Atomic force microscopy


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characterization of plant cell wall in a nano scale level using Atomic force microscopy

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Atomic force microscopy

  1. 1. Ragavi.s M.Sc PROJECT STUDENT Dept of biotechnology Alagappa university3/29/2014 1
  2. 2.  Atomic force microscopy (AFM) or scanning force microscopy (SFM) is a very high-resolution type of scanning force microscopy, with demonstrated resolution on the order of fractions of a nanometer, more than 1000 times better than the optical diffraction limit.  The AFM is one of the foremost tools for imaging, measuring, and manipulating matter at the nanoscale.  The information is gathered by "feeling" the surface with a mechanical probe. Piezoelectric elements that facilitate tiny but accurate and precise movements on (electronic) command enable the very precise scanning. In some variations, electric potential can also be scanned using conducting cantilevers. 3/29/2014 2
  3. 3.  The AFM consists of a cantilever with a sharp tip (probe) at its end that is used to scan the specimen surface.  The cantilever is typically silicon or silicon nitride with a tip radius of curvature on the order of nanometers. When the tip is brought into proximity of a sample surface, forces between the tip and the sample lead to a deflection of the cantilever according to Hooke's law. 3/29/2014 3
  4. 4.  This states that "within the limits of elasticity the strain produced by a stress of any one kind is proportional to the stress". The stress at which a material ceases to obey Hooke's Law is known as the limit of proportionality.  Typically, the deflection is measured using a laser spot reflected from the top surface of the cantilever into an array of photodiodes. 3/29/2014 4
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  6. 6. 3/29/2014 6 HOW IT WORKS
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  8. 8. 3/29/2014 8 Single Molecule Study Laboratory, College of Engineering and Nanoscale Science and Engineering Center, University of Georgia, Athens, GA 30602, USA
  9. 9.  The crystalline cellulose distributions on natural and pretreated plant cell wall surfaces were specifically characterized by the well-established recognition imaging technique.  Functionalization of AFM Tip  The AFM tip was first coated with a magnetic material followed by a thin gold layer.  The pre-coated tip was functionalized with the help of CBM3a molecule which was linked by a cross linker known as PEG 3/29/2014 9
  10. 10. • (a) AFM tip modification and recognition imaging process; • (b) topography and recognition signal division by PicoTREC controller; • (c) example of topography and recognition images generated by separated signals. Yellow strips: sketch of crystalline cellulose; black strips: recognition signal of crystalline cellulose in topography image; green marks: other components which do not have specific interactions with functionalized AFM tip and show no recognition signals in the recognition image. 6µm/s 3/29/2014 10
  11. 11.  Topography images (a-f) recognition images (g-l) of natural poplar (P), switchgrass (SG), and corn stover (CS).  (a, c, e, g, i, k) show the representative surface area mainly covered by lignin.  (b, d, f, h, j, l) show the representative surface area mainly covered by crystalline cellulose. TOPOGRAPHY IMAGES RECOGNITION IMAGES 3/29/2014 11
  12. 12.  g, i, k - absence of specific interaction between non-cellulose and CBM molecule.  b, d, f - extensively covered by crystalline cellulose micro fibrils.  h, j, l - strong recognition signals were detected in the corresponding recognition images. 3/29/2014 12
  13. 13. The pretreated sample was then washed by DI water for 5 times and finally dried in air at 45°C for 24 h. The reaction was stopped by cooling down the tube to room temperature in cold DI water. The sealed pressure tube was heated in a heating block on a hot plate at 135°C for 20 min. 0.1 g biomass of each species was pre-soaked in dilute sulfuric acid (0.03% w/w, 2 mL) for 30 min in a 20 mL glass pressure tube . Milled biomass has a size of 200 to 250µm were selected, 3/29/2014 13
  14. 14. The pretreated samples were subsequently delignified. 0.02 g sodium chlorite and 40 μL glacial acetic acid was added into pretreated biomass water slurry (3% solid, 1 mL). The reaction was taken at 80°C for 1.5 h with gentle stirring. After cooling down, the bleached sample was washed 8 times with DI water followed by centrifugation at 10,000 rpm for 10 min and then was dried in air at 45°C for 24 h. 3/29/2014 14
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  17. 17.  The crystalline cellulose distributions on the plant cell wall surface were quantitatively determined based on recognition signal distribution.  For each biomass sample 5 different surface areas in average were imaged. Over 20 sample pieces were imaged and 100 recognition images in size of 1 μm×1 μm were randomly selected for the RAP calculation.  Recognition images of each sample were divided into maximum 7 types based on surface components of pretreated cell wall before and after delignification. 3/29/2014 17
  18. 18. 3/29/2014 18 Measurementof recognition area percentage (RAP) on plant cell wall surfaces
  19. 19. FromType B to G, the amount and size of surface agglomerates gradually decreased 3/29/2014 19 This difference denoted that the lignin locating on the surface of pretreated plant cell walls was extensively removed during the delignification process, therefore the crystalline cellulose underneath was exposed and recognized by the CBM3a-modified AFM tip.
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  21. 21.  Enzymatic hydrolysis of lignocellulosic biomass is a critical process for biofuel production.  This process is greatly hindered by the natural complexity of plant cell walls and limited accessibility of surface cellulose by enzymes.  A more profound understanding of surface cellulose distributions before and after pretreatments at single- molecule level is in great need. So here they are using AFM technique.  This study provides a better understanding of surface structural changes after pretreatment such as lignin relocation, re-precipitation, and crystalline cellulose distribution, and can lead to potential improvements of biomass pretreatment.3/29/2014 21
  22. 22.  Based on AFM recognition imaging and area percentage calculations, normally 17-20% of plant cell wall surfaces were covered by crystalline cellulose before pretreatment and this coverage increased to 23-38% after dilute acid pretreatment under different temperature and acid concentrations.  When the plant cell walls were pretreated with 0.5% sulfuric acid, the crystalline cellulose surface distribution of 23% on poplar, 28% on switchgrass, and 38% on corn stover was determined as an optimized result at 135°C.  Compared to bulk component analysis, this method exhibits advantages in providing detailed surface information of plant cell walls in single molecule level. 3/29/2014 22
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