Atomic Force Microscopy in Cell Biology Episode 1 Part 10 potx

184 Braga and Ricci Fig. 2. Atomic force pictures of Helicobacter pylori and E. coli. The bar at the bottom right of each picture corresponds to 500 nanometers. (A) Common morphology of H. pylori without exposure to antibiotic. (B) Example of the different perspectives obtained by means of computer processing (C–E) Different alterations induced in H. pylori by Imaging of Bacteria Treated With Antibiotics 185 5. Care must be taken when choosing the adhesive used for fixing the glass slide to the sample holder. Avoid using thick double-sided adhesive tape, as this can expand for a long time after pressure and thus cause instability in the vertical position of the tip. The specially produced sticky tabs made by different manu- facturers are fine. 6. To obtain the best results, it is necessary to be thoroughly familiar with the char- acteristics of different cantilevers and tips, how these can be used and how they suit to the different kinds of samples investigated. For high-resolution work, tip sharpness is essential: tip properties can vary significantly within the same batch of cantilevers. Fine tuning of the feedback loop and set point, together with the chosen scan speed, is critical for good surface tracking. 7. As mentioned above, AFM offers different imaging modes for investigating the sample. There is the contact mode in which the tip of the probe makes soft physi- cal contact with the sample, which should be used with harder and stiffer materi- als than biological samples as it can easily give rise to undesirable effects as the result of tip-to-sample interactions (Fig. 2L). Tip pressure can indent and deform the sample surface, and lateral forces can stretch the sample, drag away loosely bound fragments, or even detach the whole bacterium from the substrate (3). These drawbacks of the contact AFM mode are overcome by using the intermit- tent-contact mode, also called tapping mode. In this case, the AFM feedback loop constantly dampens the high-frequency oscillations of the vibrating cantile- ver due to the tip coming into contact with the surface for a very short time (16). For this reason, indentation effects are less invasive, lateral forces are greatly reduced, and a high lateral resolution can be maintained. In the third noncontact mode, small amplitude and high-frequency oscillations induced on the cantilever allow the feedback control loop to maintain the tip-to-sample distance within the range of attractive Van der Waals forces. Tip-to-sample interactions are greatly reduced at the expense of lateral resolution and the scanning speed (2). For biological specimens the noncontact and intermittent contact are the most suitable, although the contact mode may be used for high-resolution work on very small areas. 8. To make accurate dimensional measurements, the calibration of the AFMs piezoelectric scanner has to be periodically checked. The procedures are usually described in the instrument manual. Lateral dimension calibration is relatively straightforward, but special care must be taken when calibrating height. We used a VLSI standard calibration grid (NIST traceable) with a 100-nanometer nominal step height and an in-house developed statistical analysis procedure for calibration. Fig. 2. (continued) exposure to sub-MICs and supra-MICs of rokitamycin. (F) Example of artifacts. High-frequency oscillations and lack of bacterium surface detail can be caused by feedback instabilities induced by an electrostatically charged sample. (G) Common morphology of E. coli without exposure to antibiotic. (H–K) Different alter- ations induced in E. coli by exposure to sub-MICs and supra-MICs of cefodizime. (L) Example of artefact produced by excessive tip-to-sample interactions in contact mode. 186 Braga and Ricci Fig. 3. Atomic force pictures of Bacillus cereus and Streptococcus pyogenes. The bar at the bottom right of each picture corresponds to 1 µm. (A) Example of untreated common rod-shaped morphology with flagella of B. cereus. (B) Morphostructural Imaging of Bacteria Treated With Antibiotics 187 9. The images may sometimes be blurred as a result of poor washing procedures, an electrostatic charge on the specimen, improper feedback parameter settings, debris on the tip, or an eroded tip (Fig. 2F). 10. The images of spherical bacteria, such as S. aureus, will suffer from little lateral resolution along the perimeter because of the perpendicular direction of analysis. In general, the shape of the tip and its lateral walls will limit the detection of steep elevated features (Fig. 4). 11. After image acquisition, the built-in software allows the rendering of the picture to be greatly improved by means of shadowing, rotation, different illumination, and different points of view (Fig. 2B). Acknowledgments We would like to thank M. Dal Sasso for preparing the bacterial samples. This study was partially supported by a grant from MIUR (60%). References 1. Heckl, W. M. (1995) Scanning the thread of life, in The human genome (Fisher, E. P. and Klose, S., eds.), R. Piper GmbH & Co. KG, Munchen, pp. 99–146. 2. Braga, P. C. and Ricci, D. (1998) Atomic force microscopy: Application to inves- tigation of Escherichia coli morphology before and after exposure to cefodizime. Antimicrob. Agents Chemother. 42,18–22. 3. Strausser, Y. E. and Heaton, M. G., (1994) Scanning probe microscopy technol- ogy and recent innovations. Am. Laboratory, May, 1–7. 4. Binning, G., Quate, C. F., and Gerber, C. (1986) Atomic force microscope. Whys. Rev. Lett. 12, 930–933. Fig. 3. (continued) alterations of B. cereus after 2-h incubation with daptomycin at 8× MIC. (C) Example of alterations induced in B. cereus after 4-h incubation with daptomycin 8 × MIC. (D) Lateral view of altered structure of B. cereus after 8-h incu- bation with daptomycin 4× MIC. (E) Example of common morphology of S. pyogenes phenotype M without exposure to antibiotic. (F–H) After 6-h of incubation with rokitamycin (2 µg/mL, abnormally enlarged cells, loss of typical chain structure, for- mation of clusters, flattening of the cells, and ghost formation were present. Fig. 4. Example of the AFM rendering of a spherical bacterium. 188 Braga and Ricci 5. Binnig, G. and Rohrer, H. (1982) Scanning tunneling microscopy. Helv. Phys. Acta. 55, 726–735 . 6. McDonnel, L. and Phelan, M. (1998) The scanned cantilever AFM: A versatile tool for industrial application. Microscopy Anal. 52, 25–27. 7. Ratneshwar, L. and Scott, A. J. (1994) Biological applications of atomic force microscopy. Am. J. Physiol. 266, C1–C21. 8. Campbell, P. A., Gordon, R., and Walmsley, D. G. (1998) Active surface modifi- cation by scanning tunneling microscopy. Microscopy Anal. 56, 25–27. 9. Lorian, V. (1986) Effect of low antibiotic concentrations on bacteria: effects on ultrastructure, their virulence and susceptibility to immunodefenses, in Antibiot- ics in Laboratory Medicine (Lorian, V., ed.), The Williams & Wilkins Co., Balti- more, pp. 596–668. 10. Lorian, V., Atkinson, B., Walushacka, A., and Kim, Y. (1982) Ultrastructure, in vitro and in vivo, of staphylococci exposed to antibiotics. Curr. Microbiol. 7, 301–304. 11. Braga, P. C. and Ricci, D. (2000 ) Detection of rokitamycin-induced morphostructural alterations in Helicobacter pylori by atomic force microscopy. Chemotherapy 46, 15-22. 12. Braga, P. C. and Ricci, D. (2002) Differences in the susceptibility of Streptococ- cus pyogenes to rokitamycin and erythromycin revealed by morphostructural atomic force microscopy investigation. J. Antimicrob. Chemother. 50, 457–460. 13. Braga, P. C., Ricci, D., Dal Sasso, M. and Thorne, G. (2002) Bacillus cereus morphostructural damage by daptomycin: atomic force microscopy investigation. Chemotherapy 14, 336-341. 14. Nagao, E. and Dvorak, J. A. (1999) Developing the atomic force microscope for studies of living cells. Intern. Lab. January, 21–23. 15. Schaus, S. S., and Henderson, E. R. (1997) Cell viability and probe-cell mem- brane interactions of XR1 glial cells imaged by atomic force microscopy. Biophys. J. 73, 1205–1214 16. Howland, R. and Benatar, L. (1997) A Practical Guide to Scanning Probe Microscopy. Park Scientific Instrument, Sunnyvale, CA, pp. 1–73. Nuclear Structure by AFM 191 191 14 Visualizing Nuclear Structure In Situ by Atomic Force Microscopy Luis Felipe Jiménez-García and María de Lourdes Segura-Valdez 1. Introduction The cell nucleus is an organelle where molecules involved in gene expres- sion are highly compartmentalized (1) in very dynamic (2) territories. This current notion of functional organization has been possible because of many studies of this organelle that included its cell and molecular organization, and where microscopy played an important role. Light and electron microscopy in conjunction with molecular approaches, such as the construction of both anti- bodies and nucleic acid probes, as well as molecular construction of genes with green fluorescent protein and fluorescence recovery after photobleaching tech- nology, in addition to high-resolution in situ hybridization and immunocy- tochemistry now offer a more complete knowledge. Structurally, the mammalian cell nucleus is organized as an organelle sur- rounded by a double layer of phospholipids called the nuclear envelope, which is interrupted by the nuclear pores. It is covered in the interior by the nuclear lamina. Within the cell nucleus, a nuclear matrix is present as a nuclear skel- eton made of several different proteins. DNA is organized as euchromatin and heterochromatin. The ribonucleoproteins are distinguished by their partici- pation in pre-mRNA or pre-rRNA metabolism. In the first case, ribonucleopro- teins are visualized as nuclear speckles by light microscopy, which correspond to interchromatin granule clusters and perichromatin fibers observed by electron microscopy. In addition, perichromatin granules are also present. All these structures are related to transcription and splicing of pre-mRNA and transport or storage of intranuclear mRNA. The nucleolus, however, is involved in pre-rRNA transcription and processing as well as ribosome assembly. There are other nuclear structures, such as the Cajal bodies, which contain factors From: Methods in Molecular Biology, vol. 242: Atomic Force Microscopy: Biomedical Methods and Applications Edited by: P. C. Braga and D. Ricci © Humana Press Inc., Totowa, NJ 192 Jiménez-García and Segura-Valdez involved in both pre-mRNA and pre-rRNA metabolism (1–8). In plants, there are two major classes of nuclei: (1) the chromocentric and (2) the reticulated types, depending on the arrangement of heterochromatin fibers (9–12). In the first type, DNA is organized as discrete clumps within the nucleoplasm. In the second one, DNA forms a reticulated strand. Lacandonia schismatica is a rare plant whose most important feature is the inverted position of the sexual organs, with the androecium in the center, sur- rounded by the gynoecium (13,14). In studying the biology of this species, we have analyzed the cell biology, including the interphase cell nucleus, which we have published previously (15–20). The cell nucleus of L. schismatica is reticulated. To explore the possibility of studying the interphase cell nucleus with high resolution, while at the same time of working in solution, we have been study- ing the nuclear structure by using an approach whereby a sample is prepared for transmission electron microscopy (TEM) and the surface of the unstained semithin sections is explored with an atomic force microscopy working in con- tact mode (17,19) to visualize the interior of the cell. It is a common observa- tion when sectioning with an ultramicrotome that trimming of the plastic blocks to get thin sections many times reveals the profile of the embedded cells over the surface. This observation suggested that even very flat surfaces have a tex- ture corresponding to cell organelles, and indeed this is the case. Therefore, because the microscope is an instrument to analyze surfaces, the surface of each section can be visualized as a representative sample of a portion of the cell interior. Similar approaches also have been used previously (21,22) to study cell structure in situ. As a first step, we have been validating this approach by observing and generating images of already known material. For these pur- poses we have been visualizing the nuclear structure of L. schismatica by using the protocols described in Subheading 3. (17,19). 2. Materials 2.1. Sample Preparation 1. 70% Glutaraldehyde (Polysciences). Toxic. Wear gloves, goggles and work in a chemical fume hood. Store at 4°C. 2. 2% Osmium tetroxide. Toxic. Wear gloves, goggles and work in a chemical fume hood. Store at 4°C. Protect from light. 3. Phosphate-buffered saline (PBS) 0.01 M, pH 7.4. Store at 4°C. 4. Deionized water. 5. 100% Ethanol, electron microscopy grade. 6. 100% Propylene oxide, electron microscopy grade. 7. Epoxy resin (glycidether 100, Merck). Plastic resin. 8. DDSA (dodecenylsuccinic anhydride) hardener (Merck). Nuclear Structure by AFM 193 9. NMA (methylnorbornene-2,3-dicarboxylic anhydride) plasticizer (Merck) 10. DMP (2, 4, 6-Tris[dimethyl-aminomethyl]phenol) accelerator (Merck) 11. Toluidine blue. 12. Clean glass slides. Keep within a chamber to avoid dust. 13. Ultramicrotome (Reicher-Jung). 14. Glass knives. 15. Diamond knife. 2.2. Microscope 1. BioScope (Digital Instruments, Santa Barbara, CA). 2. NanoScope IIIa (Digital Instruments, Santa Barbara, CA) control system and the software. 3. Inverted Diaphot 200 microscope (Nikon, NY). 4. Silicone nitride tips, 20–60 nm radius of curvature (model NP). 5. 100-µm Atomic force microscope scanner. 6. Proscan software (version 3.1, Park Scientific, 1997) 3. Methods 3.1. Sample Preparation Samples are prepared as for standard TEM or histology. For TEM, we have used several protocols, including different types of fixation with alde- hydes such as glutaraldehyde or paraformaldehyde at different concentra- tions, or with embedding media such as glycol methacrylate, London resin white, Lowicryl ® , and epoxy resin. Here, we describe the regular protocol for TEM (15,23–25) because it produced thus far the best results as far as morphology is concerned. 1. Samples are fixed with freshly made 6% glutaraldehyde from 70% vials, buff- ered with 0.01 M PBS at pH 7.4 for 2–6 h at room temperature. PBS is prepared as follows (23): dissolve 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na 2 HPO 4 , and 0.24 g of KH 2 PO 4 in 800 mL of distilled water. Adjust pH to 7.4 with HCl. Add water to 1 L. Sterilize by autoclaving. 2. After fixation, samples are washed with PBS for three times 10 min each. 3. Postfixation is performed with 1% osmium tetroxide in bidistilled water for 1–2 h. Prepare from a stock solution of 2% made from crystals in bidistilled water. 4. Dehydration is conducted with a series of graded concentration of ethanol, 10 min each of 30, 50, 70, 80, 90, and 96, and three changes of 100% ethanol. Pro- pylene oxide is then used for three times for 10 min each. 5. For preembedding, samples are placed in a mixture of 1:1 of propylene oxide and epoxy resin during 16 h at room temperature. The epoxy resin is made as a stock solution. A recommended proportion of the components for working with plants can be taken from the references (23) and will contain 9.44 g of epoxy resin, 5 g of DDSA, 10 g of NMA, and 0.2 mL of DMP. 194 Jiménez-García and Segura-Valdez Mix the components thoroughly with a glass rod avoiding making bubbles. Store at 4°C in a small jar avoiding the penetration of air by sealing with parafilm. Prepare in the chemical fume hood. Avoid contact with the skin and to inhale. See Note 1. 6. Embedding is conducted with epoxy resin for 16 h at 60°C. 7. Semithin sections of about 150–250 nm width are obtained with an ultramicro- tome working with glass or diamond knives. See Note 2. 8. Two sets of sections are placed onto an ethanol-cleaned glass slide. The sections are transferred by placing a platinum loop onto a drop of water. The sections are placed onto a glass slide. Sections are fixed to the glass by gently heating the slide. One set of sections is stained with toluidine blue and rinsed thoroughly with bidistilled water. Toluidine blue stain is prepared as a stock solution by dissolving 1% toluidine blue in 1% sodium borate, to stain the sections but not the plastic. For staining, once the slide reaches room temperature after fixing to the glass, cover the section with one or two drops of the stain and place it on a hot plate until stain starts to dry and obtain a metallic green color at the borders. Rinse the excess stain off the slide with bidistilled water. Sections are stored in a chamber to avoid dust. 3.2. Atomic Force Microscopy An atomic force microscope model Bioscope (Digital Instruments) is used for observations. The microscope is mounted on an inverted light microscope Diaphot 200 (Nikon). Observations are made in contact mode. The scan size are from 100 to 5 µm at a scan rate varying from 1.969 to 1.285 Hz. Images were generated with the NanoScope IIIa control system. Alternatively, an atomic force microscope from Park Scientific has been used (17,19). In that case, the microscope is equipped with a scanner of 100 µm and a 100-Å radius silicone nitride tip, mounted on a cantilever of 0.6 µm. A scan rate of 2–3 Hz, a force of 10 nN, and a gain of 0.5 arbitrary units have been used. Images are then generated with Proscan. (See Note 3.) For the BioScope model, it is sug- gested to follow the next steps: 1. Fix the slide onto the stage by using the vacuum system provided. 2. Localize the sample with the bright field microscope using a low-magnification objective. 3. Approach the head of the microscope manually. 4. Visualize the cantilever and the tip with the light microscope and align the tip (see Note 4). 5. Adjust parameters as scan size to 100 µm, scan rate to about 1.5 Hz, and data scale to about 400 nm. 6. Engage the microscope and scan the sample (see Note 5). 7. Generate the images using the Nanoscope IIIa software provided (Figs. 1 and 2; (see Notes 6 and 7). Nuclear Structure by AFM 195 Fig. 1. Light (A), electron (B), and atomic force (C) microscopy of cell nuclei from the tegument cells from an ovary of a flower of the plant Lacandonia schismatica. The three types of microscopes generate a similar image of the cell nuclei. However, the resolution in every case varies according to the instrument. N, nucleus; Nu, nucleolus; arrows, reticulated compact chromatin; W, cell walls; V, vacuoles; C, cytoplasm. [...]... penicillin, 10 0 mg/mL streptomycin (Gibco BRL, cat no 15 140 -12 2), and 2% heat-inactivated fetal calf serum (Gibco BRL, cat no 10 270-098) 2 For fast-growing cells or cells with a high metabolic rate (7,8): DMEM with 25 mM HEPES (Gibco BRL, cat no 32430-027), 2 mM L-glutamine, 10 0 U/mL penicillin, 10 0 mg/mL streptomycin, and 10 % heat-inactivated fetal calf serum 3 Trypan blue stain 0.4% (Gibco BRL, cat no 15 250-0 61) ... Segura-Valdez AFM Imaging of Living Cells 2 01 15 Imaging Surface and Submembranous Structures in Living Cells With the Atomic Force Microscope Notes and Tricks Filip Braet and Eddie Wisse 1 Introduction In 19 86, Binnig et al (1) revolutionized microscopy through the invention of the atomic force microscope (AFM) Subsequently, commercial instruments of this new imaging technique began to appear in the 5 yr after... 3.5.2 Loading Forces Using different loading forces on the same sample will result in images with different information (Fig 3) As outlined in Fig 3, applying low imaging forces will result in a more accurate surface visualization (Fig 3B), whereas increasing the force will result in obscuring of fine membranous details (Fig 3C) However, submembranous structures become apparent when higher loading forces... growth medium in the 35mm Petri dish is 1. 7 mL 2 Allow to stand 5 min at 15 to 30°C 3 Observe under the inverted microscope Nonviable cells stain blue and viable cells exclude the stain (11 ) 3.5 Imaging and Image Interpretation During Scanning 3.5 .1 Z-Height Limitations AFM imaging can result in artifactual images because of the limited range of the z piezo (Fig 2) For example, in Fig 2, the cells respond... et al (20 01) Further ultrastructural characterization of the intranuclear ring-shaped bodies of the plant Lacandonia schismatica J Struct Biol 13 6, 1 6 21 Mariani, T., Musio, A., Frediani, C., Sbrana, I., and Ascoli, C (19 94) An atomic force microscope for cytological and histological investigations J Microsc 17 6, 12 1 13 1 22 Morris, V J., Kirby, A R., and Gunning, A P (19 99) Atomic Force Microscopy. .. gene activity Int Rev Cytol 94, 21 56 12 Moreno Díaz de la Espina, S., Mínguez, A., Vázquez-Nin, G H., Echeverría, O M (19 92) Fine structural organization of a non-reticulated plant cell nucleus An ultracytochemical and immunocytochemical study Chromosoma 10 1, 311 –3 21 13 Martínez, E and Ramos, C H (19 89) Lacandoniaceae (Triuridales): Una nueva familia de México Ann Miss Bot Gard 76, 12 8 13 5 14 Márquez-Guzmán,... (see Note 12 ) according to the guileless of the manufacturer until the viability of the cell cultures started to decrease (see Note 13 ) 7 Typical loading forces to be used for imaging living cells should vary between 2 and 4 nN (see Note 14 ) 8 Optimize the scan rate during scanning (see Note 15 ) 9 Optimize feedback parameters during scanning (see Note 16 ) 3.3 Noncontact Imaging 1 Follow steps 1 to 6 as... homemade fluid cell in combination with a heating stage This design allows positioning of the cantilever in the optical axis of the inverted microscope, movement of the sample via the inverted microscope independently of the AFM, and minimizes cantilever drift by controlling temperatureinduced variations Time lapse images of living cells in contact (7–9) or noncontact mode (9 ,10 ) were obtained over a... of cells wanted to visualized 2 Materials 2 .1 Surface Coating for Nonadherent Cells 1 Collagen-S solution (Boehringer Mannheim, cat no 10 98292; see Note 1) 2 Sterile water 3 RPMI -16 40 with 25 mM HEPES (Gibco BRL, cat no 52400-025) 2.2 Growth Medium and Reagents 1 For slow-growing or nondividing cells (7,9): RPMI -16 40 with 25 mM HEPES (see Note 2), 2 mM L-glutamine (Gibco BRL, cat no 25030-024), 10 0... bodies: The first 10 0 years Annu Rev Cell Dev Biol 16 , 273–300 9 Lafontaine, J G (19 74) Ultrastructural organization of plant cell nuclei, in The Cell Nucleus (Busch, H., ed.), Academic Press, New York, pp 24–59 10 Jordan, E G., Timmis, J N., and Trewavas, A J (19 80) The plant nucleus, in Biochemistry of Plants (Tolbert, N E., ed.), Academic Press, New York, pp 489–588 11 Nagl, W (19 85) Chromatin organization . daptomycin: atomic force microscopy investigation. Chemotherapy 14 , 336-3 41. 14 . Nagao, E. and Dvorak, J. A. (19 99) Developing the atomic force microscope for studies of living cells. Intern January, 21 23. 15 . Schaus, S. S., and Henderson, E. R. (19 97) Cell viability and probe -cell mem- brane interactions of XR1 glial cells imaged by atomic force microscopy. Biophys. J. 73, 12 05 12 14 16 Ascoli, C. (19 94) An atomic force microscope for cytological and histological investigations. J. Microsc. 17 6, 12 1 13 1. 22. Morris, V. J., Kirby, A. R., and Gunning, A. P. (19 99) Atomic Force Microscopy for