Bionanoscopy in Biology and Medicine
Bacterial cell is practically a minimal form of a live organism. Its size does not exceed several microns. It is many times smaller than any device created by people and capable to move in space or to organize chemical reactions.
The scanning probe microscopy allows us to peep in the world of the molecular biology, microbiology and to develop practical recommendations for the molecular medicine. Biological observations in the probe microscopy (PМ) have essential specific features in comparison with other applications. Biological objects possess a low mechanical rigidity, which demands delicate scanning of their surfaces. PМ cannot observe isolated biomacromolecules, for example, freely floating in buffer solutions, but demands their fixing on a firm substrate.
In this connection, the techniques for fixing of concrete bioobjects should be tested carefully in practice. In PМ well-known are cases when hasty and unchecked actions resulted in discomfiture. Thus, the steps on a pyrolitic graphite were taken as DNA molecules, at that, the observed spatial period was wrongly compared with the alternation of separate base pairs . Subsequently it was demonstrated that DNA-like structures can be observed only on pure graphite , therefore the choice of a substrate plays a key role.
A substrate should make a minimal impact on the conformational state of the bioobjects. On the other hand, coupling with it should be strong enough to avoid a detachment of the observable object from a substrate during interaction with a microscope probe. Many objects of the molecular biology have a typical size of several nanometers, therefore the roughness of the applied substrates should be at least 10 times lower.
The traditional substrates for nano- and microobjects of biology are graphite and mica. These materials are easily chopped off by the cleavage plane, baring two absolutely pure crystal flat surfaces. Some difficulties arise with the further chemical processing of these surfaces necessary to make them suitable for adsorption of biomacromolecules or for adhesion of big objects, for example, bacteria.
In the present review first of all the methods and results of observation of concrete objects are considered, and then attention of the readers is attracted to the unique possibilities of PМ unattainable with the other methods – measurement of the mechanical properties of nucleic acids, proteins, virus particles and bacterial cells. Success in observation of larger bioobjects on the example of a structure of human hair is also discussed, and certain attention is devoted to the technologies of creation of biosensors and biochips.
Authors of the review consider it expedient to tell some words about the place occupied by PM among the other analytical methods of high resolution. The raster and transmission electronic microscopies give higher spatial resolution in observation of nucleic acids, protein molecules and virus particles in vacuum in comparison with PM in vacuum or in the air. In particular, the electronic microscopy allows us to see packaging of protein subunits of the capsule of a tobacco mosaic virus or poliomyelitis virus.
A resolution of such quality has not been reached in PM yet. However as far as observation in liquids is concerned, it has practically no competitors. Besides, the electronic microscopy transfers the width of the objects, but measurement of a height with its help is rather complicated. PM, on the contrary, ensures a direct measurement of the height of the observed objects. PM data are three-dimensional images, which include lateral sizes and height. The nuclear magnetic resonance and X-ray-structural analysis also give additional information about the structure of the biomacromolecules and relative positioning of separate atoms, components of biomacromolecules. PM cannot compete in this field. However it has no competitors when it is necessary to observe in details the structure of a dot defect or dislocation on a crystal surface. The methods of a nuclear magnetic resonance or X-ray-structural analysis allow us to read the signals from many millions of identical molecules and to judge about the structure of one of such molecules. In this sense they are integrated, while PM is a local method and can study also a single molecule or their small ensemble.
Equipment of the scanning probe microscopy
The modern medicine becomes more and more personalized. The treatment techniques inevitably should correspond to the specific features of each patient. Such conditions form a requirement for the technological complexes capable to quickly and comprehensively analyze a biological material at the level of separate cells and biomacromolecules. Thus, the scanning probe microscopy (SPM) can be applied in many directions of the modern diagnostic medicine. An effective integration of a big number of methods of analysis and their extremely high sensitivity make such a platform unique.
Today the atomic-force microscopy (AFM) is the only method, which allows us to advance microsurgery to the nanolevel, to meet the demands to accuracy, repeatability and automation of the impact on cells and give a new impetus to development of the cell technologies, including the ones for the medical practices. At the same time AFM is a modern and promising method for scientific research. Its main advantages are the following:
possibility to obtain a three-dimensional image of the examined objects with an atomic and molecular resolution;
possibility to investigate objects in a liquid environment;
high spatial accuracy and localization of the impact by the method of the power nanolithography.
The essence of the power nanolithography consists in the use of AFM probe (cantilever) as the tool for a dot impact on a surface. Although the method is widely used for creation of micro- and nano-sized reliefs on lifeless objects (basically, on synthetic polymers), its application to the live systems is limited to single experiments. Therefore, the use of AFM for microsurgical operations on cells and cellular structures is an essentially new approach in the domestic and world science.
The authors point out, that SPM are produced in Russia, USA, Germany, Japan, China and other countries. As a matter of fact, they became desktop devices used in real nanotechnologies, including in materials science, biology and medicine.
Figure 1 presents an image of SPM FemtoScan mechanical system manufactured by Center of Perspective Technologies Co. and NPP Center of Perspective Technologies Co. (www.nanoscopy.ru). It is important to notice, that in a simple contact mode the probe microscope can be considered as a profilometer with subnanometer spatial resolution (Fig.2).
Substrates for Bionanoscopy. Graphite
In order to examine an object at the atomic and molecular level it is necessary to use special atomic-smooth substrates. For this purpose the layered crystals are applied, the chip of which ensures clean and smooth surfaces. Most frequently used are crystals of mica and graphite. Graphite as a substrate has a number of important advantages:
it conducts electric current, which allows us to examine objects not only with an atomic-power microscopy, but also with a scanning tunnel, scanning resistive microscopy;
the surface of the graphite is inert, and when it is placed in water solutions, ions are not emitted from it, which could influence considerably their ionic force near a substrate.
It is necessary to conclude, that a graphite substrate has smaller influence on the objects adsorbed in it. For example, measured in AFM the height of DNA molecules on a graphite surface appears approximately twice as big, than that of the DNA molecules adsorbed on the surface of mica .
However, unlike mica, substrates from graphite are less perfect, which becomes apparent in higher frequency of occurrence of the crystal defects on a chip surface. Therefore, in order to exclude possibility of erroneous interpretation of images, it is very important, while using graphite in medical and biologic researches to have an idea about the basic types of defects on its surface. Below a brief characteristic is presented of the defects observed on the surface of a graphite chip. First, it would be appropriate to talk about the defects appearing on topographical images, and then about the flat defects which do not create a relief on a surface, but are capable to affect the process of adsorption of the bioobjects.
Among the most widespread defects of the surface of graphite are chip steps (Fig.3). They have the same direction which coincides with the chip direction. For samples of highly oriented pyrolitic graphite (HOPG) with a mosaic structure of 0.4° and 0.8° the extent of the chip steps per unit of the area of the surface equals to 1–3 μm–1. The smaller is the angle of the mosaic structure of such a graphite, the lower is the height of the chip steps. For the mosaic structure of 0.4° the share of the one-layer steps is ~50%, and for the mosaic structure of 0.8° their share is ~35%.
Some of the chip steps break in the centre of the graphite terraces. The reason for this is the exit of the screw dislocations to the surface, the lines of which are directed perpendicularly to the surface (Fig.4). It is interesting to notice, that often a screw dislocation makes the beginning of the edge dislocation, the line of which is in depth, under several atomic layers of the graphite. Possibility of observation of the transition of a screw dislocation into an edge one testifies to a small extent of the screw dislocations into the graphite’s depth.
Edge dislocations are visible in the atomic-force microscope (AFM), scanning tunnel microscope (STM) and scanning resistive microscope (SRM) as steps corresponding to the ruptures of the atomic layers of the graphite under the surface. Unlike the chip steps, the lines of the edge dislocations have fanciful forms and, as a rule, are closed in loops. The near-surface structure of HOPG is clearly visible, when big parts of relief are removed during subtraction of the surface spline from the images (Fig.5). The sizes of the loops vary from several dozens of nanometers up to several micrometers.
In many cases the height of the edge dislocations is lower than 0.3 nm which is less than the interlayer distance in the graphite. This is connected with their moving off the surface. Relief smoothing occurs rather slowly. In AFM experiments the edge dislocations were observed laying on the depth more than 4 nm. The surface density of the edge dislocations for the graphite with mosaic structure of 0.4° and 0.8° is
about 1 μm–1.
Structures in the form of fibers are observed on HOPG chips (Fig.6) rather seldom. Their diameter is not constant and decreases closer to their ends. If the fiber appears to be under the chip surface, the superficial atomic layers are bent in the place of its passage. In the area of passage of the fibers the top atomic layers can be bent by dozens of nanometers by height, at that, the width of the bent area can be more than one micrometer.
One of the most unusual defects on the graphite surface is stars (Fig.7). There are several versions of formation of such defects. A needle is sharply inserted into the surface of HOPG, and then is abruptly taken away, and together with it the top layers rise till the moment when the elastic energy of their deformation exceeds the energy of formation of cracks. However, the experiments with impression of the edge of a needle into the surface led to formation of a crater with a considerably less ordered structure. According to other hypothesis, the stars are formed when HOPG is chipped in the places where the graphite structure is strongly strained (compressed pore, inclusion of another phase with strongly differing characteristics).
The elastic energy of a defect is subsequently used for development of cracks and bend of the petals. The second version is confirmed by the circumstance that several stars can be at once in one frame.
HOPG is a polycrystal for which axes from all the crystal grains are oriented equally, however in the surface plane the orientation of the crystal grains is free. The size of a crystal grain is ~10 μm, therefore junction of the grains can occur rather often. The intergrain borders are observed by means of SТМ in the form of threads with height from 0.1 up to 0.3 nm and diameter of 2–5 nm. In AFM the intergrain borders are observed rarely, because in many cases their structure is flat. The intergrain borders can be observed by means of the microscopy of lateral forces.
It is interesting to point out, that the chip steps are not interrupted when they cross an intergrain border. Fig.8 demonstrates a juncture of three grains. In the area of the juncture the borders meet at the angles close to 120°. These borders have a periodic structure. The wider is the rotation angle between the neighboring grains, the less is the border period. On the graphite surface borders were observed with the period up to 25 nm.
In the places of passage of the dislocation lines on HOPG surface there is no change of the height of the surface, therefore, usually, in the atomic-force microscopy the given defects are not observed. However if measurements are done with SТМ or REM, then on atomic-smooth terraces it is possible to discover structures in the form of a set of strips. An example is shown in Fig.9. Occurrence of the given defects is connected with a graphite deformation. On an undistorted surface of HOPG the dislocation lines are observed rarely. If before a survey we bend a thin slice of a graphite, the content of the dislocation lines in it will increase considerably.
A dislocation line consists of two partial dislocations, between which a defect of packaging is located. The width of the strips usually varies from 15 up to 65 nm. In the fastening sites on the steps or intergrain borders the dislocation lines widen up to 60–70 nm. In SТМ the heights corresponding to the dislocation lines vary from 0.08 up to 0.18 nm. The contrast of the dislocation lines can be inverted, that is, the area of a packaging defect can be either above or below the neighboring areas. This effect is still unexplained. It can be due to the change of the state of the tip of a needle in the process of scanning. In many cases the dislocation lines are observed by whole groups with periods from 26 up to 170 nm.
Dislocation grids are formed in the areas of crossing of the dislocation lines with two different directions. Just like the dislocation lines, they are not visible in AFM, but can be detected by means of SТМ and REМ. A dislocation grid consists of triangles.
Sides of the triangles correspond to partial dislocations, delimiting the areas of the packaging defect and of the defect-free graphite. The triangles containing a packaging defect have concave borders. During scanning the contrast of the dislocation grids can be inverted just like in case with the dislocation lines. For example, in Fig.10 the dark triangles correspond to the packaging defects. As a rule, in STM images the triangles’ height is less than 1 nm.
Hexagonal superlattices (moires) are formed, when the atomic layers of the graphite turn in relation to each other (Fig.11). In most cases the areas with a superlattice are limited by the chip steps and intergrain borders. The less is the angle of rotation of the atomic layers of the graphite in relation to each other, the longer is the moire period.
The symmetry of the moires corresponds completely to the symmetry of the crystal lattice of the graphite. Moires can be observed by means of SТМ and REM. For SТМ images the heights of moires vary from 0.05 up to 0.7 nm and periods – from 1.7 up to 44 nm. In AFM the moires are not visible, because the surface remains flat.
According to estimations, in normal conditions concentration of the dot defects on the surface of HOPG of different quality can make from 0 up to 20 μm–2. They are visible as bright stains on STM images. When a needle approaches the surface, the defects become invisible, because in the given conditions the main contribution to tunneling is made by the volume states of the graphite.
The local density of the states near the site of a surface with the passed atom (vacancy) increases, therefore the vacancies can be easily visualized by means of SТМ, but for AFM at a room temperature and in the air they are invisible. Figure 12 demonstrates the dot defects created on the surface of the graphite by the method of a local anode oxidation.
To be continued in the next issue