Life Science

19 Dec 2013

Live Cell Volume Measurement of SICM

Cell Volume Measurement of Scanning Ion Conductance Microscopy Myunghoon Choi (Research Product Management of Park Systems, Seoul, Korea) Ion conductance microscopy (SICM)1 is a useful tool for obtaining non-invasive images of cell surface topography.  Recently, it has been used for imaging live cells in culture medium2. Cell volume is one of the important factors for cell research because the regulation of cell volume is fundamental to cellular homeostatic mechanism3. SICM can provide accurate volume measurement of cells due to its ability to acquire quantitative dimension information. Furthermore, it can calculate cell volume changes over time caused by physiological environment or its cell status changes. In this paper, live HEK293T cell was successfully imaged and its volume changes monitored using Park SICM. Human embryonic kidney(HEK) 293T cell was cultured on glass-coverslip in DMEM(dulbeco’s modified eagle medium) with 10% FBS(fetal bovine serum) containing 1% penicillin at 37°C in a 5 % CO2 humidified atmosphere (Multi Gas Cell Incubator, SANYO, JAPAN). Then, the grown cell coverslip was transferred to the Live Cell Chamber installed in the Park XE-Bio (Park Systems). While the cell was being cultured in the live cell chamber (37°C, 5% CO2, and >70% humidity), a live cell’s images were acquired by SICM-ARS mode4. The pipette used for the SICM was pulled from borosilicate glass (O.D.,1.0 mm, I.D., 0.58 mm, length 90 mm;Warner Instruments, US) using a CO2-laser-based micropipette puller (P-2000,Sutter Instruments, Novato, US). The inner and outer diameters of the pipette were about 100 nm and 200 nm, respectively. Figure 1 (a) Sequential HEK 293T live cell images. It took approx. 0.6 hour to obtain each image, and a total of 3.3 hours for 6 images. (b) Volume change of HEK 293T: the initial cell volume was 5008 μm3; 2.6 hours later it shrank to 3645 μm3.   Cell Volume Analysis Method Using Park XEI image processing software, the cell volume was calculated from the SICMimage data. After the cell area had been  selected, the volume of the chosen area was calculated automatically. Figure 2 Illustration of the process for the cell volume measurement.  (a) Park SICM image of HEK cell (0.6h) loaded at main frame of Park XEI software. (b) Cell area selected as pink, marked as ‘1’. (c) The volume of selected cell area (pink, 1) calculated as 5395 μm3. Figure 1 is an example that explains how well SICMmeasures cell’s morphology in detail. The SICM images show the whole cell shape clearly including its delicate structures. It even shows the filopodia (the small projections around the cell) distinctively–something that is difficult to be distinguished using optical microscopes. In addition, the quantitative dimension information from the SICM allows the calculation of the cell volume precisely as shown in Figure 2. The cell volume measurement method by SICM gives more quantitative and accurate results compared to the method that monitors reagent like ions or fluorescence dyes with quantitative fluorescence microscopy5 or ion-sensitive microelectrodes6. Hence, the SICM method gives useful information to cell researches related with hypertrophy.  For instance, the quantitative volume measurement provides a new method to analyze and gauge the changes in the cell structure of cardiomyocyte that are frequently accompaniedby the changes in its volume7.   Reference 1P. K. Hansma, B. Drake, O. Marti, S. A. Gould, C. B. Prater,  Science 243, 641 (1989). 2Y. E. Korchev, C. L. Bashford, M. Milovanovic, I. Vodyanoy, M. J. Lab,  Biophys. J. 73, 653 (1997). 3M. Miragoli, A. Moshkov, P. Novak, A. Shevchuk, V.O. , et al.,   J. R. Soc. Interface doi:10.1098 (2010). 4C. Myunghoon, K. Ahram,  XE-Bio Application Note 1., Park Systems (2012) 5Crowe, W.E., Altamirano, J., Huerto, L.&Alvarez-Leefmans, F.J.,  Neuroscience 69, 283-296 (1995) 6Alvarez-Leefmans, F.J., Gamino, S.M. & Reuss, L.  J. Physiol. 458, 603-619 (1992) 7Dhalla, N.S., Saini-Chohan, H.K., Rodriguez-Leyva, D., Elimban, V.k Dent, M.R. & Tappia, P.S.  Cardiovasc. Res. 81, 429-438 (2009)

19 Dec 2013

SICM Image of Suspended Collagen Fibrils

SICM Image of Suspended Collagen Fibrils                                                                                                   Tatsuo Ushiki and Masato Nakajima (Niigata University, Japan)   Ion conductance microscopy (SICM) is useful for obtaining contact-free images of the surface topography and has been used for imaging cultured cells under liquid conditions. The Approach-Retract-Scan (ARS) mode is a new mode of SICM, which is especially powerful for imaging samples with steep slopes.  To examine the applicability of the ARS mode for imaging biological samples with greater height gaps, dense collagen fibril networks were examined by SICM. Sample preparation Collagen fibrils were obtained from the tail tendon of adult Wistar rats and stored in physiological saline with 1-10 % tymol (2-isopropyl-5-methylphenol) at 4 °C for a minimum of 1 day. A small piece of the tendon was then stretched on the surface and air dried overnight, followed by immersion of the sample in physiological saline. SSICM imaging was made in the ARS mode using XE-Bio System Figure 1 Dense collagen fibril networks imaged by the ARS/hopping SICM mode SICM. Line profile analysis is based on the red line indicated in the SICM image.Conclusion Figure 1 is an example of SICM imaging of collagen fibril networks using the ARS mode. The width of the individual fibrils varied from 50 to 470 nm. The section profile also showed that the width of the collagen fibril indicated by green arrows was 277 nm and the height difference between the glass surface and the fibril was 2606 nm. This result indicates that some fibrils are suspended over the glass substrate during scanning for SICM imaging. Thus, the ARS mode of SICM has the advantage of minimizing (or free from) the loading force, which is unavoidable in AFM.  Reference 1P. K. Hansma, B. Drake, O. Marti, S. A. Gould, C. B. Prater, Science 243, 641 (1989). 2Y. E. Korchev, C. L. Bashford, M. Milovanovic, I. Vodyanoy, M. J. Lab,  Biophys. J. 73, 653 (1997). 3P. Novak, C. Li, A. I. Shevchuk, R. Stepanyan, M. Caldwell, S. Hughes, T. G. Smart,, et al.. Nat. Methods 6, 279 (2009). 4T. Ushiki, M. Nakajima, M. Choi, S.-J. Cho, F. Iwata. Micron (2012, in press)

19 Dec 2013

Single Strand DNA Molecules

NC-AFM Imaging of Bio-molecular Samples Deoxyribonucleic Acid (DNA) is a polymer chain that stores hereditary information of species. It is made from repeating units called nucleotides. In its natural state, the chain is 2.2 nm ~ 2.6 nm, and one nucleotide unit is 0.33 nm long. Although each individual repeating unit is very small, DNA polymers can be very large molecules containing millions of nucleotides. While X-ray crystallography and electron microscopy are the main tools used to resolve the structure of DNA in 3-dimensions, scanning probe techniques have gradually gained importance in the field of high resolution DNA imaging due to their ease of sample preparation (in-necessity to crystallize the DNA prior to imaging) as well as the possibility of functional studies (e.g., study of protein–DNA interaction processes) under physiological conditions. In preparation for AFM imaging, DNA samples are usually immobilized onto a stiff, flat substrate surface. The negatively charged backbone of the DNA can be utilized for immobilization onto charged substrates such as freshly cleaved mica via electrostatic interactions. After the DNA molecules are adsorbed onto the substrate, the surface can be washed, air-dried, and promptly imaged. Alternatively, the imaging process can be carried out in physiological solutions. Publications Using the XE-AFM on DNA Molecules Journal: Nucleic Acids Research, 33(20) November 2005. 6515. Title: Synthesis of novel poly(dG)–poly(dG)–poly(dC) triplex structure by Klenow exofragment of DNA polymerase I Authors:  A. Kotlyar, N. Borovok1, T. Molotsky1, D. Klinov, B. Dwir and E. Kapon;  System: Park Systems XE-100 Abstract: The extension of the G-strand of long (700 bp) poly(dG)–poly(dC) by the Klenow exo-fragment of DNA polymerase I yields a complete triplex structure of the H-DNA type. Atomic force microscopy morphology imaging shows that the synthesized structures lack single-stranded fragments and have approximately the same length as the parent 700 bp poly(dG)–poly(dC). CD spectrum of the polymer has a large negative peak at 278 nm, which is characteristic of the poly(dG)–poly(dG)–poly(dC) triplex. The polymer is resistant to DNase and interacts much more weakly with ethidium bromide as compared with the double-stranded DNA.   Fig. 2. AFM images of (A) synthesized poly(dG–dG)–poly(dC) molecules , (B) ~700 bp parent poly(dG)–poly(dC) and (C) poly(dG–dG)–poly(dC) molecules co-deposited with a linear plasmid pSK+ DNA on mica. Statistical length analyses of single, well-separated molecules from several images at several areas are shown in the inserts to (A) and (B).

19 Dec 2013

DNA Oligonucleotides

Picoforce Measurements of Single DNA Interaction Figure 1. The chemical attachment process of 50-mer DNA oligonucleotides involves self-assembled monolayers (SAMs) of DNA-functionalized dendrons. The dendron used  in this study was 9-anthrylmethyl N-({[tris({2-[({tris-[(2-carboxyethoxy)methyl]methyl}amino)carbonyl]ethoxy}methyl)-methyl]amino}carbonyl)propyl carbamate. Sample courtesy of Prof. J.W Park (Dept. of Chemistry, POSTECH)   Using force spectroscopy, interactions between single molecules can be observed by monitoring the attractive or “jump-in” forces associated with molecular binding events. Molecular binding experiments presented by Jung et al., (2007) were repeated using the XE-100 under the guidance of Prof. Park. With the oligonucleotide sequences immobilized on the dendron-modified surface, the F-d curve shows association and dissociation of the DNA duplex. The force constant of the cantilever was calibrated in solution for every measurement by the thermal tune method, the variation of which is 12~15 pN/nm. Figure 2. (A) A typical force-distance curve of the interaction between a DNA probe and the complementary 50-mer DNA. (B) The histogram of unbinding forces acquired from the force-distance curves. The mean value of 64 pN with a narrow distribution shows that AFM can be a useful tool to study the interactions between a single DNA molecule and other single DNA molecules, RNA molecules, and proteins.   Reference Journal: J. Am. Chem. Soc. 2007, 129 (30), 9349–9355. Title: Dendron Arrays for the Force-Based Detection of DNA Hybridization Events Authors: Yu Jin Jung, Bong Jin Hong, Wenke Zhang, Saul J. B. Tendler, Philip M. Williams, Stephanie Allen, and Joon Won Park

19 Dec 2013

Embryonic Stem Cell

Elastic Properties – F-d Spectroscopy Fig 1a.  AFM force-distance (F-d) curve on cover glass in air.  The AFM was equipped with a super luminescence diode (SLD) and a cantilever with a spring constant of 0.06 K (N/m). The red line indicates the tip extension towards the sample (①②) and blue line indicates the tip retraction away from the sample (③④⑤). Downward cantilever deflection of the blue line indicates the adhesion force between the tip and sample (③④). b. Contact mode image of fixed human embryonic stem (hES) cells (45x45um). The arrow indicates the point where F-d curve (c) was obtained.  c. The F-d curve of the hES cell. The attraction due to the meniscus force, shown in Fig 1a,  is eliminated by performing the measurement in liquid.  The gradual slope of the curve in the constant compliance region is related to the elastic properties of the cell. The mechanical properties of biological systems play a major role in cellular processes. By recording and analyzing the F-d spectroscopy measurements by AFM, the elastic properties of biological systems can be determined quantitatively and reproducibly. Figs. 1a and c demonstrate the differences in force curves on stiff and soft samples. The deflection rises slower due to the deformation of the soft sample, which causes the gradual increase in the slope of the curve. Mechanical properties of many different cell types including glial cells, platelets, cardiocytes, macrophages, endothelial cells, epithelial cells, fibroblasts, bladder cells and more have been determined using F-d spectroscopy. Fig 2a. F-d curve of the H-band (a), the structure between the Z and H bands (b) and the Z-band (c). The differences in the slopes of curves indicate the different elastic properties of the structures. b. Red dots (a,b,c) indicate the positions where F-d curves were taken. c. The structure of muscle fiber. (Image courtesy of Prof. N. Rozlosnik, DTU Nanotech, Denmark)   Relevant Publications Using the XE-AFM       -  Noemi Rozlosnik, 2006 Nanomechanics of Single Muscle Fibers by AFM, International Nano-Conference(ICN+T), Basel(CH)       -  Mette Christensen, et al., 2009 Injection of marinade with actinidin increases tenderness of porcine M. biceps femoris and affects myofibrils and connective tissue, J. of the Science of Food and Agriculture, Vol. 89:1607-14      -  Manfred Radmacher, 2002 Measuring the Elastic Properties of Living Cells by the Atomic Force Microscope. Methods in Cell Biology Vol. 68:67-90

19 Dec 2013

Mouse Sperm Cells

Excellent to Assess the Morphological Features of Sperm Cells Sperm morphology is assessed routinely as part of standard laboratory analysis in the diagnosis of human male infertility. It is well-known that the morphology of fertile sperm is significantly different from that of infertile sperm.  Optical microscopy has been used to observe the morphological features of sperm cells, but requires sample staining and offers only limited resolution.  In contrast, atomic force microscopy doesn’t need any sample staining. Furthermore, it provides much higher resolution images than optical microscopy.   Figure 1. High resolution AFM Images of mouse sperm cells. (a) Topography of sperms (45 μm scan size), (b) error image of sperm (25 μm scan size) and (c) 3D rendering of topography image of sperm head (11 μm scan size).   Figure 1 and 2 show high resolution AFM images of mouse sperm cells. Mouse sperm cells were extracted from the mouse testis tissue and fixed with 1% glutaraldehyde and 1% paraformaldehyde in phosphate buffered saline. Then, they were attached onto the poly-L-lysine coated glass slide and it was washed out with filtered distilled water twice. Figure 2. High resolution AFM images of mouse sperm cells. (a) Error image of sperm head (11 μm scan size), (b) 3D rendering of topography image of sperm tail (5 μm scan size) and (c) error image of sperm tail (5 μm scan size).   The images were obtained using contact mode AFM in air. A gold-coated silicon nitride cantilever was used to image the sample. Its spring constant is approximately 0.06 N/m.  AFM images can be quantitatively analyzed using XEI software. The dimensions and features of sperm cell parts can be measured and evaluated with the strict criteria for sperm morphology.  The Park AFM is expected to become an excellent tool to assess the morphological features of sperm cells.

19 Dec 2013

Liposomes and Vesicles

Bio Atomic Force Microscope: Liposomes and Vesicles AFM imaging as a Tool to Visualize Liposomes and Vesicles Lipid bilayer formed by the fusion of phospholipid vesicles is invaluable to researchers in the study of the characteristics and behavior of membrane-bound proteins, cell membrane and signal transduction. Phospholipid vesicles or liposomes, however, are also important to the study of secretory vesicles. Figure 1. AFM image of liposomes in liquid imaged with the XE-100 (2 μm size scan) Non-Contact mode.   A vesicle is a relatively small and enclosed compartment, separated from the cytosol by at least one lipid bilayer. The vesicle is the simplest organelle in the cell. These vesicles are used for chemical reaction chambers, enzyme storage, and transporters.  Vesicle trafficking is a common source of manifestation of many human pathologies. Dysfunction in vesicle trafficking in targeting and delivery of specific proteins to the cell surface have serious consequences and have been implicated in diseases such as Alzheimer’s, cystic fibrosis, type II diabetes and polycystic kidney diseases, just to name a few.  AFM can be utilized to study the size dynamics of vesicles in isolated preparations in liquid. The swelling dynamics of vesicles and interaction with target membranes can be visualized with high resolution. Phospholipid vesicles or liposomes can mimic secretory vesicles such as zymogen granules or synaptic vesicles by incorporating vesicular proteins. Fundamental studies on vesicular membrane ligand-receptor interaction, membrane adhesion and fusion, and numerous other biophysical phenomena can be studied. By using specific substrates such as titanium oxide, which resists lipid absorption, AFM can image liposomes. Non-Contact mode of Park AFM is specially useful for imaging ultra-soft materials like liposomes in liquid. Figure 2. 3D rendering of AFM images of lliposomes in liquid with the XE-100 (2 μm size scan) Non-Contact mode.   Figure 3. AFM topography and error images of lliposomes in liquid with the XE-100 (2 μm size scan) Non-Contact mode.

19 Dec 2013

Lipid Vesicle and Bilayer

AFM Imaging as a Tool to Visualize Bilayer Formation from Lipid Vesicles Figure 1. AFM image of lipid vesicles and bilayer in liquid imaged with the XE-100 (3 μm scan size).   Phospholipid bilayers are important in biology because they form the basic structure of the plasma membranes of all living cells. The plasma membrane surrounding the living cell serves several functions, such as control of solute permeability and recognition events.  AFM is an ideal tool for studying both physical and chemical characteristics of those membranes at a highly localized level since it can uniquely generate high resolution images in liquid. Lipid bilayer is usually deposited onto the substrate using so-called vesicle fusion technique. Figure 1 shows a topographic image of partially flattened vesicles and lipid bilayers. The image was obtained using contact mode AFM in buffer solution. When the vesicles hit a suitable solid surface, they may adsorb, break up, and spread to form a bilayer on a hydrophilic surface. The fused vesicles produce flat domains as shown in Figure 2, 3 and eventually will cover more of a solid surface. Figure 2.  Line profile of lipid vesicles and bilayer on mica surface 3 μm scan size).   The sequential vesicle fusion process such as adsorption, rupture and spreading can be studied at each stage using AFM. Besides dynamic processes, AFM can also be used to image lipid-protein mixed phospholipid bilayer for further studies. It is also possible to separate different kinds of phospholipids mixture into distinct domains since there is a difference in height or structure between the species.  Due to its excellent resolution, the Park AFM is useful for fundamental studies of lipid membranes such as dynamics, structure, and biomolecular interactions. Figure 3. 3D rendering of AFM images of lipid vesicles and bilayer in liquid with the XE-100 (2 μm, 0.8 μm scan size).

19 Dec 2013

Human Astrocytoma Cells

Useful to Study Morphology and Physical Properties of Cells Derived from Human Brain Tumor       Figure 1. AFM Images of human astrocytoma cells. (a) Topography image (50 μm scan size), (b) topography image (40 μm scan size) and (c) error image (55 μm scan size).   Human astrocytes, one type of glial cells, are major components of the brain tissue, which supports neurons and blood vessels in the brain. Astrocytoma cells are derived from brain tumor whose origin is in the astroglial lineage.     Figure 2. AFM images of human astrocytoma cells. (a) Topography image (70 μm scan size), (b) FMM amplitude image (70 μm scan size), and (c) living cell in PBS (33 μm scan size).   Figure 1 and 2 show AFM images of human astrocytoma cells, U373 MG in PBS. The cells were cultured in DMEM supplemented with 10% FBS in 35 φ T/C dish. Before imaging, the cells were washed with PBS twice and fixed with 1% glutaraldehyde and 1% paraformaldehyde in PBS. Then, the cells were washed with PBS once more and the dish was filled with 3mL PBS.    The images were obtained using contact mode AFM in liquid. A gold-coated silicon nitride cantilever was used to image the sample. Its spring constant is approximately 0.06 N/m.    Remarkably, the center image in Figure 2 shows the amplitude image of FMM (Force Modulation Mode). Fine details of cell surface were displayed in the figure. The image in Figure 2 (c) is taken from living astrocytoma cell in PBS.    It is also possible to investigate physical properties of cells using FMM or a F-d curve. Functionalizing the AFM tip with antibodies of the specific receptor existing on the cell surface will allow the user to obtain antibody recognition images of the cells.    The Park AFM is expected to become a useful tool for studying morphology and physical properties of cells.  

18 Dec 2013

AFM and Confocal Fluorescence Microscopy can be Complementary Tools

Since its invention in 1986, atomic force microscopy (AFM) has been used to visualize the surface topographies of various samples by using the tip-sample interaction force. Due to its ability to operate in liquid environments, the AFM has had a significant impact in biological science. Figure 1 shows an AFM image of astrocytes, which are the major component cells of brain tissue. Astrocytes are fixed with 2% formaldehyde and 2% glutaraldehyde in PBS and imaged in phosphate buffered saline. Figure 1. AFM image of astrocytes taken with XE-120 (75 μm scan size). Astrocytes are fixed with 2% formaldehyde and 2% glutaraldehyde in PBS and imaged in PBS.   AFM is a powerful tool for imaging the surfaces of cellular membranes, but biomedical research also requires observation of molecules inside the cells. In this aspect, confocal fluorescence microscopy can complement AFM imaging. Confocal fluorescence microscopy has become an essential tool in many areas of biological and medical research. Figure 2 shows confocal fluorescence microscopy image of astrocytes which is visualized with FITC-conjugated GFAP-specific antibody. Figure 2. Confocal fluorescence microscopy image of astrocytes. GFAP is visualized with FITC-conjugated antibody (green).   Confocal fluorescence microscopy has various applications in biomedical research; for instance, FRET (fluorescence resonance energy transfer), FRAP (fluorescence recovery after photo-bleaching), FISH (fluorescence in situ hybridization), etc. Confocal fluorescence microscopy can be also extended to more advanced optical tools such as FLIM (fluorescence lifetime imaging microscopy), FCS (fluorescence correlation spectroscopy), TPE (Two-photon excitation) microscopy, SHG (second harmonic generation) microscopy, etc. • AFM is a powerful tool for imaging the surface of cellular membranes. • Confocal fluorescence microscopy obtains optical sectioning images inside the cell.