PROBE MICROSCOPY WORKSHOP AT THE PHYSICAL DEPARTMENT OF LOMONOSOV MOSCOW STATE UNIVERSITY
. Scanning probe microscopy is a simple method to study materials, including polymers and biopolymers at nanometre resolution in liquid and air. Probe microscopy allows of visualizing the surface structure of samples, assess their conformation, adhesion and adsorption on different substrates. The method does not require time-consuming sample preparation, it is sufficient t to just place a sample on an atomically smooth substrate, such as graphite and mica. Probe microscope skills and ability to process the data present an important step in the training programme for young professionals. To develop these competences, the Physical department of MSU is continuously improving and upgrading the probe microscopy workshop. The workshop is composed of eight laboratory work sessions on key topics and includes training in both basic and advanced skills in microscope handling, processing and interpretation of the probe microscopy data.
Since its invention, the scanning probe microscope (SPM) has been used in many areas of materials science and microelectronics, to characterise solid surfaces and visualise topography of microcircuits and electronic devices. The main interest present the studies of biological objects using SPM . The non-invasive nature of the method makes it an ideal tool for studying living systems. The main problem of its use while studying biological objects is that it is necessary to keep the minimal probe impact on the object surface under study.
The current laboratory workshop consists of eight lab exercises on key topics and includes an initial introduction to image acquisition and processing techniques, followed by a detailed study of four samples: graphite, polymer (butadiene styrene block copolymer), nucleic acids and bacterial cells:
Basics of the scanning probe microscope;
Scanning probe microscopy image processing;
Scanning probe microscopy: taking three-dimensional images. Part 1;
Scanning probe microscopy: three dimensional imaging. Part 2;
Scanning tunneling microscopy. Visualization of atomic lattice of graphite;
Scanning probe microscopy of block copolymers;
Scanning probe microscopy of nucleic acids;
Scanning probe microscopy of bacterial cells.
In the laboratory work, the state-of-the-art FemtoScan Online software is used to control the microscope and analyse the data obtained . All laboratory tasks have a practical orientation. Thus, evaluation of morphological features of bacterial cells is particularly important when studying the effect of biocidal drugs on cells. Basic morphological characteristics of a bacterium can be obtained using built-in algorithms. Different methods, such as threshold filtering, can be used to distinguish individual bacterial cells in an image. Figure 1 shows an AFM image of E. coli, the cells were fixed in formalin for fifteen minutes, then washed off formalin and PBS and applied onto the mica surface.
Bacterial cell characteristics obtained:
bacterial volume = 0.226 µm3;
shape factor 1 = 0.08 (ratio of radius of circumference of equivalent area to radius of circumference of equivalent perimeter), indicating that the perimeter of the object is irregular. For a circular object, the shape factor 1 is equal to unity;
shape factor 2 = 1.4 (ratio of twice the length of the skeleton of the object to its perimeter), characterises the elongated shape of the bacterium;
mean height = 131 nm (mean value of heights on all lines occupied by the object);
root-mean-square value of the surface roughness is 12 nm.
Probe microscopy provides useful information about the morphometric characteristics of red blood cells, which can be used for diagnostic purposes. In particular, erythrocyte volume is an important parameter that characterises the oxygen supply. A deviation from the norm can lead to hypoxia.
Using software, the morphometric parameters of the erythrocyte can be calculated by plotting the cross section and 3D view . Figure 2 shows an erythrocyte with a diameter of 8 μm, a height of 500 nm and a volume of 12 μm3.
Membrane shape, stiffness and roughness are also important for the diagnosis of pathologies. In , AFM was used to examine freshly isolated erythrocytes from women with early pregnancy loss (EPL); their erythrocytes differed in both roughness and Young’s modulus, and showed a trend towards a decrease in cell morphometric parameters (cell size and surface roughness) and membrane elasticity as compared with values of non-pregnant and healthy pregnant women. Accelerated erythrocyte aging was expressed by an earlier appearance of spicular and globular shaped cells, a decrease in membrane roughness and elasticity with age-related evolution.
In this way, the workshop not only provides students with useful skills in working with a microscope, but also teaches them how to analyse samples from a medical point of view. The laboratory work is carried out by both undergraduate and graduate students. Descriptions of the laboratory work can be found in detail on the website of Lomonosov Moscow State University . Currently, the task description of laboratory practical work "Atomic force microscopy: determination of persistence length of polymers and biopolymers" is under development. Organization of students’ work in the workshop, including recording of the tasks, storage of the results of work, schedule is carried out with the help of a portal with authorized and identifiable access: SPM laboratory workshop . The portal is based on the freely distributed open source system Moodle.
Provision of the workshop with a modern microscope enables practical training at a significantly higher level, and one of the most suitable options, both in terms of technical characteristics and ease to use, is a FemtoScan multifunctional scanning probe microscope. It is a multifunctional scanning probe microscope with the world’s first remote control and data analysis technology implemented via the Internet. This allows full-scale measurements to be made from any computer connected to a local area network or the Internet with an unlimited number of authorised network users being able to access experiment data in real time and carry out independent analysis, processing and construction of three-dimensional images.
When a full parallel workshop is ongoing, where all workshop labs can be performed online or offline at the same time, the number of microscopes should be the same as the number of labs. In this case, each of the microscopes is ready for observation of a specific pre-selected and preset specimen. In a sequential workshop, the optimum number of microscopes is one, and preferably at least two. In this case, switching from one examination to another will not take extra time.
The study was completed with the financial support of the Foundation for the Promotion of Innovation, Project No. 71108 and the JSC "Endor", Moscow, Russia.
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Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.