THE COMBINED LOM/AFM STUDY OF HUMAN BLOOD CELLS

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1 THE COMBINED LOM/AFM STUDY OF HUMAN BLOOD CELLS Pavel KEJZLAR 1,2, Eva MACAJOVÁ 1, Lukáš VOLESKÝ 1,2, Lenka BERGEROVÁ 3 1 Department of Material Science, Technical University of Liberec, Studentska 1402/2, , Liberec 2 Laboratory of Analytical Methods, Centre for Nanomaterials, Advanced Technologies and Innovation, Technical University of Liberec, , Liberec 3 Department of Clinical Haematology, Liberec Regional Hospital, Liberec Abstract The present work deals with the methodology and usage of a light optimal microscopy connected with atomic force microscopy for the study of different human blood cells. Through the connection of these different microscopic methods it is possible to obtain a detailed 3D image of a real sample surface supplemented by a possibility of mechanical properties (adhesion or toughness) measurement. All measurements are performed in the area of interest selected by the light optical microscopy. Keywords: Haematology, Light optical microscopy, Atomic force microscopy, Direct Overlay, Blood cells 1. INTRODUCTION Haematology is a major sub-branch of internal medicine that deals with the physiology, pathology, etiology, diagnosis, treatment, prognosis and prevention of blood-related disorders. It deals also with a study of haematopoietic organs and blood formation under physiological and pathological conditions of the whole organism. The main task of haematology is given by the very nature of blood, the only organ in the body, which comes into the direct contact with almost all tissues of the body and thus reflects their changes. Blood is a bodily fluid that delivers necessary substances to the cells and transports metabolic waste products away from those same cells. In the human body ml of blood falls on the each kilogram, therefore it occupies about 1/13 of the total weight. Of this amount, approximately % consists of blood cells and % is plasma. The most abundant cells in vertebrate blood are red blood cells which contain haemoglobin and distribute oxygen. White blood cells help to resist infections and parasites. Platelets are important in the clotting of blood [1-6]. One microliter of blood contains about 4.3 to 5.8 million erythrocytes, 4 to 7 thousands of leukocytes and 100 to 300 thousands of thrombocytes [2]. Red blood cells, or erythrocytes, are most common type of blood cell. These cells cytoplasm is rich in haemoglobin that can bind oxygen and is responsible for the blood s red colour. Mature red blood cells lack a nucleus and most organelles, in order to accommodate maximum space for haemoglobin. The erythrocytes are marked by glycoproteins that define the different blood types. The proportion of blood occupied by red blood cells is referred to as the haematocrit (normally about 45 %) [1-6]. White blood cells (leukocytes) are a part of the body s immune system. They remove old cells and cellular debris, as well as attack pathogens and foreign substances. Haematology science involves the five types of white blood cells neutrophils, monocytes, lymphocytes, eosinophils and basophils. They all have many things in common, but they are distinct in their form and function. A major distinguishing feature of some leukocytes is the presence of granules; white blood cells are often characterized as granulocytes or agranulocytes. Granulocytes (polymorphonuclear leukocytes) are characterized by the presence of differently staining granules in their cytoplasm when viewed under light microscopy. These granules (usually

2 lysozymes) are membrane-bound enzymes that act primarily in the digestion of endocytosed particles. There are three types of granulocytes: neutrophils, basophils, and eosinophils, which are named according to their staining properties. Agranulocytes (mononuclear leukocytes): are characterized by the apparent absence of granules in their cytoplasm. The agranulocytes include lymphocytes, monocytes, and macrophages [1-8]. Platelets, also called thrombocytes, are small, disk shaped clear cell fragments which are derived from fragmentation of precursor megakaryocytes. Thrombocytes are generally responsible for blood clotting. Thrombocytes are very brittle. If the vessel cell is damaged, they hit against the edge of damaged vessel, shatter and thromboplastin, a substance responsible for blood clotting, is released from their cytoplasm [1-8]. An exact haematological diagnosis, in which we use the information obtained from the blood analyser and light optical microscope, tell us about whether is the assessed sample physiological or pathological. The pathological specimens can be assessed in what stage of the disease the patient is. Application of advanced analytical methods such as scanning probe microscopy and adequate research in this area opens up new treatment options not only in haematology, but also in cardiology, neurology, etc. The purpose of this work is to outline the possibilities and potential of combination of currently used light optical microscopy with high resolution atomic force microscopy. 2. EXPERIMENT 2.1 Sample preparation For the experiment the blood smears were prepared on glass slides. The peripheral blood was collected from the finger. After drying the smear was fixed by a May-Grünwald solution which contains a methanol. For a light optical microscopy the sample was stained by a Romanowsky-Giemsa method. Between the individual steps, the sample was flushed by distilled water. The sample used for AFM measurement was only fixed by methanol. 2.2 Experimental equipment For the light optical microscopy (LOM) was used an inverted microscope Zeiss AXIO Observer A1 (Fig. 1A). It was configured for observation of transparent samples in a transmitted light. The LOM is equipped with a special stand that allows three-point establishment of an atomic force microscope (AFM) head (Fig.1B). This enables simultaneous analysis of the area of interest by the both methods. Detailed imaging was realized by the use of atomic force microscope JPK Nanowizard III. AFM analysis were performed in non-contact (AC) mode, the used cantilever was Nanosensors CPPP-NCHAuD-30. The software used for AFM control was JPK SPM Desktop The results were processed in JPK Dataprocessing software. For the correlation of both, LOM and AFM images, a SW module Direct Overlay was used.

3 Fig. 1: A) Inverted light optical microscope with AFM head. B) A special stand allowing three-point establishment of the AFM head. 2.3 Experimental procedure The blood smear was placed to the holder on the special stage (Fig. 1B), then the AFM head was established on the light optical microscope. Area of interest was selected by a camera connected to LOM optics. Before the analysis, calibration between AFM- and LOM image has been performed. Transformation between optical images coordinates and AFM scan coordinates is calculated by the use of recognition of the cantilever in 25 predefined grid positions. All the magnification, rotation, stretching and nonlinearity are calculated solely from the cantilever images. After this simple semiautomatic procedure, the apparatus is ready for detailed AFM analysis. 2.3 Experimental results Erythrocytes Thrombocytes Leukocytes Fig. 2: A) LOM image of the stained blood smear. Two big cells containing nuclei are leukocytes (neutrophils), non-nuclear cells are erythrocytes, the smallest objects are thrombocytes. B) LOM image of non-stained blood smear overlayed by AFM scans.

4 Fig. 2A shows typical light optical image of stained blood smear in transmitted light. Erythrocytes can be seen as pink, disc-shaped non-nuclear cells. The big cells in the middle showing blue stained nucleus are leukocytes, especially neutrophils. The small violet segments are platelets. In Fig. 2B is an example of LOM image of non-stained blood smear overlayed by some AFM scans. In the Fig. 3 there are detailed images of the erythrocyte s surface in taken different modes. Scanned area was about 1.5 x 1.5 µm. The left image shows detailed topology. In the Fig. 3B, which was taken in phase contrast we can clearly see glycoproteins on its surface. On the right side is Fig. 3C showing a material contrast based on different suppleness. Fig. 3: Detailed erythrocyte surface. A) Height image; B) Phase image; C) Amplitude image Fig. 4 shows a core of neutrophil. On the left side, LOM image is overlayed by height AFM scan, on the right side is a detail in phase contrast. Very fine surface structure is clearly visible. Fig. 4: LOM image of neutrophil s nucleus overlayed by AFM height image. B) Detailed Lock-in Phase AFM image.

5 In Fig. 5 are AFM scans of a neuthrophil. In phase contrast, there are visible microvilli-like tips serving as receptors. Fig. 5: AFM image of neutrophil. A) Height image; B) Lock-in Phase. In Fig. 6 there is a height image of thrombocyte; on the right side is its 3D reconstruction. Thrombocyte. A) Height image; B) 3D reconstruction. 3. CONCLUSIONS The images taken by LOM usually show a small difference from real dimensions. This is caused due to aberrations arising from the lenses and mirrors of the light optical microscope. If we use the DirectOverlay feature, all of these imperfections are compensated, because the DirecOverlay uses the automatically

6 recognized cantilever positioning to map the optical image and calibrates it based on precise positioning of linearized AFM piezos. This option is important for comparison of AFM and LOM images during imaging or for precise offline overlays. Light optical microscopy is an intensively used method in haematology for identification and evaluation of individual blood cells. The main advantages of this method are speed and ability to use different contrast methods (different colouring techniques, phase contrast, DIC ). On the other hand, atomic force microscopy gives the opportunity for the detailed study with a resolution up to individual molecules level. In addition to the detailed surface structure imaging, the measurement of mechanical properties is possible. This allows us better understand to the physiological mechanisms occurring in the body. The method allows microscopic study of both, dried specimens on air and living cells in physiological saline. ACKNOWLEDGEMENT The research was supported by the SGS project Innovation in Material Engineering and by the project CxI CZ.1.05/2.1.00/ LITERATURE [1] Robert B. Tallitsch, et al. Human anatomy (5th ed.). San Francisco: Pearson/Benjamin Cummings. (2006). ISBN [2] Pecka M. Laboratorní hematologie v přehledu I-III. Český Těšín : Tiskárna FINIDR. (2002). ISBN: [3] [4] [5] [6] [7] [8]