Scanning Electron Microscopy

Elizabeth R. Fischer1, Bryan T. Hansen1, Vinod Nair1, Forrest H. Hoyt1, David W. Dorward1

1 Electron Microscopy Unit, Research Technologies Branch, Rocky Mountain Laboratories, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 2B.2
DOI:  10.1002/9780471729259.mc02b02s25
Online Posting Date:  May, 2012
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Abstract

Scanning electron microscopy (SEM) remains distinct in its ability to allow topographical visualization of structures. Key elements to consider for successful examination of biological specimens include appropriate preparative and imaging techniques. Chemical processing induces structural artifacts during specimen preparation, and several factors need to be considered when selecting fixation protocols to reduce these effects while retaining structures of interest. Particular care for proper dehydration of specimens is essential to minimize shrinkage and is necessary for placement under the high‐vacuum environment required for routine operation of standard SEMs. Choice of substrate for mounting and coating specimens can reduce artifacts known as charging, and a basic understanding of microscope settings can optimize parameters to achieve desired results. This unit describes fundamental techniques and tips for routine specimen preparation for a variety of biological specimens, preservation of labile or fragile structures, immune‐labeling strategies, and microscope imaging parameters for optimal examination by SEM. Curr. Protoc. Microbiol. 25:2B.2.1‐2B.2.47. © 2012 by John Wiley & Sons, Inc.

Keywords: scanning electron microscopy; immune‐labeling; EM specimen preparation; critical‐point drying; sputter coating; specimen fracturing; microwave‐processing; cryo‐SEM quantum dots

     
 
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Table of Contents

  • Introduction
  • Safety Considerations
  • Basic Protocol 1: Chemical Preparative Techniques for Preservation of Biological Specimens for Examination by SEM
  • Alternate Protocol 1: Practical Considerations for the Preparation of Soft Tiissues
  • Alternate Protocol 2: Removal of Debris from the Exoskeleton of Invertebrates
  • Alternate Protocol 3: Fixation of Colonies Grown on Agar Plates
  • Alternate Protocol 4: Stabilization of Polysaccharide Structures with Alcian Blue and Lysine
  • Alternate Protocol 5: Preparation of Non‐Adherent Particulates in Solution for SEM
  • Support Protocol 1: Application of Thin Layer of Adhesive on Substrate to Improve Adherence
  • Support Protocol 2: Poly‐L‐Lysine Coating Specimen Substrates for Improved Adherence
  • Support Protocol 3: Microwave Processing of Biological Specimens for Examination by Conventional SEM
  • Basic Protocol 2: Critical‐Point Drying Specimens
  • Alternate Protocol 6: Chemical Alternative to Critical‐Point Drying
  • Basic Protocol 3: Sputter Coating
  • Alternate Protocol 7: Improved Bulk Conductivity Through “OTOTO”
  • Basic Protocol 4: Immune Labeling Strategies
  • Alternate Protocol 8: Immune Labeling Internal Antigens with Small Gold Probes
  • Alternate Protocol 9: Quantum Dot or Fluoronanogold Preparation for Correlative Techniques
  • Basic Protocol 5: Exposure of Internal Structures by Mechanical Fracturing
  • Basic Protocol 6: Anaglyph Production from Stereo Pairs to Produce 3‐D Images
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Chemical Preparative Techniques for Preservation of Biological Specimens for Examination by SEM

  Materials
  • Eukaryotic cells grown in suspension or on solid substrate: Thermanox coverslips, silicon chips, aclar film (aclar typically comes in sheets, which can be cut or punched to desired size), or transwell membrane filters (available from Ted Pella, Electron Microscopy Sciences, Falcon, and other sources)
  • Physiologically appropriate buffer [e.g., Hank's buffered saline solution (HBSS) or phosphate‐buffered saline (PBS)]
  • Primary fixative: typically 2.5% GA (see recipe) and/or 4% paraformaldehyde (PFA; see recipe) in 0.1 M sodium cacodylate (CAC) or phosphate buffer (PB; see recipe), pH 6.8 to 7.4
  • Rinsing buffer (e.g., 0.1 M CAC or PB; see reciperecipes)
  • Secondary fixative: typically 1% OsO 4 in dH 2O or reduced osmium (see recipe) with potassium ferrocyanide [0.5% OsO 4/0.8% K 4Fe(CN) 6] in dH 2O or 0.1 M CAC
  • Distilled water (dH 2O)
  • Dehydrating agent, typically ethanol or acetone
  • Fine‐tipped forceps
  • Scissors
  • Mounting substrate (e.g., silicon chip, coverslip, or membrane filter)
  • Container for processing (e.g., 24‐cell well plate, microcentrifuge tube, PTFE basket)
  • Water/ethanol‐proof marking pen
  • Parafilm or tape, optional
NOTE: All procedures are carried out at room temperature unless noted otherwise.

Alternate Protocol 1: Practical Considerations for the Preparation of Soft Tiissues

  • Excised tissues
  • 1% Tannic acid (TA) in dH 2O
  • 1% to 2% Uranyl acetate (UA) in dH 2O
  • Dissecting tools (e.g., scalpel, micro‐scissors, or razor blade)
  • Dish for dissection

Alternate Protocol 2: Removal of Debris from the Exoskeleton of Invertebrates

  Materials
  • Tick or mite
  • 70% ethanol
  • Acetone
  • Xylene
  • Sonicator
  • 1.5‐ml glass vials with tightly sealed caps or 1.5‐ml microcentrifuge tubes or appropriate container
  • Fine‐point forceps or tweezers

Alternate Protocol 3: Fixation of Colonies Grown on Agar Plates

  • Colonies grown on agar plates
  • Scalpel or razor blade
  • 25‐ to 90‐mm glass or plastic petri dish

Alternate Protocol 4: Stabilization of Polysaccharide Structures with Alcian Blue and Lysine

  • Colonies grown on agar plates or in suspension
  • Alcian blue/lysine fixative (see recipe)
  • 0.1 M sodium cacodylate (CAC), pH 7.2 (can be purchased as a powder or pre‐made from a variety of microscopy sources, including Ted Pella, Electron Microscopy Sciences, Poly Sciences, etc.)

Alternate Protocol 5: Preparation of Non‐Adherent Particulates in Solution for SEM

  Materials
  • Particulates/macromolecules in water or volatile salt solution at desired concentration
  • Appropriate mounting substrate

Support Protocol 1: Application of Thin Layer of Adhesive on Substrate to Improve Adherence

  • Acetone
  • Carbon conductive mounting tabs (available from microscopy suppliers, including Ted Pella, Electron Microscopy Sciences, and Ladd Research)
  • Microcentrifuge tubes, acetone‐resistant such as polypropylene (PP) or high‐density polyethylene (HDPE)
  • Tweezers
  • Petri dish lid or similar cover

Support Protocol 2: Poly‐L‐Lysine Coating Specimen Substrates for Improved Adherence

  • 0.1 % poly‐L‐lysine in solution
  • Small glass beaker
  • Coverslip holder for drying

Support Protocol 3: Microwave Processing of Biological Specimens for Examination by Conventional SEM

  • Specimens on SEM appropriate substrate (e.g., silicon chips, coverslips, etc.) in fixative
  • Appropriate buffer
  • Suitable container (e.g., 24 cell well tissue culture plate)
  • Laboratory wattage‐controllable microwave oven with load cooler, vented into a fume hood
NOTE: Do not fill liquid solutions over 1 cm in height to prevent inadequate microwave oven penetration (Sanders, see Internet Resources).

Basic Protocol 2: Critical‐Point Drying Specimens

  Materials
  • Silicon chip, pretrimmed coverslip, or transwell membrane (see protocol 1)
  • 100% dry ethanol
  • Critical‐point dryer (CPD)
  • Specimen container
  • Solvent‐resistant container: e.g., polyethylene specimen cup (PE)
  • Fine‐tipped forceps
  • Scalpel
  • CO 2 siphon tank

Alternate Protocol 6: Chemical Alternative to Critical‐Point Drying

  • Specimen adhered to chip or coverslip
  • Hexamethyldisilazane (HMDS)
  • Laboratory microwave oven with controllable wattage, optional
  • Filter paper

Basic Protocol 3: Sputter Coating

  Additional
  • Appropriately fixed and dried specimen (see Basic Protocols protocol 11 and protocol 102)
  • Microscope‐specific stage mount (e.g., aluminum stubs)
  • SEM specimen storage box
  • SEM stub tweezers
  • Conductive double‐sided adhesive tape and/or conductive paint
  • Fine‐tip tweezers
  • Sputter coater
  • Desiccator

Alternate Protocol 7: Improved Bulk Conductivity Through “OTOTO”

  Materials
  • Cells
  • Fixative: 2% to 4% EM‐grade paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB), pH 6.8 to 7.4
  • Rinsing buffer (e.g., 0.1 M CAC or PB; see reciperecipes)
  • Blocking buffer [e.g., serum‐free bovine serum albumin (BSA) in 0.1 M Tris, PB, or PBS buffer (see reciperecipes)]
  • Primary antibodies or anti‐serum (e.g., rabbit or mouse anti‐sera)
  • Secondary antibodies (e.g., goat anti‐rabbit or mouse) conjugated to electron dense probe (e.g., 10 to 40 nm colloidal gold)
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 10010‐023)
  • Silicon chips
  • 24‐well plates
  • Fine‐tipped tweezers, optional

Basic Protocol 4: Immune Labeling Strategies

  • Hank's buffered saline solution (HBSS)
  • 2% to 4% paraformaldehyde (PFA)
  • 0% to 2.5% glutaraldehyde (GA; see recipe)
  • Periodate‐lysine‐PFA (PLP) fixative (see recipe)
  • Phosphate‐buffered saline [PBS; 50 mM PB (see recipe)/150 mM NaCl], pH 7.4
  • Glycine
  • Saponin (prepare fresh)
  • 1% bovine serum albumin (BSA)/PBS
  • Phosphate buffer (PB; see recipe)
  • Small diameter (<1.5 nm) gold conjugates: Nanoprobes Nanogold or Aurion Ultrasmall gold
  • Deionized water
  • Nanoprobes or Aurion's R‐Gent silver enhancement kit

Alternate Protocol 8: Immune Labeling Internal Antigens with Small Gold Probes

  Materials
  • Samples adhered to silicon chip or coverslip
  • Appropriate buffer (e.g., PBS or HBSS)
  • 4% paraformaldehyde (PFA)/0.1% GA in PBS
  • Blocking buffer: e.g., 2% (w/v) globulin‐free BSA (Sigma, cat. no. A7638) in PBS
  • Primary antibody (e.g., rabbit or mouse anti‐sera)
  • Secondary antibody conjugate (e.g. goat anti‐rabbit 655‐nm quantum dots; Invitrogen or eBioscience)

Alternate Protocol 9: Quantum Dot or Fluoronanogold Preparation for Correlative Techniques

  Materials
  • Fixed and dried specimen (e.g., see Basic Protocols protocol 11 and protocol 102)
  • Mild adhesive tape (e.g., Scotch tape) or syringe or micro‐dissection scissors
  • Eyelash brush
  • Additional reagents and equipment for mounting the specimen on SEM stub ( protocol 12)

Basic Protocol 5: Exposure of Internal Structures by Mechanical Fracturing

  Materials
  • Stereo pair
  • Computer with imaging program, e.g., Adobe Photoshop
  • Red/Blue 3‐D glasses
NOTE: PC control key short cuts are utilized in the example below; Mac users should use the command key.
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Figures

  •   FigureFigure 2.B0.1 Thermanox or aclar materials can easily be trimmed or punched to desired dimensions using scissors or punch tool for adaptation to size requirements for subsequent processing steps.
  •   FigureFigure 2.B0.2 (A) Removal of prescored silicon wafer followed by application of specimen in suspension (B) and, (C) proper dispersal of fluids.
  •   FigureFigure 2.B0.3 Low‐magnification SEM image of Ornithodoros hermsi tick (A), and higher magnification images of uncleaned (B, D, E) compared with cleaned tick exoskeleton (C, F). Scale bars as indicated.
  •   FigureFigure 2.B0.4 SEM of Hms+ and Hms‐ Yersinia pestis grown as bacterial lawns on agar plates. The Hms+ colonies showed retention of an extracellular substance (A), while the mutant treated with the same alcian blue‐lysine fixative mixture did not (B). Scale bars = 0.5 µm.
  •   FigureFigure 2.B0.5 Agarose bead with dendrocytes adhered to silicon chip precoated with thin layer adhesive. Scale bar = 25 µm.
  •   FigureFigure 2.B0.6 Placement of a chip in specimen vessel used in a Bal‐Tec CPD.
  •   FigureFigure 2.B0.7 Membrane filter insert removed from a 24‐well tissue culture plate (A) is quickly transferred and immersed for trimming (B) and the membrane is removed with fine‐tipped forceps (C) for placement in CPD container.
  •   FigureFigure 2.B0.8 Carefully place the lid on the specimen chamber and make sure it is properly threaded and closed tightly as this chamber reaches high pressure.
  •   FigureFigure 2.B0.9 HeLa cells grown on silicon chips were dehydrated using either a CPD (A) or HMDS (B). Specimens were coated with 80 Å of Ir. Scale bar = 1 µm.
  •   FigureFigure 2.B0.10 Double‐sided adhesive tab placeed on aluminum SEM stub (A) and removal of protective layer with fine‐tipped forceps (B).
  •   FigureFigure 2.B0.11 Transwell membrane filters curl up into a scroll after drying in a CPD (A). After placing on the adhesive, it can simply be rolled across the tape (cell layer rolls inward) (B) and gently apply pressure for improved contact. Silver paint can be CAREFULLY applied to increase contact of membrane to adhesive (C).
  •   FigureFigure 2.B0.12 Conductive paint is carefully applied with a brush to form complete contact with the underlying surface. If the substrate hangs slightly over the stub, extra paint can be applied on the bottom side of the coverslip.
  •   FigureFigure 2.B0.13 Silver paint was carefully applied after sputter‐coating tissues mounted on prepared SEM stubs to improve contact between tissue and conductive surface.
  •   FigureFigure 2.B0.14 Specimen appearance after coating with 75 Å of Ir by visual or SEM examination of Salmonella‐infected polarized HeLa cells grown on transwell membrane filters (A, B), non‐adherent red blood cells infected with malarial parasites settled on a silicon chip (C, D), or macrophages grown on an aclar coverslip (E, F) prior to mounting on double‐sided carbon tape, and stabilized further with Leitsilber silver paint. Scale bars (B, D) = 1 µm and E = 10 µm.
  •   FigureFigure 2.B0.15 Chlamydia‐infected HeLa cells were prepared by OTOTO and left uncoated for examination (A, B), minimally coated with 20 Å of Cr, (C, D) or conventionally processed and coated with 75 Å of Ir (E, F). Scale bars = 0.5 µm.
  •   FigureFigure 2.B0.16 Yersinia pestis labeled with 20 nm gold, imaged at a nominal magnification of 35,000× (A) compared with Staphylococcus epidermidis labeled with 10 nm gold and visualized at a nominal magnification of 60,000× (B). Scale bars = 0.5 µm.
  •   FigureFigure 2.B0.17 Bacteria in ovarian tissue labeled with Nanogold and silver enhanced for 15 min to improve visualization by SEM. Scale bar = 0.5 µm.
  •   FigureFigure 2.B0.18 Q‐dot labeling and detection by SEM. HeLa cells were infected with Chlamydia trachomatis elementary bodies, probed with anti‐chlamydial rabbit serum, and labeled with anti‐rabbit Q‐dots (565‐nm peak emission). Panels AC demonstrate examination of clustered elementary bodies on the surface of infected HeLa cells by fluorescence/light microscopy, TEM, and SEM, respectively. Scale bars = as designated.
  •   FigureFigure 2.B0.19 Scotch tape is gently applied to cell layer grown on Thermanox coverslips.
  •   FigureFigure 2.B0.20 Macrophages infected with Francisella tularensis fractured with adhesive tape to expose internalized bacteria (A), Yersinia pestis attached to internal spines of a flea proventriculus exposed by disruption with syringe tip (B), and malarial parasites invading the apical surface of a mosquito midgut after exposure using a micro dissection scissor (C). Scale bar = 1 µm.
  •   FigureFigure 2.B0.21 Aligned stereo pair.
  •   FigureFigure 2.B0.22 Images converted to RGB mode.
  •   FigureFigure 2.B0.23 Red channel removed from left image.
  •   FigureFigure 2.B0.24 Red image from right image copied on to left image.
  •   FigureFigure 2.B0.25 Selection of the RGB channel shows overlay of the new red channel, creating an anaglyph for 3‐D viewing using red/blue glasses.
  •   FigureFigure 2.B0.26 Chlamydia‐infected HeLa cells high‐pressure frozen and freeze fractured after fixing with PFA only (A) or with 2.5% GA/0.1% MG and OTOTO treatment (B). Scale bar = 5 µm.
  •   FigureFigure 2.B0.27 Polarized epithelial cells grown on membrane filters after drying and coating, revealed damage to the monolayer (A) and at higher magnification damage to the membrane was evident (B). Scale bar = 0.5 µm.
  •   FigureFigure 2.B0.28 Protein globules adhered to an untreated silicon chip were either rotary‐coated (tilted ± 90 degrees, 360 degree rotation) with 40 Å of Ir (A) or shadowed at a 15 degree fixed angle with 20 Å of Pt, followed by rotary coating ± 85 degrees with 20 angstroms of C (B). Scale bar = 0.5 µm.
  •   FigureFigure 2.B0.29 Immune‐labeled bacteria demonstrating excessive background labeling in the out‐of‐focus regions (A), moderate background labeling (B), or minimal levels of background labeling (C). Scale bars = 0.5 µm.
  •   FigureFigure 2.B0.30 Immune‐labeled Staphylococcus epidermidis prepared by conventional EM techniques demonstrates detachment of structure of interest. Scale bar = 0.25 µm.
  •   FigureFigure 2.B0.31 Chlamydia‐infected HeLa cells were immune‐labeled intracellularly for antigens against a bacterial surface protein using Nanogold followed by silver enhancement and viewed by either cryo‐SEM (A) or TEM (B). Scale bar = 0.5 µm.
  •   FigureFigure 2.B0.32 Macrophages grown on aclar coverslips, conventionally prepared and coated with 75 Å of Ir were imaged by SEM at 2 kV (A), 5 kV (B), or 10 kV (C). Scale bar = 1 µm.
  •   FigureFigure 2.B0.33 Amyloid protein fibrils were processed by OTOTO and then left uncoated (A), or coated with 20 Å of Cr and examined at 5 kV (B), 10kV (C), 30 kV (DE). Also examined at 30 kV were fibrils coated with 15 Å of Pt and 20 Å of C (F). Scale bars = 0.05 µm.
  •   FigureFigure 2.B0.34 HIV‐infected T lymphocyte imaged with 5 kV at a working distance of 5 mm (A) or 17 mm (B). Scale bar = 1 µm.
  •   FigureFigure 2.B0.35 Images of a macrophage show astigmatism in the x direction (A), y direction (B), corrected (C), corrected and focused (D). Scale bar = 2 µm.
  •   FigureFigure 2.B0.36 Mouse dendritic cells (A) and Plasmodium‐infected red blood cells (B) imaged at 2 kV display minor signs of charging seen as flattening or streaking, respectively. The macrophage imaged at 2 kV in (C) shows more extreme charging, rendering the image useless. Scale bars = 0.5 µm.
  •   FigureFigure 2.B0.37 HeLa cell co‐infected with VZV and Streptococcus. Scale bar = 1.5 µm.
  •   FigureFigure 2.B0.38 Staphylococcus epidermidis immune‐labeled for biofilm localization shown in both SE (A) and mixed SE and BSE (B) imaging mode to allow visualization of gold particles. Scale bar = 0.5 µm.

Videos

Literature Cited

   Boyd, A. and Franc, F. 1981. Freeze‐drying shrinkage of glutaraldehyde fixed liver. J. Micros. 122:75‐86.
   Boyd, A. and Maconnachie, E. 1979. Freon 113 freeze‐drying for SEM. Scanning 2:164‐166.
   Braet, F., deZanger, R., and Wisse, E. 1997. Drying cells for SEM, AFM and TEM by hexamethyldisilazane: A study on hepatic endothelial cells. J. Micros. 186:84‐87.
   Brown, W.J. and Farquhar, M.G. 1989. Immunoperoxidase methods for the localization of antigens in cultured cells and tissues by electron microscopy. Methods Cell Biol. 31:553‐569.
   Corwin, D. 1979. An improved method for cleaning and preparing ticks for examination with the electron microscope. J. Med. Entomol. 16:352‐353.
   DeLeo, F.R. and Otto, M. 2008. Bacterial pathogenesis: methods and protocols. In Methods in Molecular Biology, vol. 431 (D.W. Dorward, ed.) pp. 173‐187. Humana Press, Totowa, New Jersey.
   Dixon, B.R., Petney, T.N., and Andrews, R.H. 2000. A simplified method of cleaning Ixodid ticks for microscopy. J. Micros. 197:317‐319.
   Fassel, T.A., Mozdiak, P.E., Sanger, J.R., and Edminston, C.E. 1997. Paraformaldehyde effect on ruthenium red and lysine preservation and staining of the staphylococcal glycocalyx. Microscop. Res. Tech. 36:422‐427.
   Giberson, R.T. and Demaree, R.S. Jr. 2002. Microwave Techniques and Protocols. Humana Press, Totowa, New Jersey.
   Goldstein, J.I., Newbury, D.E., Echlin, P., Joy, D.C., Fiori, C., and Lifshin, E. 2002. Scanning Electron Microscopy and X‐Ray Microanalysis. Plenum Press, New York and London.
   Hayat, M.A. 1993. Stains and Chemical Methods. Plenum Press, New York and London.
   Karnovsky, M.J. 1965. A formaldehyde‐glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27:137A.
   Lawton, J.R. 1989. An investigation of the fixation and staining of lipids by a combination of malachite green or other triphenylmethane dyes with glutaraldehyde. J. Micros. 154:83‐92.
   Lee, J.T.Y. and Chow, K.L. 2011. SEM sample preparation for cells on 3D scaffolds by freeze‐drying and HMDS. Scanning 33:1‐14.
   Luft, J.H. 1971. Ruthenium red and violet. Anatom. Record 171:347‐377.
   Nation, J.L. 1983. A new method using hexamethyldisilazane for preparation of soft insect tissues for scanning electron‐microscopy. Stain Technol. 58:347‐351.
   Palade, G.E. 1951. A study of fixation for electron microscopy. J. Exp. Med. 95:285‐297.
   Sabatini, D.D., Bensch, K., and Barrnett, R.J. 1963. Cytochemistry and electron microscopy: The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell. Biol. 17:19‐58.
Key References
   Glauert, A.M. 1974. Fixation, Dehydration, and Embedding of Biological Specimens. Elsevier, Amsterdam.
  The following books are excellent resources for general EM preparative techniques covering general principle for conventional and immunological preparation.
   Hayat, M.A. 1989. Colloidal gold: Principles, Methods, and Applications. Volumes 1‐3. Academic Press, San Diego.
   Hayat, M.A. 2000. Principles and Techniques of Electron Microscopy: Biological Applications. Cambridge University Press, New York.
Internet Resources
   http://www.tedpella.com/
  The above Web sites are some common sources for electron microscopy supplies, reagents and useful tips for immune‐labeling and microwave oven processing.
   http://www.emsdiasum.com/microscopy/
  M. Sanders at the University of Minnesota. A University site containing particularly useful microwave oven theory and protocols.
   http://www.2spi.com/
  Advanced Microscopy Facility, University of Victoria. A University site containing particularly useful microwave oven theory and protocols.
   http://www.ebsciences.com/
  Above are additional resources for commercially available antibodies and small gold probes
   http://www.cbs.umn.edu/ic/
  Anaglyph method from Bob Mannle's Web site, Micro Format.
   http://www.stehm.uvic.ca/docs/prep/microwave/protocols.php
  Adobe Photoshop.
   http://www.invitrogen.com
   http://www.nanoprobes.com/
   http://www.aurion.nl/products/gold_sols.php
   http://www.ebioscience.com/
   http://supercolorviewerpaper.com/advanced3‐d.html
   http://www.adobe.com/products/photoshop.html
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