Basic Principle Of Scanning Probe Microscopy Biology Essay

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Scanning probe microscopy (SPM) is a family of microscopy which is used to measure surface images and surface properties by scanning the surface of the samples [2, 3]. Since the inception of SPM in 1982, large numbers of papers are devoted to extend its types and applications.

Until the year 2012, there are almost 30 established types of scanning probe microscopy, including atomic force microscopy (AFM) [4], scanning tunneling microscopy (STM) [5], kelvin probe force microscopy (KPFM) [6] and scanning thermal microscopy (SThM) [7]. Of all these techniques, AFM and STM are the most commonly used ones, especially for measuring roughness of samples.

Considering its advantages, SPM is used to measure the surface properties in many research fields, such as semiconductor applications, organic applications, biological applications and polymer applications [3]. SPM is well suitable for measuring the characterization of semiconductor materials, the surface topography, and defect content [8]. Harris et al. [9, 10] used low-temperature scanning near-field optical microscopy (SNOM), which is a new developed type of SPM, studying the spectroscopies of single, nanometer dimension, cleaved edge overgrown quantum wires.

Recent studies about SPM also show the possibility of producing high resolution images of biomolecules. Although STM is the first one to be applied to biological fields, the limitation is also obvious that it is not suited for analyzing some biomolecules such as deoxyribonucleic acid (DNA), which are not electrically conductive. On the contrary, AFM, which was invented in 1986, is widely used for imaging both electrically conductive and nonconductive samples in biological fields [4]. Just because of its high resolution and other advantages, a number of studies are taken to analyze the three-dimensional surface properties of biological samples from micrometer scale to the atomic scale by AFM [11-13].

In AFM, a sharp tip (probe) is attached to a cantilever-type spring at its end. The images of AFM are taken by scanning the sample surface in response to the force between the tip (probe) and sample [14]. Commonly, the cantilever is made of silicon or silicon nitride in the scale of nanometers. Depending on different samples and modes, forces which are measured between the tip and sample can summarized as follows (Fig. 1): van der Waals forces, capillary forces, ionic repulsion forces, elastic forces, frictional forces, chemical binding forces, and magnetic and electrostatic forces, etc. [15]. To measure the deflection, some different methods such as laser or piezoresistive AFM cantilevers are used. In addition, the modes of AFM, including contact mode, non-contact mode, dynamic contact mode and tapping mode, depend on its applications.

2. Scope and Motivations

In this book chapter [1], three applications of AFM are introduced in biological fields. One application is that AFM is used for measuring visualization of biological samples. On the other hand, much attention has been paid to the study of the physical properties of biological samples. Finally, the possible use of AFM as a manipulation tool is also described and the dissections of samples have been performed. Additionally, the results of biomaterials such as DNA molecules, chromosomes and collagen fibrils obtained by AFM are shown for further comparison and discussions.

Although the images obtained by AFM are similar to SEM in some respects, some advantages of AFM in biological studies are summarized in the chapter [1]. AFM can provide three-dimensional information of samples in the images, which makes them similar to SEM images. The high resolution of AFM shows the potential to analyze the three-dimensional characterization of samples from the micrometer scale to the atomic scale. Comparing with other microscopy such as STM and SEM, AFM can directly measure the samples which are not electrically conductive. Thus, no metal coat or any other conductive treatment is needed for the nonconductive samples. Moreover, AFM could overcome the shortcomings of SEM, that is, it can observe samples not only in a vacuum but also in air or liquid environment, and it can also provide quantitative height information of biological samples.

3. Methods and Results

3.1 Measuring visualization of biological samples

AFM is the most competitive method to obtain the visualization information of biological samples among all the types of SPM techniques. Many relevant experiments [11, 16] are taken and the shape of several macromolecules is obtained by AFM [17]. Although the resolution of AFM images hasn't shown the predicted level, which may be caused by the unevenness of the samples, the preparation procedure is less complicated than the similar technique transmission electron microscopy (TEM).

3.1.1 Biomolecules

The measuring methods and visualization results of two examples (DNA molecules and collagen type I molecules) are shown in this section.

The DNA preparation for AFM is to deposit it on the substrate such as mica, sapphire and graphite. However, previous studies show that there exists a serious problem that DNA molecules cannot bind strongly to the substrate. The common method to solve it is to treat the surface with Mg2+ and other divalent and trivalent cations [17]. In this experiment [1], the mice substrate was treated with N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid (HEPES)-Mg2+ buffer and result image is shown in Fig. 2 (left). Actually the experiment also found that DNA molecules can bind to the mica substrate even in distilled water. Meanwhile, the operation mode was chosen as a dynamic mode or intermittent contact mode. Comparing with TEM images, the height information obtained by AFM images shows that the DNA height is usually about 1 nm or less and the width ranges from 8 to 20 nm.

The images showed in Fig. 2 (right) provide the collagen type I molecules information of height and width as 0.5-1 and 6-10 nm, respectively. At high magnification, at the ends of the molecules we can clearly see the globular bulges.

3.1.2 Isolated Intracellular and Extracellular Structures

In this section, the AFM imaging of chromosomes and collagen fibrils is shown.

Both the images of dry and wet chromosomes can be obtained by AFM using a dynamic mode. The preparation of dry chromosomes is usually the same with the standard method for light microscopy. The images in Fig. 3 show that chromosomes are composed of the highly condensed chromatids, and the fibers of which is about 50-60 nm thick. However, the preparation of imaging wet chromosomes is easier because the images can be obtained in liquid environments. Due to the softness of chromosomes in liquid environments, the operator should carefully adjust the interaction force to the minimum degree.

A bunch of dry collagen fibrils with a diameter of about 40nm are observed and the results show that the height and width are 35-40 nm and 100nm, respectively.

3.1.3 Living Cells and the Movement

To better observe the living cells and their movement in liquid environments by AFM, two pretreatments should be done. One is that living cells should be attached to the substrate firmly. The other is to expose the chamber to 5% CO2/95% air or to perfuse fresh culture medium into the chamber, so that the pH in the chamber is suitable. The same as the operation of imaging chromosomes, the interaction force should be carefully adjust to the weakest one. Using the contact mode, the results of AFM provide the information of the contour of living cells as well as the fixed ones. And also, variable deflection mode is used to obtain the undulation of the cell surface and the some cell processes.

3.1.4 Combination of AFM and SNOM

AFM combined with scanning near-field optical microscopy (SNOM), which can obtain both topographic and fluorescence images of biological samples simultaneously, is competitive in biological fields.

A Study by Yoshino et al. [18] shows the successful image results of a single DNA fiber at 100-nm resolution gotten by SNOM/AFM.

3.2 Measuring physical properties of biological samples

3.2.1 Evaluation Methods

In order to measure physical properties of biological samples, two modes, which are useful for soft material, are described: force mapping mode and force modulation mode.

The force mapping mode is generally used to measure the local Young's modulus of soft materials [19] by analyzing the recorded force versus distance curve. The relation between the depth and force F is shown as follows:

where k is the spring constant of the cantilever, Z0 is the sample height under zero loading force. By combining the theory of Hertz and the fitting curve of Sneddon's model, the applied loading force F can by calculated by

where E is the Young's modulus, v is the Poisson ratio of the sample, and R is the radius of the cantilever tip. If the thickness of the sample is comparable to the indentation, the model is invalid, and a new model proposed by Dimitriadis et al. is chosen.

The force modulation mode is able to measure both local elasticity and viscosity of samples. Young's modulus E and the viscosity coefficient can be expressed as follows:

where C1 and C2 are instrument constants including the spring constant of the cantilever, is the amplitude ratio and is the phase lag of the cantilever deflection.

3.2.2 Examples for Sample Measurements

The physical properties obtained by AFM from chromosomes, single cells and cell colonies are described in this section.

The experiment of mapping mitotic human chromosomes was taken in phosphate-buffered saline (PBS) solution and the cantilever was chosen with a length of 85 , a width of 20 and a spring constant of 0.5 N/m. The curve (3) was used to analyze the physical properties. Fig. 4 shows both the topography and elasticity images of the human chromosome by AFM using the force mapping mode. The height of the chromosome is 150-360 nm.

On the other hand, the researchers took experiments to measure living fibroblasts (NIH-3T3) using the force modulation mode. The pretreatment for NIH-3T3 is complicated. The cell suspension should firstly be plated on a glass petri dish precoated with fibronection. Then the samples were incubated in the HEPES buffer for 1 hour to keep the pH constant during the measurement. From the stiffness image, the local stiffness can good agree with that obtained by force mapping mode for an identical cell [20].

After the similar preparations of the samples, the stiffness distribution and time-lapse images of the colony were measure using a new developed wide-range scanning probe microscope. The results indicate that the colony moves like a single cell.

3.2.3 Combination of AFM with Other Techniques

Combination of AFM with other techniques can fully take advantage of all the methods, being more suited for investigating physical properties of living systems. For example, stretching devices can be used to examine the response of living cells caused by external mechanical stimuli, fluorescence observation for green fluorescent protein (GFP)-actin is used to examine the reconstruction of the stress fibers after the deformation.

3.3 AFM as a manipulation tool

AFM has the potential to be a manipulation tool owing to its direct contact with the sample. Depending on the different size of the sample, the mode should be chosen differently. For example, an experiment [21] was taken to dissect the DNA molecules using the contact mode by increasing the force applied by the AFM tip in a nonvacuum (liquid) environment. However, when dissecting the larger biological structures such as chromosomes, the contact mode is not suitable. Thus, a dynamic force mode has been chosen for such thick and wide samples [22].

4. Problems

From the results obtained from the experiments, we find the width is always much larger than their height. This phenomenon can be explained by the convolution effect of the probe tip shape. This effect cannot be ignored that the radius of the probing tip is generally about 10 nm when the cantilevers are commercially purchased. Much research [23] has been taken to apply carbon nanotube as a new kind of probe material to overcome this shortcoming. These carbon nanotube probes are also make it possible to dissect biomolecules at the nanoscale.

Although the predicted resolutions of AFM for imaging are rather high, the molecular structures of softer biological materials can only be achieved at lower resolutions. The potential development of AFM is how to increase the resolutions of images to the predicted ones.

Meanwhile, during the process of measurement, the scan speed for AFM imaging of living cells is quite slow, under 20 /s. That means it takes 2min or more to get a single image with a 40 -40 scan area. This speed only allows examining dynamic events of living cells on a time scale of minutes, including cell movement and secretory and excretory events. As a result, how to develop AFM to a high-speed one, which is expected to become a powerful tool for investigating sequential process of biological events occurring on a less time level, has attracted much more attention than before.

How to attach living cells to the glass surface during scanning process. It is difficult to operate the tip accurately at the steep slope of the high sample.

The values of the viscosity on the nanoscale have not been compared with those obtained by other methods.