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 The Aim of the Study.
The aim of the study is to examine deformation of erythrocytes and changes in membrane structure during virus-erythrocyte interaction. This investigation shows the potential of AFM when used to examine blood cells in different physiological conditions (disease, drug action etc.)
Preparation of the Sample
The procedure of biological sample preparation is of critical importance. Two basic procedures are used for examination in the air.
Smear method. A simple blood smear is suitable for examination. It is prepared on a slide with a standard clinical laboratory method. A 5 mkl-aliquot of blood is placed onto a slide and smeared with another slide. The smear thickness decreases along a direction of smearing. The atomic force microscope SolverP47BIO has light inverting microscope as an integral part, the latter can be used for preliminary analysis of the sample. We apply it to find the areas, where blood cells are arranged in one layer and are accessible for studying. The sample is ready to research and does not require additional manipulations.
Cell sedimentation from a suspension. We use the suspension of erythrocytes in a buffer (phosphate or anyone) with pH 7.0. Cells are fixed by adding paraformaldehyde to a final concentration of 2% for 2 or more hours; then they are put into distilled water up to the concentration of 108 cell/ml. A 5 mkl-aliquot is placed onto a slide and dried in the air. The slide is attached to the microscope stage by double-sided stick tape.
Preparation for Measurement
RBC can be examined in the air or a liquid buffer. Imaging erythrocytes in buffer under physiological conditions is a more complex method. According to [1,2] drying of erythrocytes practically does not change their shape and membrane structure. It is in dried samples that the spectrin membrane skeleton was imaged [1]. Dry mode allows registering the distortion of fine membrane structure while spatial resolution in a liquid medium is limited [3]. That’s why we use the dry mode.
Both IC mode and contact mode are suitable for measurements in the air. The error mode can be used in both cases with similar results. IC mode allows simultaneous imaging in phase contrast, which gives data on the object’s elasticity, and the contact mode allows imaging in the lateral force (friction force) mode and measuring the object’s elasticity (in the mode of Force-distance curves). In this case, IC mode is employed to examine erythrocytes in the air.
When the membrane images with high resolution are obtained, it is useful to examine two images:
In HIGH and MAG (error mode).In our case, the phase contrast mode did not give additional information and was not employed in the air.
Cantilevers
We use NSG11cantilever in the intermittent contact mode and CSC11 cantilever in the contact mode. Dry erythrocytes are stable samples and not very sensitive to the tip chosen. Results with similar quality were obtained in the intermittent contact and contact mode with different cantilevers. For the liquid medium, it is very important to choose the softest cantilever to decrease the interaction forces between the tip and the sample. Erythrocytes can break away in scanning.
Special Measurement Conditions
We use a few virus-RBC pairs with viruses of different families and erythrocytes obtained from different sources. Viral suspension was mixed with erythrocytes at 4-6îC to reduce the rate of cell-virus interaction. The reaction was stopped by addition of paraformaldehyde to a final concentration of 2%.
Measurement Procedure
IC mode in the air was used. The quality of cells was estimated in a Biolam light inverted microscope that is a part of SOLVERP47BIO. The scan size was 70x70 mkm, therefore, the first scanning showed some tens of erythrocytes. This enabled to estimate deformations of RBC and to choose the object for detailed examination. We performed simultaneous registration of HIGHT and MAG signals, when the fine membrane structure was studied.
Results:
Analysis, Processing, Problems and Prospects
Overall scans in IC mode show that the smear method of preparation can deform the cells. Nevertheless this is a fast and simple method. It displays adequate results of size distribution of cells. We can see other sorts of blood cells in the smears, observe the so-called “rouleaus” described in low circulating blood and are seen in a light microscope. Sedimentation method is more flexible in use, but its application can lead to Artifacts of preparation.
Comparison of overall scans of control and experimental samples gives an overview of deformation of erythrocytes during their interaction with viral particles. For instance, avian erythrocytes do not vary during interaction with parvovirus and influenza virus while simian erythrocytes deform very fast and very strongly. The Error mode allows detecting small details of the membrane structure against an abrupt slope of the erythrocyte surface. This mode enables to identify defects of the membrane surface after the virus action. On a control sample, we can see the net of the membrane peptide described [1,2]. Influenza virus particles are visualized on the surface of chicken erythrocytes. According to publish data, the sorption on a substrate and drying of erythrocytes do not change the shape of erythrocytes and their surface structure. The comparison of deformations of RBC induced by different effects (chemicals, ionic strength or pH of solutions, disease, action of viral particles, mechanical deformation) (see [8]) enables to understand physical and chemical reactions that take place in each specific case. This information helps to develop the therapies and to create effective drugs and vaccines.
AFM study of blood can be used for direct diagnostics of blood diseases [9].
References
1.Minoru Takeuchi,* Hiroshi Miyamoto,# Yasushi Sako,§ Hideo Komizu,# and Akihiro Kusumi Structure of the Erythrocyte Membrane Skeleton as Observed by Atomic Force Microscopy Biophys J, May 1998, p. 2171-2183, Vol. 74, No. 5.
2. Zhang, P. C., Bai, C., Huang, Y. M., Zhao, H., Fang, Y., Wang, N. X. and Li, Q. (1995). "Atomic force microscopy study of fine structures of the entire surface of red blood cells." Scanning Microsc. 9(4): 981-989; discussion 1009-1010
3. Nowakowski R, Luckham P, Winlove P Imaging erythrocytes under physiological conditions by atomic force microscopy.
Biochim Biophys Acta 2001 Oct 1;1514(2):170-6
4. Zaitsev B.N., Durymanov A.G., Generalov V.M. Atomic Force Microscopy of the Interaction of Erythrocyte Membrane and Virus Particles. Proc. Intern. Workshop "Scanning Probe Microscopy-2002", p. 211-213. Nizhny Novgorod, 03.03-06.03.2002
5. Ohta Y, Okamoto H, Kanno M, Okuda T Atomic force microscopic observation of mechanically traumatized erythrocytes.. Artif Organs 2002 Jan;26(1):10-7
6. Cheng Y, Liu M, Li R, Wang C, Bai C, Wang K. Gadolinium induces domain and pore formation of human erythrocyte membrane: an atomic force microscopic study. Biochim Biophys Acta 1999 Oct 15;1421(2):249-60
7. Girasole, M., Cricenti, A., Generosi, R., Congiu-Castellano, A., Boffi, F., Arcovito, A., Boumis, G. and Amiconi, G. (2000). "Atomic force microscopy study of erythrocyte shape and membrane structure after treatment with a dihydropyridinic drug." Appl. Phys. Letters. 76(24): 3650-3652.
8. Girasole M, Cricenti A, Generosi R, Congiu-Castellano A, Boumis G, Amiconi G. Artificially induced unusual shape of erythrocytes: an atomic force microscopy study. J Microsc 2001 Oct;204(Pt 1):46-52
9. O'Reilly M, McDonnell L, O'Mullane J. Quantification of red blood cells using atomic force microscopy. Ultramicroscopy 2001 Jan;86(1-2):107-12
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