Scaning investigation microscopy ( SPM ) is a household of microscopy which is used to mensurate surface images and surface belongingss by scanning the surface of the samples [ 2, 3 ] . Since the origin of SPM in 1982, big Numberss of documents are devoted to widen its types and applications.
Until the twelvemonth 2012, there are about 30 established types of scanning investigation microscopy, including atomic force microscopy ( AFM ) [ 4 ] , scanning burrowing microscopy ( STM ) [ 5 ] , kelvin investigation force microscopy ( KPFM ) [ 6 ] and scanning thermic microscopy ( SThM ) [ 7 ] . Of all these techniques, AFM and STM are the most normally used 1s, particularly for mensurating raggedness of samples.
Sing its advantages, SPM is used to mensurate the surface belongingss in many research Fieldss, such as semiconducting material applications, organic applications, biological applications and polymer applications [ 3 ] . SPM is good suited for mensurating the word picture of semiconducting material stuffs, 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, analyzing the spectrometries of individual, nanometer dimension, cleaved border overgrown quantum wires.
Recent surveies about SPM besides show the possibility of bring forthing high declaration images of biomolecules. Although STM is the first one to be applied to biological Fieldss, the restriction is besides obvious that it is non suited for analysing some biomolecules such as deoxyribonucleic acid ( DNA ) , which are non electrically conductive. On the contrary, AFM, which was invented in 1986, is widely used for imaging both electrically conductive and nonconducting samples in biological Fieldss [ 4 ] . Just because of its high declaration and other advantages, a figure of surveies are taken to analyse the 3-dimensional surface belongingss of biological samples from micron graduated table to the atomic graduated table by AFM [ 11-13 ] .
In AFM, a crisp tip ( investigation ) is attached to a cantilever-type spring at its terminal. The images of AFM are taken by scanning the sample surface in response to the force between the tip ( investigation ) and sample [ 14 ] . Normally, the cantilever is made of Si or Si nitride in the graduated table of nanometres. Depending on different samples and manners, forces which are measured between the tip and sample can sum up as follows ( Fig. 1 ) : new wave der Waals forces, capillary forces, ionic repulsive force forces, elastic forces, frictional forces, chemical binding forces, and magnetic and electrostatic forces, etc. [ 15 ] . To mensurate the warp, some different methods such as optical maser or piezoresistive AFM cantilevers are used. In add-on, the manners of AFM, including contact manner, non-contact manner, dynamic contact manner and tapping manner, depend on its applications.
2. Scope and Motivations
In this book chapter [ 1 ] , three applications of AFM are introduced in biological Fieldss. One application is that AFM is used for mensurating visual image of biological samples. On the other manus, much attending has been paid to the survey of the physical belongingss of biological samples. Finally, the possible usage of AFM as a use tool is besides described and the dissections of samples have been performed. Additionally, the consequences of biomaterials such as DNA molecules, chromosomes and collagen filaments obtained by AFM are shown for farther comparing and treatments.
Although the images obtained by AFM are similar to SEM in some respects, some advantages of AFM in biological surveies are summarized in the chapter [ 1 ] . AFM can supply 3-dimensional information of samples in the images, which makes them similar to SEM images. The high declaration of AFM shows the possible to analyse the 3-dimensional word picture of samples from the micron graduated table to the atomic graduated table. Comparing with other microscopy such as STM and SEM, AFM can straight mensurate the samples which are non electrically conductive. Therefore, no metal coat or any other conductive intervention is needed for the nonconducting samples. Furthermore, AFM could get the better of the defects of SEM, that is, it can detect samples non merely in a vacuity but besides in air or liquid environment, and it can besides supply quantitative height information of biological samples.
3. Methods and Consequences
3.1 Measuring visual image of biological samples
AFM is the most competitory method to obtain the visual image information of biological samples among all the types of SPM techniques. Many relevant experiments [ 11, 16 ] are taken and the form of several supermolecules is obtained by AFM [ 17 ] . Although the declaration of AFM images has n’t shown the predicted degree, which may be caused by the variability of the samples, the readying process is less complicated than the similar technique transmittal negatron microscopy ( TEM ) .
The measurement methods and visual image consequences of two illustrations ( DNA molecules and collagen type I molecules ) are shown in this subdivision.
The DNA readying for AFM is to lodge it on the substrate such as isinglass, sapphire and black lead. However, old surveies show that there exists a serious job that DNA molecules can non adhere strongly to the substrate. The common method to work out it is to handle 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 consequence image is shown in Fig. 2 ( left ) . Actually the experiment besides found that DNA molecules can adhere to the isinglass substrate even in distilled H2O. Meanwhile, the operation manner was chosen as a dynamic manner or intermittent contact manner. Comparing with TEM images, the height information obtained by AFM images shows that the DNA tallness is normally about 1 nanometers or less and the width scopes from 8 to 20 nanometers.
The images showed in Fig. 2 ( right ) provide the collagen type I molecules information of tallness and breadth as 0.5-1 and 6-10 nanometer, severally. At high magnification, at the terminals of the molecules we can clearly see the ball-shaped bumps.
3.1.2 Isolated Intracellular and Extracellular Structures
In this subdivision, the AFM imagination of chromosomes and collagen filaments is shown.
Both the images of dry and wet chromosomes can be obtained by AFM utilizing a dynamic manner. The readying of dry chromosomes is normally the same with the standard method for light microscopy. The images in Fig. 3 show that chromosomes are composed of the extremely condensed chromatids, and the fibres of which is about 50-60 nm midst. However, the readying 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 set the interaction force to the minimal grade.
A clump of dry collagen filaments with a diameter of about 40nm are observed and the consequences show that the tallness and breadth are 35-40 nanometers and 100nm, severally.
3.1.3 Living Cells and the Motion
To better detect the life cells and their motion in liquid environments by AFM, two pretreatments should be done. One is that populating cells should be attached to the substrate steadfastly. The other is to expose the chamber to 5 % CO2/95 % air or to perfuse fresh civilization medium into the chamber, so that the pH in the chamber is suited. The same as the operation of imaging chromosomes, the interaction force should be carefully adjust to the weakest 1. Using the contact manner, the consequences of AFM provide the information of the contour of populating cells every bit good as the fixed 1s. And besides, variable warp manner is used to obtain the wave of the cell surface and the some cell procedures.
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 at the same time, is competitory in biological Fieldss.
A Survey by Yoshino et Al. [ 18 ] shows the successful image consequences of a individual Deoxyribonucleic acid fibre at 100-nm declaration gotten by SNOM/AFM.
3.2 Measuring physical belongingss of biological samples
3.2.1 Evaluation Methods
In order to mensurate physical belongingss of biological samples, two manners, which are utile for soft stuff, are described: force function manner and force transition manner.
The force function manner is by and large used to mensurate the local Young ‘s modulus of soft stuffs [ 19 ] by analysing the recorded force versus distance curve. The relation between the deepness and force F is shown as follows:
where K is the spring invariable of the cantilever, Z0 is the sample tallness under zero lading force. By uniting the theory of Hertz and the fitting curve of Sneddon ‘s theoretical account, the applied burden force F can by calculated by
where Tocopherol 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 indenture, the theoretical account is invalid, and a new theoretical account proposed by Dimitriadis et Al. is chosen.
The force transition manner is able to mensurate both local snap and viscousness of samples. Young ‘s modulus Tocopherol and the viscousness coefficient can be expressed as follows:
where C1 and C2 are instrument invariables including the spring invariable of the cantilever, is the amplitude ratio and is the phase slowdown of the cantilever warp.
3.2.2 Examples for Sample Measurements
The physical belongingss obtained by AFM from chromosomes, individual cells and cell settlements are described in this subdivision.
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 breadth of 20 and a spring invariable of 0.5 N/m. The curve ( 3 ) was used to analyse the physical belongingss. Fig. 4 shows both the topography and snap images of the human chromosome by AFM utilizing the force function manner. The tallness of the chromosome is 150-360 nanometer.
On the other manus, the research workers took experiments to mensurate populating fibroblasts ( NIH-3T3 ) utilizing the force transition manner. The pretreatment for NIH-3T3 is complicated. The cell suspension should foremost be plated on a glass petri dish precoated with fibronection. Then the samples were incubated in the HEPES buffer for 1 hr to maintain the pH invariable during the measuring. From the stiffness image, the local stiffness can good hold with that obtained by force function manner for an indistinguishable cell [ 20 ] .
After the similar readyings of the samples, the stiffness distribution and time-lapse images of the settlement were step utilizing a new developed wide-range scanning investigation microscope. The consequences indicate that the settlement moves like a individual cell.
3.2.3 Combination of AFM with Other Techniques
Combination of AFM with other techniques can to the full take advantage of all the methods, being more suited for look intoing physical belongingss of populating systems. For illustration, stretching devices can be used to analyze the response of life cells caused by external mechanical stimulation, fluorescence observation for green fluorescent protein ( GFP ) -actin is used to analyze the Reconstruction of the emphasis fibres after the distortion.
3.3 AFM as a use tool
AFM has the possible to be a use tool owing to its direct contact with the sample. Depending on the different size of the sample, the manner should be chosen otherwise. For illustration, an experiment [ 21 ] was taken to dissect the Deoxyribonucleic acid molecules utilizing the contact manner by increasing the force applied by the AFM tip in a nonvacuum ( liquid ) environment. However, when dissecting the larger biological constructions such as chromosomes, the contact manner is non suited. Therefore, a dynamic force manner has been chosen for such thick and broad samples [ 22 ] .
From the consequences obtained from the experiments, we find the breadth is ever much larger than their tallness. This phenomenon can be explained by the whirl consequence of the investigation tip form. This consequence can non be ignored that the radius of the examining tip is by and large approximately 10 nanometers when the cantilevers are commercially purchased. Much research [ 23 ] has been taken to use C nanotube as a new sort of investigation stuff to get the better of this defect. These C nanotube investigations are besides make it possible to dissect biomolecules at the nanoscale.
Although the predicted declarations of AFM for imaging are instead high, the molecular constructions of softer biological stuffs can merely be achieved at lower declarations. The possible development of AFM is how to increase the declarations of images to the predicted 1s.
Meanwhile, during the procedure of measuring, the scan velocity for AFM imagination of life cells is rather slow, under 20 /s. That means it takes 2min or more to acquire a individual image with a 40 -40 scan country. This velocity merely allows analyzing dynamic events of populating cells on a clip graduated table of proceedingss, including cell motion and secretory and excretory events. As a consequence, how to develop AFM to a high-velocity one, which is expected to go a powerful tool for look intoing consecutive procedure of biological events happening on a less clip degree, has attracted much more attending than earlier.
How to attach populating cells to the glass surface during scanning procedure. It is hard to run the tip accurately at the steep incline of the high sample.
The values of the viscousness on the nanoscale have non been compared with those obtained by other methods.