If you want to do a good job, you must first sharpen your tools, and over the decades, many modern instruments for surface structure analysis have been developed. Such as transmission electron microscopy (TEM), scanning electron microscopy (SEM), field electron microscopy (FEM), field ion microscopy (FIM), low energy electron diffraction (LEED), Auger spectrometer (AES), photoelectron spectroscopy (ESCA), electron probe, etc. These techniques play an important role in the study of various fields of surface science. ButAny technology will have certain limitations in its applicationFor example, diffraction methods such as LEED and X-ray diffraction require the sample to have a periodic structure, the resolution of optical microscopy and SEM is not enough to resolve surface atoms, high-resolution TEM is mainly used for bulk phase and interface study of thin layer samples, FEM and FIM can only detect the atomic structure and two-dimensional geometric properties on the tip of a needle with a radius of less than 100nm, and the sample preparation technology is complex, so it can be used as a sample research object is very limited; There are also surface analysis techniques, such as X-ray photoelectron spectroscopy (ELS), which can only provide spatially averaged electronic structure information; Some techniques can only obtain indirect results, and they also need to be fitted with trial and error models. In addition, some of the above-mentioned analytical techniques have special requirements for the measurement environment, such as vacuum conditions. In 1985, IBM's Binning and Stanford University's Quate developed the Atomic Force Microscope (AFM), which complemented the STM and could be used to measure the surface of any sample, whether conductive or not. Table 1 Comparison of the performance indexes of microscopy techniques
Background of electron microscopy
1932:Transmission electron microscopy（transmission electron microscope，tem）。German physicists Ernst and Luska created the first electron microscope. This microscope is imaged by emitting electrons through extremely thin sections of the specimen and is very useful for observing the internal structure of cellsTEM can magnify specimens up to 500,000 times.
1965:Scanning electron microscopy（scanning electron microscope, sem）。It emits an electron beam onto the surface of the specimen (rather than through it) and then forms a detailed three-dimensional image of the specimen's appearanceSEM can magnify specimens up to 150,000 times. 1981:Scanning tunneling microscopy（scanning tunneling microscopy，stm）；IBM-Zurich's Binnig and Rohrer invented that scientists could observe individual molecules on the outer layer of cells by detecting electron imaging escaping from the surface of a specimenSTM can magnify specimens up to 1 million times. In 1985,Atomic force microscopy（atomic force microscope，afm）。Binnig, Gerber and Quate develop the first atomic force microscope. In the 70s of the 20th century, it was proposedCryo-electron microscopy(Cryo-electron microscopy, Cryo-Tem), December 5, 2013, University of California, San Francisco, USA, with near-atomic resolution (34 angstroms), which identified the structure of TRPV1, a membrane protein that plays a central role in pain and heat perception, marking the official entry of cryo-EM into the era of "atomic resolution". AFM principle
The principle of AFM is relatively simple, it is "groping" with a tiny probeThe surface of the sampleto get information.
As shown in **, when the tip of the needle approaches the sample, the force on the tip of the needle causes the cantilever to deflect or change the amplitude. This change in the cantilever is detected by the detection system and then converted into an electrical signal that is transmitted to the feedback system and imaging system, and a series of probe changes are recorded during the scanning processImages of the sample surface information can be obtained.
AFM was developed on the basis of STM. The difference is that instead of using the electron tunneling effect, it uses the Van der Waals force interaction between atoms to represent the surface properties of the sample.
Suppose two atoms one is at the tip of the probe in the cantilever and the other is on the surface of the sample, the force between them will change with the change of distance, and the relationship between the force and the distance is shown in the figure below, when the atoms are very close to each other, the repulsion of each other's electron clouds is greater than the attraction between the nucleus and the electron cloud, so the whole resultant force is shown as the effect of repulsion, on the contrary, if the two atoms are separated by a certain distance, the effect of the electron cloud repulsion is less than the attraction between each other's nuclei and the electron cloud, Therefore, the whole resultant force behaves as a gravitational force. Atomic force microscopy uses the subtle relationship between atoms to show the appearance of atoms.
AFM imaging mode
(1) Contact type
Contact AFM is a repulsive mode in which the probe tip makes "physical contact" with the sample for flexibility, and when the tip is gently swept across the sample surface, the force of the contact causes the cantilever to bend, resulting in a surface pattern of the sample. Since it is a contact scan, the surface of the sample may be bent when it comes into contact with the sample. After multiple scans, the tip or sample becomes passivated.
Features: Typically, contact mode produces a stable, high-resolution image. But this patternNot suitable for studying biological macromolecules, samples with low modulus of elasticity, and samples that are easily moved and deformed.
(2) Contactless
In the non-contact mode, the tip of the needle vibrates above the surface of the sample and is never in contact with the sample, and the detector detects long-range forces such as van der Waals forces and electrostatic forces that do not damage the sample.
A stiffer cantilever is required (to prevent contact with the sample). The resulting signal is smaller and requires a more sensitive device, and while this mode increases the sensitivity of the microscope, it has lower resolution than both contact and tapping modes when the distance between the tip and the sample is longer.
Features: Due to the non-contact state, it is better for studying soft or elastic samples, and there is no passivation effect on the tip or sample surface, but it doesThere is a miscalculation. This mode is relatively difficult to operate, is generally not suitable for imaging in liquids, and has few applications in biology.
(3) Tapping
The microcantilever vibrates at its resonant frequency, and the oscillating tip gently taps the surface, intermittently in contact with the sample. When the needle tip is not in contact with the sample, the microcantilever oscillates freely at maximum amplitude. When the tip of the needle comes into contact with the sample surface, the amplitude of the microcantilever is reduced by the spatial obstruction, although the piezoelectric ceramic disc excites the microcantilever oscillation with the same energy. The feedback system controls the amplitude of the microcantilever to be constant, and the tip of the needle moves up and down with the undulation of the surface to obtain topography information.
Similar to non-contact AFM, it is closer to the sample surface than non-contact. There is less chance of damaging the sample than contact (without side force, friction or dragging).
The resolution of the tapping mode is as good as the contact mode, and since the contact time is very short, the interaction force between the tip and the sample is very small, typically 1 piconewton (pn) and 1 nanonewton (nn), the reduction in resolution and the destruction of the sample due to the shear force are almost gone, soIt is suitable for soft samples such as biological macromolecules and polymersImaging studies are performed.
AFM sample preparation and testing
(1) Sample preparation process
When AFM is prepared, there is no requirement for whether the sample is conductive or not, so the measurement range is relatively wide.
Fig.1 AFM sample flowprocess
(2) Test and result analysis
Take graphene oxide AFM results as an example:
Fig.2 AFM pattern of GO
Examples of AFM application techniques
AFM can operate in a variety of environments such as atmosphere, vacuum, low and high temperatures, different atmospheres and solutions, and is not limited by the conductive properties of the sample, so it has gained a wider range of applications than STM. The main uses are:(1) High-resolution imaging of conductors, semiconductors and insulator surfaces, three-dimensional topography observation
For example, AFM has 01-0.High resolution of 2nm with a resolution of about 0 in the vertical direction01nm。Although the lateral resolution of AFM and scanning electron microscopy (SEM) is similar, both AFM and SEM techniquesThe most basic difference is that there are different characterizations when dealing with specimen depth variations. Since the undulating state of the surface can be accurately obtained in the form of numerical values, AFM can analyze the overall surface image to obtain parameters such as roughness, granularity, average gradient, pore structure and pore size distribution of the sample surface, and can also perform rich three-dimensional simulation display of the topography of the sample, making the image more suitable for human intuitive vision.
Fig.3. AFM diagram of silica anti-reflection film and AFM diagram of grating
Figure 3a shows the atomic force image of the silica anti-reflection film obtained under contact mode, and the three-dimensional topography of its surface can also be seen. Figure 3b is an AFM image of a grating, where high aspect ratio structures, such as trenches and holes, are typically measured during semiconductor processing to determine the depth and width of etching. These can only be measured under SEM by cutting the sample along the cross-section. AFM can be measured non-destructively and then returned to the production line.
(2) High-resolution imaging of biological samples and organic membranes
For example, high-speed atomic force microscopy (HS-AFM) can be used to observe the activity of antimicrobial peptides on membranes at the molecular level.
Fig.4 Changes in antimicrobial peptides observed by AFM
As can be seen from Figure 4, after several tens of minutes of exposure, the observable structure has changed, and imaging observes a "depression", a circular area with reduced film thickness, which, together with oligomers (Figure 4a), changes at an instantaneous velocity (0.7±0.5 ms) on the membrane. In addition, dimple clusters were observed (Figure 4C), which had not previously been described in the literature, slightly reminiscent of the cubic phase, statically located on the membrane, although the internal dynamics were rearranged on a time scale of a few seconds (Figure 4D).
The imaging speed of the AFM depends on the scanning time of each pixel. When a particle moves rapidly relative to the scanning speed of the microscope, the particle traverses several pixels in the time it takes the microscope to scan one pixel, in which case it is impossible to define each frame of the pixel in which the particle is located; The particle is not broken up in the image. In order to resolve the particles, the particles must reside in an area of one pixel for at least the time required for the microscope to scan the pixel.
Surface chemical reaction studies
(4) Nanoprocessing and manipulation, observation of the self-assembly process of biological samples.
Fig.5 The effect of A5 self-assembly on PS-rich membranes in the presence of Ca2+ was observed by HS-AFM
(5) Ultra-high-density information storage
(6) Intermolecular forces and molecular dynamics
Fig. 6 AFM monitors conformational changes with millisecond temporal resolution
As shown in Figure 6, AFM can be used to characterize the single-molecule dynamics of wild-type bacterial rhodopsin under continuous light and short pulses, and its conformational changes can be monitored with millisecond temporal resolution.
(7) Tribology and various mechanical studies
(8) Testing and quality control
Atomic force microscopy has:The following advantages:
1) The sample does not need to be conductive;
2) Able to work in a variety of environments (such as vacuum, atmosphere, liquid, low temperature, etc.);
3) It can obtain a high-resolution three-dimensional image of the surface of the object;
4) It can operate on single cells and single molecules, such as punching holes in the cell membrane, cutting chromosomes, etc.