CN105842484A - SICM amplitude modulation imaging mode scanning device and method - Google Patents
SICM amplitude modulation imaging mode scanning device and method Download PDFInfo
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- CN105842484A CN105842484A CN201510023158.5A CN201510023158A CN105842484A CN 105842484 A CN105842484 A CN 105842484A CN 201510023158 A CN201510023158 A CN 201510023158A CN 105842484 A CN105842484 A CN 105842484A
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Abstract
The invention relates to an SICM (Scanning Ion Conductance Microscope) amplitude modulation imaging mode scanning device, comprising a signal generator, a patch clamp amplifier, an effective value conversion circuit, a controller, a probe, an XY nano platform, and a Z-direction nano piezoelectric ceramic element, wherein the signal generator, the patch clamp amplifier, the effective value conversion circuit and the controller are connected in sequence. A method provided by the invention comprises the following steps: the signal generator outputs an alternating current signal to the patch clamp amplifier; the compensation capacitance of the patch clamp amplifier is adjusted; and the effective value conversion circuit calculates the effective value of current amplitude, and feeds the effective value of amplitude back to the controller, and the controller controls the height of the probe in real time according to the effective value of amplitude to realize sample scanning. According to the invention, the alternating current amplitude is used as a feedback value, so the defects of a direct-current mode such as direct-current drift and vulnerability to electrical noise are overcome. Higher modulation frequency is achieved compared with the traditional alternating-current mode, and scanning is accelerated.
Description
Technical field
The present invention relates to the quick high stability noninvasive imaging technology of a kind of low cost, specifically a kind of base
Scanning means in the novel imaging pattern amplitude modulation pattern of scanning Ion Conductance Microscope (SICM) technology
And method.
Background technology
Scanning Ion Conductance Microscope (scanning ion conductance microscope:SICM) is that scanning is visited
A member in pin microscope family, flows through the ion current of ultra micro glass tubing probe, it is possible at sample by detection
Product surface realizes contactless force scanning, and then the pattern of study sample and character.SICM has non-force contact nothing
Damage scanning, imaging resolution high, and the feature such as probe is easily prepared, be particularly suitable for studying physiological condition
Under active somatic cell.Have seminar at present raw to live bodies such as myocardial cell, renal epithelial cell, neurocytes
Thing sample carries out Nano grade morphology observations.
But all there are some defects in existing SICM imaging pattern.DC mode can be by DC shift, external electrical
Gas interference effect and cannot work long hours, therefore real work application less.AC mode utilizes some
The AC compounent of frequency of vibration is as feedback, and stability has obtained large increase, but due to the sound of feedback quantity
Answering the restriction of the vibrated frequency of speed low (1-2kHz), scanning speed is slower.And we organize the scan pattern of proposition
-phase modulation pattern, changes into alternating voltage by driving DC voltage, with the current component of alternating voltage homophase
As feedback quantity, this pattern possesses the high stability advantage of AC mode, and overcomes piezoelectric ceramic vibration
The shortcoming that frequency is low, scanning speed is improved.But phase modulation pattern must detect with driving voltage is same
The current signal of phase, it is therefore necessary to depend on this instrument costly of lock-in amplifier.
Summary of the invention
The invention provides scanning means and the side of the novel scan pattern-amplitude modulation pattern of a kind of SICM
Method.This novel scan pattern is a kind of low cost replacement scheme of phase modulation pattern.
The technical solution used in the present invention is: the amplitude modulation(PAM) imaging mode scan device of a kind of SICM, bag
Include signal generator, patch clamp amplifier, RMS-DC converter circuit, controller, probe, XY nano platform
With Z-direction nanometer piezoelectric ceramics；
Described signal generator, patch clamp amplifier, RMS-DC converter circuit, controller are sequentially connected with；Institute
State patch clamp amplifier to be connected with probe；Probe is fixed on Z-direction nanometer piezoelectric ceramics and is positioned at XY nanometer
Above platform；Described controller is connected with XY nano platform, Z-direction nanometer piezoelectric ceramics.
Described RMS-DC converter circuit uses RMS conversion chip, the input of chip to amplify with patch-clamp
The current output terminal of device connects, and outfan is connected with controller.
A kind of amplitude modulation(PAM) imaging mode scan method of SICM, comprises the following steps:
Signal generator output AC signal, makees after the applied voltage end decay 10 times of input diaphragm pincers amplifier
It is two interelectrode drive voltage signal；
The compensation capacitance of regulation patch clamp amplifier, to optimal value, then increases drive voltage signal and makes diaphragm
The current signal of pincers amplifier output reaches the threshold value close to RMS-DC converter circuit input limit value, and inputs extremely
RMS-DC converter circuit；
RMS-DC converter circuit extraction goes out the virtual value of current signal amplitude and feeds back to controller, controller according to
This virtual value keeps the setting height of probe tip distance sample surfaces, then controls XY nano platform fortune
Dynamic, it is achieved the scanning of sample.
Described drive voltage signal frequency is 5～50kHz, and amplitude is 10～200mV.
Described capacitance compensation value is 3～10pF.
Described optimal value is that drive voltage signal is constant, current signal amplitude virtual value minimum time capacitance compensation value.
Advantages of the present invention with good effect is:
1, the present invention use alternating current amplitude as value of feedback, therefore can overcome DC shift under DC mode,
Easily by shortcomings such as electrical noise are affected；Have more higher modulating frequency than conventional AC pattern, can realize accelerating
The purpose of scanning.
2, with low cost.When capacitance compensation (patch clamp amplifier carries function) thoroughly compensates, total current IacAbout
Equal to in-phase current Isol, now may utilize IacThe virtual value of amplitude controls the motion of probe.
3, the RMS-DC converter circuit of the present invention can extract the amplitude virtual value of total current signal, uses self-control
Circuit board completes, be a kind of low cost seek virtual value method.
When 4, the present invention regulating capacitance compensation value, use when drive voltage signal is fixed, current signal width
Capacitance compensation value during value virtual value minimum, so can make current effective value (the control input letter of control system
Number) sensitivity adjusted the distance is the highest, now system controls optimum, and scanning accuracy also can reach best.
Accompanying drawing explanation
Fig. 1 is the structural principle schematic diagram of the present invention；
Fig. 2 a is capacitance compensation process undercompensation schematic diagram of the present invention；
Fig. 2 b is that capacitance compensation process of the present invention just compensates schematic diagram；
Fig. 2 c is capacitance compensation process overcompensation schematic diagram of the present invention；
Fig. 3 is the change schematic diagram of capacitance compensation process feedback amount Iac of the present invention and distance Curve；
Fig. 4 is the scanning result figure under amplitude adjusted pattern of the present invention；
Wherein (a) two-dimensional appearance figure；(b) Two-dimensional current Error Graph；The contour line of a certain tangent plane of (c) two-dimensional appearance；
(d) three-dimensional appearance figure；
Fig. 5 is RMS-DC converter circuit theory diagrams.
Detailed description of the invention
The invention will be further described below in conjunction with the accompanying drawings.
The present invention is the novel scanning imagery pattern of SICM: the probe of SICM is positioned at inverted light mirror
In (Ti-S, Nikon company), in probe, include the submicron rank electric platforms in three directions
(9062-XYZ-PPP, New Focus company), XY nano platform (P517.3CD, PI Corp.), and Z
Sample XY direction can be carried to nanometer piezoelectric ceramic actuator (P-753.31C, PI Corp.) .XY nano platform to receive
Rice level Motion, scope is 100 μ m 100 μm, and glass tubing probe is with Z-direction piezoelectric ceramics Nano grade
Move up and down.
As it is shown in figure 1, the exchange of signal generator (AFG3022B, Tektronix company) output (5-50kHz)
Signal, signal amplitude 100-1000mV is applied to the applied voltage end of patch clamp amplifier.Patch clamp amplifier
Being set to amplification 0.1mV/pA, cut-off is filtered into 100kHz.Regulation capacitance compensation amount, electric capacity is thoroughly mended
After repaying, patch clamp amplifier (ImonitorOutfan) after the electric current that exports gives RMS-DC converter circuit, through having
Valid value switching current calculates the virtual value of current amplitude.This virtual value, as feedback quantity, is given on controller
Capture card (PCI-6251, NI company), controller is to run the computer of linux system after real time implementation, controls
Device obtains exporting controlled quentity controlled variable through PID arithmetic, then comes through Z-direction piezoelectric ceramics amplifying circuit (E504, PI Corp.)
Output drives Z-direction piezo ceramic motion.
Fig. 1 is the structured flowchart of the present invention.Patch clamp amplifier earth terminal is molten with the electrolyte immersing planchet
Ag/Agcl electrode (reference electrode) in liquid be connected, sound end then be positioned at perfusion electrolyte solution glass tubing
Ag/Agcl electrode (working electrode) in probe is connected.It is total that circuit between sound end and earth terminal can be reduced to one
Resistance Rsol composes in parallel with total capacitance Ctotal, and patch clamp amplifier capacitor compensating circuit is by electric capacity Ccomp
Form with operational amplifier.As electric capacity CcompWhen being adjusted to optimal, electric current IacPut through the output of I-V changer
Standby current I after greatmonitor(Imonitor Yu Iac is simple proportionate relationship) can pass through RMS-DC converter
Circuit is obtained amplitude and then controls the distance between probe-sample.
Fig. 5 is RMS-DC converter circuit theory diagrams, and RMS-DC converter circuit is in the hope of virtual value chip AD536
Based on chip, use dual power supply, Fig. 5 seen from external circuit connection.External communication signal i.e. monitors
Electric current ImonitorInputted by the pin 1 of AD536.Again by AD536 chip internal through calculating from pin 6
The virtual value of output current signal amplitude.Chip internal is made up of four parts successively: seek absolute value circuit
(ABSOLUTE VALUE), square divider (SQUARER DIVIDER), mirror current source
(CURRENT MIRROR) and output buffer (BUF) form.R1 is that amplification adjusts
(SCALE FACTOR ADJUST) and R4 are that direct current biasing adjusts (OFFSET ADJUST), output letter
Number VOUTWith input signal VINRelation be represented by
Avg [] is computing of averaging.
Electric capacity C1 can carry out isolated DC process to input signal, removes the direct current biasing in input signal.
Electric capacity CAVCan be used to regulate the time constant of circuit average treatment, this value is the biggest, and output virtual value is the most steady
Fixed, but response time can be elongated.
The regulation of circuit shown in R1, R4 can be used to improve the accuracy to the detection of input signal real effective.
Fig. 2 a～2c is the capacitance compensation process schematic of the present invention.As it is shown in figure 1, Icomp leads to electric capacity
Road electric current Icap is contrary, and all with driving voltage UacQuadrature in phase.And the size of Icomp has with Ccomp
Following relation
Icomp=Icap×(A1-1)Ccomp/Ctotal (1)
Icomp is for compensating electric current, and Icap is electric capacity channel current, A1For the gain of operational amplifier, Ccomp
For compensating capacitance, Ctotal is the electric capacity of electric capacity passage.Icomp and Ccomp is can be seen that from equation (1)
It is directly proportional.Along with the value of Ccomp changes, capacitance compensation process can be divided into three situations.1. undercompensation is the most firm
Compensate well 3. overcompensation.Such as Fig. 2 b, when just compensating, Icomp is equal to Icap, and now Iac obtains minimum
Value (equal to Isol)；Such as Fig. 2 a, during undercompensation, Icomp is less than Icap, and Iac is along with Ccomp increase
Reduce；On the contrary, such as Fig. 2 c, during overcompensation, Icomp is more than Icap, and now Iac is along with Ccomp increase
And increase.
Fig. 3 is the empirical curve change of Iac and distance under capacitance compensation process difference degree of compensation in the present invention,
I.e. IacAmplitude virtual value and probe/sample interval from curve with the change schematic diagram of compensation dosage.Ccomp takes
During value optimum (for 5.65pF), for just compensating situation, now Iac sensitivity of adjusting the distance is optimal, now can connect
It is bordering on the sensitivity of Isol；And when Ccomp is less than or greater than 5.65pF, is respectively at undercompensation and mends with crossing
Repay situation.During undercompensation, along with Ccomp increases (near optimum 5.65pF), Iac sensitivity of adjusting the distance increases
Add.This point can by Isol during 5.4pF adjust the distance sensitivity be better than 5.1pF time situation be verified.
During overcompensation, now increasing (away from optimal value 5.65pF) along with Ccomp, Iac sensitivity of adjusting the distance subtracts
Low, during 6.5pF, sensitivity is better than 6.2pF situation and demonstrates this point.Therefore when Ccomp value optimum, Iac
Sensitivity is the highest, and along with Ccomp deviates optimal value, Iac sensitivity is gradually lowered.
Fig. 4 is the scanning result under amplitude modulation pattern of the present invention.Modulating frequency is 30kHz, electric capacity
Compensation dosage is 6.2pF, rate of scanning 0.75Hz, and feedback control set-point is the 96% of reference signal.In Fig. 4
(a) two-dimensional appearance figure；(b) two dimension Iac Error Graph；The contour line of a certain tangent plane of (c) two-dimensional appearance figure；(d)
Corresponding three-dimensional appearance figure.
Applying alternating voltage is as pumping signal, after capacitance compensation, obtains and flows through electricity in glass tubing probe
Stream amplitude controls the distance between probe sample as feedback quantity, finally realizes under micro-nano object liquid environment
Quick, long-time lossless high resolution observations.
Concretely comprise the following steps:
1. the external voltage end of signal generator output AC voltage signal input diaphragm pincers amplifier, exchange
Voltage signal be frequency be 5～50kHz, amplitude is 10～200mV.Patch clamp amplifier is by additional exchange
Voltage attenuation 10 times is after-applied between reference electrode and working electrode, can therefore produce exchange change between two electrodes
Current signal Iac, patch clamp amplifier can amplify current signal I by I-V changerac, and export amplification
After signal Imonitor(being fixing proportionate relationship with Iac) gives RMS-DC converter circuit.
2. the compensation capacitance Ccomp of patch clamp amplifier is regulated to optimal value.I.e. keep reference electrode with
Driving voltage between working electrode is constant, and regulation compensates capacitance；Simultaneously monitor the output of RMS-DC converter circuit,
When being trough when output valve minimum, capacitance compensation terminates.
The capacitance compensation value of described patch clamp amplifier is 3～10pF.When drive voltage signal keeps constant, regulation
Capacitance compensation value also synchronizes standby current amplitude virtual value, and when virtual value minimum, capacitance compensation value is optimal.
3. increase generator output voltage and then increase driving voltage UacAmplitude, until patch-clamp is put
Big device output electric current ImonitorThe threshold value set within reaching the input peak signal limit value of RMS-DC converter circuit
(such as the 98% of limit value), it is ensured that ImonitorNot yet reach the input peak signal limit value of RMS-DC converter circuit with
Exempt to damage RMS-DC converter circuit.
4. the virtual value amount of RMS-DC converter circuit extracts current amplitude, defeated as feedback quantity using this virtual value
Deliver to the input of controller, in controller, be provided with reference value, obtain output valve by PID arithmetic, pass through
The amplification of piezoelectric ceramics power amplification circuit drives the z of carrying probe to move up and down to piezoelectric ceramics, finally makes
Feedback quantity reaches a certain setting value (such as the 96% of reference value), and now z characterizes sample to the position of piezoelectric ceramics
Product are at the topographical height of this point, and then XY platform moves in doing two dimensional surface with sample line by line, and probe
Along sample distance fixed above fluctuations, the movement locus recording piezoelectric ceramics then can depict sample
The Complete three-dimensional feature image of product.
Embodiment one
1. with wide 5 μm, the AFM demarcation silicon materials standard grid of deep 200nm (P/N 498-000-026,
Digital Instruments company) as master mold, face up, pour into 10:1 PDMS (Sylgard 184,
Dow Corning company) host stir with sclerosing agent after mixed liquor；Evacuation 20 minutes again so that
Mixed liquor bubble-free；Then it is placed in 70 DEG C, heating plate (PC-600, Corning company) top to bakee 4-5 hour；
Finally the PDMS layer after solidification is torn from silicon materials standard grid, now press close to the PDMS of Si-gate lattice
Face, by fine structure complementary with grid on the marking, so obtains PDMS material grid sample.
2. having marking structure to face up in PDMS sample, bottom surface is stained with in Φ 35mm culture dish, utilizes micro-
Phosphate buffer (PBS) is injected in culture dish by pipettor, and liquid level more than PDMS sample surfaces 2mm is
Most preferably；
3. glass tubing probe by borosilicate microelectrode capillary glass tube through program control laser draw instrument (P2000/G,
Sutter Instrument company) same program drawn, and tip internal radius is about 75nm, glass tubing probe
Interior perfusion PBS solution, with hands play gently the afterbody of glass tubing several under, it is ensured that most advanced and sophisticated bubble-free, then will
Ag/Agcl electrode is inserted, and another Ag/Agcl electrode immerses in the electrolyte solution in culture dish；
4. signal generator output AC signal, frequency is 5～50kHz, and amplitude is 100mV.And patch-clamp
Inside amplifier, external alternating voltage is decayed after 10 times as drive voltage signal, be applied to two Ag/Agcl
Between electrode, the electric current flowed through between two electrodes is linked into RMS-DC converter circuit after amplifying, and now electric current is put
Large gain is set to 0.1mV/pA, and cut-off filtering is set to 100kHz.By RMS-DC converter circuit output signal
It is transported to the input of controller；
5. regulating electric platforms by inverted microscope CCD, electric platforms slightly approaches sample, soon with probe
Close to time stop electric platforms；
6. monitor that true rms circuit output signal (is equivalent to IacAmplitude virtual value), and regulate CcompValue, makes defeated
Stop when going out signal minimum, then increase Uac；
The most carefully approaching beginning, detailed process is: piezoelectric ceramics platform with probe at the uniform velocity with the speed of 100nm/ms
Degree approaches sample, and monitors true rms circuit output signal in real time, until monitoring that signal drops to set-point (ginseng
Examine the 96% of signal) stop.Change, then piezoelectricity still it is not detected by when piezoelectric ceramics platform moves to range
Ceramic platform rollback is to initial position, and then electric platforms declines the distance approximating piezoelectric ceramics stroke, then
Repeat above-mentioned thin approximation step.
The most now probe and sample distance about glass tubing inside radius distance.Then about probe rollback 1 μm,
The most fine-tuning CcompMake true rms circuit output signal minimum.Now CcompReach optimal value.Further
Increase UacMake IacMaximize.Ultimate current amplifier capacitance compensation value is 6.2pF, is applied to patch-clamp and amplifies
The alternating voltage amplitude of device is 1500mV, and now drive voltage signal amplitude is 150mV, now feedback quantity
Value is updated to reference signal last look.Control point is set to the 96% of reference signal, regulates pid parameter,
After glass tubing probe approaches sample, finally scanning sample with the sweep speed of 0.75Hz, sweep limits is 30 μm
×30μm.See the SICM amplitude modulation pattern of Fig. 4 scanning result to PDMS grid.
Claims (6)
1. the amplitude modulation(PAM) imaging mode scan device of a SICM, it is characterised in that: include that signal occurs
Device, patch clamp amplifier, RMS-DC converter circuit, controller, probe, XY nano platform and Z-direction nanometer
Piezoelectric ceramics；
Described signal generator, patch clamp amplifier, RMS-DC converter circuit, controller are sequentially connected with；Institute
State patch clamp amplifier to be connected with probe；Probe is fixed on Z-direction nanometer piezoelectric ceramics and is positioned at XY nanometer
Above platform；Described controller is connected with XY nano platform, Z-direction nanometer piezoelectric ceramics.
2. the amplitude modulation(PAM) imaging mode scan device of a kind of SICM as described in claim 1, its feature
It is: described RMS-DC converter circuit uses RMS conversion chip, and the input of chip is put with patch-clamp
The current output terminal of big device connects, and outfan is connected with controller.
3. the amplitude modulation(PAM) imaging mode scan method of a SICM, it is characterised in that comprise the following steps:
Signal generator output AC signal, makees after the applied voltage end decay 10 times of input diaphragm pincers amplifier
It is two interelectrode drive voltage signal；
The compensation capacitance of regulation patch clamp amplifier, to optimal value, then increases drive voltage signal and makes diaphragm
The current signal of pincers amplifier output reaches the threshold value close to RMS-DC converter circuit input limit value, and inputs extremely
RMS-DC converter circuit；
RMS-DC converter circuit extraction goes out the virtual value of current signal amplitude and feeds back to controller, controller according to
This virtual value keeps the setting height of probe tip distance sample surfaces, then controls XY nano platform fortune
Dynamic, it is achieved the scanning of sample.
4. the amplitude modulation(PAM) imaging mode scan method of a kind of SICM as described in claim 3, its feature
Being that described drive voltage signal frequency is 5～50kHz, amplitude is 10～200mV.
5. the amplitude modulation(PAM) imaging mode scan method of a kind of SICM as described in claim 3, its feature
It is that described capacitance compensation value is 3～10pF.
6. the amplitude modulation(PAM) imaging mode scan method of a kind of SICM as described in claim 3, its feature
Be described optimal value be that drive voltage signal is constant, current signal amplitude virtual value minimum time capacitance compensation value.
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| CN109387670A (en) * | 2017-08-07 | 2019-02-26 | 中国科学院沈阳自动化研究所 | A kind of SICM voltage modulated imaging device and method based on difference noise reduction |
| CN109884345A (en) * | 2019-02-25 | 2019-06-14 | 燕山大学 | The method of image quality is characterized under AFM tapping-mode |
| CN110333371A (en) * | 2019-06-21 | 2019-10-15 | 武汉科技大学 | A kind of gold nano-probe preparation facilities and preparation method thereof |
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Application publication date: 20160810 |