlling the displacement of the scanner along the X axis in accordance with the X scan signal. A driving circuit for controlling displacement of the scanner along the Y-axis in accordance with the Y-scanning signal, wherein the calculation display unit is based on the X-scanning signal and the Y-scanning signal in addition to the Z control signal; 2. The scanning probe microscope according to claim 1, wherein information on the surface of the sample is obtained by calculation. The transducer has a vibrator that generates ultrasonic waves according to an input excitation signal, and an ultrasonic wave propagation unit that propagates the ultrasonic waves generated by the vibrator. The ultrasonic wave propagation portion has an output end for emitting ultrasonic waves to the outside, and at least the output end of the ultrasonic wave propagation portion is located in the liquid held on the liquid stage. Alternatively, the scanning probe microscope according to claim 2. The scanning probe microscope according to claim 1, wherein the ultrasonic wave propagation unit has a horn for amplifying the ultrasonic wave. The scanning probe microscope according to claim 1, wherein the ultrasonic wave propagation unit is made of a polymer material. The scanning probe microscope according to any one of claims 1 to 5, wherein the ultrasonic wave propagation unit is fixed to the liquid stage via an anti-vibration rubber. The scanning probe microscope according to any one of claims 1 to 5, wherein the transducer is held by a scanner, and the ultrasonic wave propagation part of the transducer also serves as a sample stage. The scanner includes a Z scanner that moves (samples) the sample along the Z axis with respect to the cantilever. The Z scanner also functions as a transducer of the transducer, and the scanning probe microscope uses a Z control signal and a Z control signal. The scanning probe microscope according to claim 7, further comprising an adder for adding the excitation signal, wherein an output signal of the adder is input to the Z drive circuit. The scanning probe microscope according to any one of claims 1 to 8, wherein the excitation signal generation unit is a self-excited oscillation circuit that generates an excitation signal based on a displacement signal from a displacement detection sensor. 10. The self-excited oscillation circuit includes a phase shift circuit for advancing the phase of the displacement signal from the displacement detection sensor by 90 degrees, and an auto gain control circuit for keeping the level of the output signal of the phase shift circuit constant. 2. The scanning probe microscope according to 1. The cantilever excitation means further includes an excitation control circuit that adjusts an excitation signal from the excitation signal generation unit, the excitation control circuit includes a phase shift circuit that advances the phase of the displacement signal from the displacement detection sensor by 90 degrees, A gain adjustment circuit that applies a predetermined gain to the output signal of the shift circuit; and an addition circuit that adds the excitation signal from the excitation signal generation unit to the output signal of the gain adjustment circuit. The scanning probe microscope according to any one of claims 1 to 8, which outputs an output signal as an excitation signal. The scanning probe microscope according to any one of claims 1 to 8, wherein the excitation signal generation unit outputs an excitation signal adjusted based on a displacement signal from the displacement detection sensor. The excitation signal generation unit includes a phase shift circuit for advancing the phase of the displacement signal from the displacement detection sensor by 90 degrees, an auto gain control circuit for keeping the level of the output signal of the phase shift circuit constant, and a predetermined signal for the output signal of the phase shift circuit. 13. The scanning probe microscope according to claim 12, further comprising: a gain adjustment circuit that applies the gain of (1), and an addition circuit that adds an output signal of the auto gain control circuit to an output signal of the gain adjustment circuit.

Images