ISSN 0006-3509, Biophysics, 2011, Vol. 56, No. 6, pp. 1069–1073. © Pleiades Publishing, Inc., 2011. Original Russian Text © G.V. Maksimov, N.A. Brazhe, A.I. Yusipovich, E.Yu. Parshina, O.V. Rodnenkov, A.B. Rubin, G.G. Levin, V.A. Bykov, 2011, published in Biofizika, 2011, Vol. 56, No. 6, pp. 1099–1104. 1069 INTRODUCTION A topical task in nanobiotechnology is to design integrated methods of cell microscopy that would assess not only the morphology of subcellular nano- structures but also their functional state. Here we dis- cuss the merits and specific features of integrating atomic force microscopy (AFM), laser interference microscopy (LIM), and Raman scattering microscopy (RSM) in erythrocyte studies. AFM is used to determine the geometric dimen- sions and relief of the cell. LIM allows examining low- contrast biological objects without using dyes; to add, knowing the refractive index, one can thereby deter- mine the content and distribution of substance in the cell [1]. With RSM, it is possible to assess not only the distribution of but also the conformational changes in the molecules of interest by the features of Raman spectra. Each of these methods separately is also used to help detect various pathological states. The shape and volume of fixed erythrocytes estimated by AFM [2] is a diagnostic trait for a number of pathologies [3]. LIM has revealed a significant increase in erythrocyte area and volume in patients with ischemic heart disease [4], which appears to be connected with a change in plasma membrane viscosity and charge, as well as with water accumulation in the erythrocyte because of acti- vated Na/H exchange. Altered conformation of hemoglobin (Hb) revealed by RS in cardiovascular patients [5] and in experimental rat brain ischemia [6] is supposed to result from changes in the membrane characteristics and redistribution of Hb in erythrocytes. Combination of AFM, LIM and RSM would thus permit assessing the change in geometry and redistri- bution of cytoplasmic compartments as well as the dis- tribution and conformation of Hb hemoporphyrin. Visualization of the distribution of organelles and plasma proteins, Hb in particular, provides valuable information on cell functioning, which can be used for studying the mechanisms of oxygen transport by erythrocytes and fine interactions among cell organelles. To add, joint use of these methods allows substantial reduction of the required sample volume. The erythrocyte is a widespread object in biomedi- cal research. Many drugs are introduced into the bloodstream, so it is important to be able to analyze their impact of erythrocyte Hb. It is now customary to distinguish two fractions: free cytosolic Hb c and mem- brane-associated Hb m ; the latter makes ~0.5% of the total. In present-day notions, Hb m interacts with the cytosolic part of the AE1 transporter (band 3 protein), as does ankyrin of the submembrane cytoskeleton (Fig. 1). Changes in the properties of the plasma membrane affect the Hb m conformation. Therefore, it is supposed that early erythrocyte pathologies may be detected by conformational alterations in submembrane Hb. It is such nanoscale alterations in Hb m that are revealed by surface-enhanced Raman scattering (SERS) [7], which is an excellent supplement to AFM и LIM. CELL BIOPHYSICS Use of Nanoparticles for Studying the Conformations of Submembrane Hemoglobin G. V. Maksimov a , N. A. Brazhe a , A. I. Yusipovich a , E. Yu. Parshina a , O. V. Rodnenkov b , A. B. Rubin a , G. G. Levin c , and V. A. Bykov d a Biological Faculty, Moscow State University, Moscow, 119991 Russia b Russian Cardiology Research-and-Practice Complex, Moscow, 121552 Russia c All-Russia Research Institute for Optical and Physical Measurements, Moscow, 119361 Russia d NT-MDT Co., Zelenograd, Moscow 124482 Russia Received June 9, 2011 Abstract—The advantages and specific features of integrated application of atomic force microscopy, laser interference microscopy, and Raman microscopy in the study of erythrocytes are discussed. For successful application of Raman microscopy in the surface-enhanced mode, use was made of silver colloids. The depen- dence of the enhancement of Raman signals on silver nanoparticle size is demonstrated. The use of developed methods in clinical diagnostics is discussed. Keywords: erythrocyte, hemoglobin, Raman spectroscopy, silver colloids, diagnostics. DOI: 10.1134/S0006350911060121