Microscopy Applied In Biomass Characterization



This chapter serves as an introduction to the major types of microscopy that are applied to the characterization of lignocellulosic biomasses. The covered techniques include optical microscopies (light, Raman, and confocal microscopy), scanning probe microscopy, and electron microscopy. This chapter provides a general description of the principles, advantages and drawbacks, type of information that can be obtained using the different microscopic techniques, and includes a wide range of examples on the use of such techniques to characterize lignocellulosic biomass samples before and after pretreatments. Finally, some of the reviewed microscopic techniques were used to visualize samples of wheat straw nodes before and after acid and alkali pretreatments. This chapter is designed to help scientists select the best microscopic technique to study biomass feedstocks with recalcitrant natures.


Alkali pretreatment Atomic force microscopy Dilute acid pretreatment Electron microscopy Optical microscopy 



Financial support was received from the CB CONACYT project (Grant No. CB-2011/168921), the Red Temática del Hidrógeno Subprograma de Producción de Hidrógeno (Grant No. 252003), and the CONACYT project Apoyo al Fortalecimiento y Desarrollo de la Infraestructura Científica y Tecnológica (Grant No. 250738).


  1. Abraham E, Deepa B, Pothan LA, Jacob M, Thomas S, Cvelbar U, Anandjiwala R (2011) Extraction of nanocellulose fibrils from lignocellulosic fibres: a novel approach. Carbohydr Polym 86:1468–1475CrossRefGoogle Scholar
  2. Abud Y, Costa LT, de Souza W, Sant’Anna C (2013) Revealing the microfibrillar arrangement of the cell wall surface and the macromolecular effects of thermochemical pretreatment in sugarcane by atomic force microscopy. Ind Crop Prod 51:62–69CrossRefGoogle Scholar
  3. Akin DE (1979) Microscopic evaluation of forage digestion by rumen microorganisms – a review. J Anim Sci 48:701–710CrossRefGoogle Scholar
  4. Akin DE, Rigsby LL, Sethuraman A, Morrison WH, Gamble GR, Eriksson KE (1995) Alterations in structure, chemistry, and biodegradability of grass lignocellulose treated with the white rot fungi Ceriporiopsis subvermispora and Cyathus stercoreus. Appl Environ Microbiol 61:1591–1598Google Scholar
  5. Binnig G, Rohrer H (1982) Scanning tunneling microscopy. Helv Phys Acta 55:726–735Google Scholar
  6. Blancaflor EB, Gilroy S (2000) Plant cell biology in the new millennium: new tools and new insights. Am J Bot 87:1547–1560CrossRefGoogle Scholar
  7. Bogner A, Jouneau PH, Thollet G, Basset D, Gauthier C (2007) A history of scanning electron microscopy developments: towards “wet-STEM” imaging. Micron 38:390–401CrossRefGoogle Scholar
  8. Bohning JJ (1998) The Raman effect. American Chemical Society Indian Association for the Cultivation of Science, KolkataGoogle Scholar
  9. Børsheim KY, Bratbak G, Heldal M (1990) Enumeration and biomass estimation of planktonic bacteria and viruses by transmission electron microscopy. Appl Environ Microbiol 56:352–356Google Scholar
  10. Bozzola JJ (2001) Electron microscopy eLS. John Wiley & Sons, New York, NYGoogle Scholar
  11. Bumbrah GS, Sharma RM (2015) Raman spectroscopy – basic principle, instrumentation and selected applications for the characterization of drugs of abuse. J Forensic Sci (in press)Google Scholar
  12. Butt HJ, Wolff EK, Gould SAC, Dixon B, Peterson CM, Hansma PK (1990) Imaging cells with the atomic force microscope. J Struct Biol 105:54–61CrossRefGoogle Scholar
  13. Cappella B, Dietler G (1999) Force-distance curves by atomic force microscopy. Surf Sci Rep 34:1–104CrossRefGoogle Scholar
  14. Clarke K, Li X, Li K (2011) The mechanism of fiber cutting during enzymatic hydrolysis of wood biomass. Biomass Bioenerg 35:3943–3950Google Scholar
  15. Conchello JA, Lichtman JW (2005) Optical sectioning microscopy. Nat Methods 2:2920–2931CrossRefGoogle Scholar
  16. Corrales RCNR, Mendes FMT, Perrone CC, Sant’Anna C, Souza W, Abud Y, Bon EPS, Ferreira V (2012) Structural evaluation of sugar cane bagasse steam pretreated in the presence of CO2 and SO2. Biotechnol Biofuels 5:1–8CrossRefGoogle Scholar
  17. Crawford NC, Nagle N, Sievers DA, Stickel JJ (2016) The effects of physical and chemical preprocessing on the flowability of corn stover. Biomass Bioenerg 85:126–134CrossRefGoogle Scholar
  18. Davidson RS, Choudhury H, Origgi S, Castellan A, Trichet V, Capretti G (1995) The reaction of phloroglucinol in the presence of acid with lignin-containing materials. J Photochem Photobiol A Chem 91:87–93CrossRefGoogle Scholar
  19. Dhiman SS, Haw JR, Kalyani D, Kalia VC, Kang YC, Lee JK (2015) Simultaneous pretreatment and saccharification: green technology for enhanced sugar yields from biomass using a fungal consortium. Bioresour Technol 179:50–57CrossRefGoogle Scholar
  20. Ding SY, Himmel ME (2006) The maize primary cell wall microfibril: a new model derived from direct visualization. J Agric Food Chem 54:597–606CrossRefGoogle Scholar
  21. Donohoe BS, Vinzant TB, Elander RT, Pallapolu VR, Lee YY, Garlock RJ, Balan V, Dale BE, Kim Y, Mosier NS, Ladisch MR, Falls M, Holtzapple MT, Sierra R, Shi J, Ebrik MA, Redmond T, Yang B, Wyman CE, Hames B, Thomas S, Warner RE (2011) Surface and ultrastructural characterization of raw and pretreated switchgrass. Bioresour Technol 102:11097–11104CrossRefGoogle Scholar
  22. Dornez E, Holopainen U, Cuyvers S, Poutanen K, Delcour JA, Courtin CM, Nordlund E (2011) Study of grain cell wall structures by microscopic analysis with four different staining techniques. J Cereal Sci 54:363–373CrossRefGoogle Scholar
  23. Ghaffar SH, Fan M (2015) Revealing the morphology and chemical distribution of nodes in wheat straw. Biomass Bioenerg 77:123–134CrossRefGoogle Scholar
  24. Gierlinger N (2010) Raman imaging of plant cell walls. In: Thomas D, Olaf H, Jan T (eds) Confocal Raman microscopy. Series in optical sciences no. 158. Springer, Berlin, pp 225–235CrossRefGoogle Scholar
  25. Gunning PA (2013) Light microscopy: principles and applications to food microstructures. Woodhead Publishing Limited, SawstonGoogle Scholar
  26. Haigler CH, Brown RM Jr, Benziman M (1980) Calcofluor white ST Alters the in vivo assembly of cellulose microfibrils. Science 210:903–906CrossRefGoogle Scholar
  27. Haque MA, Barman DN, Kang TH, Kim MK, Kim J, Kim H, Yun HD (2013) Effect of dilute alkali pretreatment on structural features and enhanced enzymatic hydrolysis of Miscanthus sinensis at boiling temperature with low residence time. Biosyst Eng 114:294–305CrossRefGoogle Scholar
  28. Hell J, Donaldson L, Michlmayr H, Kraler M, Kneifel W, Potthast A, Rosenau T, Böhmdorfer S (2015) Effect of pretreatment on arabinoxylan distribution in wheat bran. Carbohydr Polym 121:18–26CrossRefGoogle Scholar
  29. Holopainen-Mantila U, Marjamaa K, Merali Z, Käsper A, de Bot P, Jääskeläinen A-S, Waldron K, Kruus K, Tamminen T (2013) Impact of hydrothermal pre-treatment to chemical composition, enzymatic digestibility and spatial distribution of cell wall polymers. Bioresour Technol 138:156–162CrossRefGoogle Scholar
  30. Igarashi K, Uchihashi T, Koivula A, Wada M, Kimura S, Okamoto T, Penttilä M, Ando T, Samejima M (2011) Traffic jams reduce hydrolytic efficiency of cellulase on cellulose surface. Science 333:1279–1282CrossRefGoogle Scholar
  31. Jean-Marie E (2016) Microscopy: light microscopy and histochemical methods. Encyclopedia of food and health. Elsevier, AmsterdamGoogle Scholar
  32. Ji Z, Ding DY, Ling Z, Zhang X, Zhou X, Xu F (2014) In situ microscopic investigation of plant cell walls deconstruction in biorefinery. In: Méndez-Vilas A (ed) Microscopy: advances in scientific research and education. Formatex, Badajoz, pp 426–433Google Scholar
  33. Johnson D, Hilal N, Bowen WR (2009) Basic principles of atomic force microscopy. In: Bowen WR, Hilal N (eds) Atomic force microscopy in process engineering. Elsevier, Amsterdam, pp 1–30CrossRefGoogle Scholar
  34. Karp EM, Resch MG, Donohoe BS, Ciesielski PN, O’Brien MH, Nill JE, Mittal A, Biddy MJ, Beckham GT (2015) Alkaline pretreatment of switchgrass. ACS Sustain Chem Eng 3:1479–1491CrossRefGoogle Scholar
  35. Keegstra K (2010) Plant cell walls. Plant Physiol 154:483–486CrossRefGoogle Scholar
  36. Kim SJ, Hyeon JE, Jeon SD, Choi GW, Han SO (2014) Bi-functional cellulases complexes displayed on the cell surface of Corynebacterium glutamicum increase hydrolysis of lignocelluloses at elevated temperature. Enzyme Microb Technol 66:67–73CrossRefGoogle Scholar
  37. Kirby AR, Gunning AP, Waldron KW, Morris VJ, Ng A (1996) Visualization of plant cell walls by atomic force microscopy. Biophys J 70:1138–1143CrossRefGoogle Scholar
  38. Lara-Vázquez AR, Quiroz FR, Sánchez A, Valdez-Vazquez I (2014) Particle size and hydration medium effects on hydration properties and sugar release of wheat straw fibers. Biomass Bioenerg 68:67–74CrossRefGoogle Scholar
  39. Li W, Yan L, Yang J (2006) AFM study of crystalline cellulose in the cell walls of straw. Polym Int 55:87–92CrossRefGoogle Scholar
  40. Li C, Knierim B, Manisseri C, Arora R, Scheller HV, Auer M, Vogel KP, Simmons BA, Singh S (2010) Comparison of dilute acid and ionic liquid pretreatment of switchgrass: biomass recalcitrance, delignification and enzymatic saccharification. Bioresour Technol 101:4900–4906CrossRefGoogle Scholar
  41. Lou B, Peng B, Rong N, Li Y, Chen H, Sree KS, Gao Q, Varma A (2014) Root and root endophytes from the eyes of an electron microscopist. In: Asunción M, Ajit V (eds) Root Engineering, soil biology, vol 40. Springer, New York. NY, pp 469–486Google Scholar
  42. Luterbacher JS, Walker LP, Moran-Mirabal JM (2013) Observing and modeling BMCC degradation by commercial cellulase cocktails with fluorescently labeled Trichoderma reseii Cel7A through confocal microscopy. Biotechnol Bioeng 110:108–117CrossRefGoogle Scholar
  43. Ma J, Zhou X, Ma J, Ji Z, Zhang X, Xu F (2014) Raman microspectroscopy imaging study on topochemical correlation between lignin and hydroxycinnamic acids in Miscanthus sinensis. Microsc Microanal 20:956–963CrossRefGoogle Scholar
  44. Marton L (1934) Electron microscopy of biological objects. Nature 133:911CrossRefGoogle Scholar
  45. McCartney L, Marcus SE, Knox JP (2005) Monoclonal antibodies to plant cell wall xylans and arabinoxylans. J Histochem Cytochem 53:543–546CrossRefGoogle Scholar
  46. Morris VJ, Gunning AP, Kirby AR, Round A, Waldron K, Ng A (1997) Atomic force microscopy of plant cell walls, plant cell wall polysaccharides and gels. Int J Biol Macromol 21:61–66CrossRefGoogle Scholar
  47. Nanotechnology Now, 2011. Press release: Hitachi launches world’s highest resolution FE-SEM Accessed 20 Jan 2016
  48. Nwaneshiudu A, Kuschal C, Sakamoto FH, Anderson RR, Schwarzenberger K, Young RC (2012) Introduction to confocal microscopy. J Invest Dermatol 132, e3CrossRefGoogle Scholar
  49. O'Brien TP, Feder N, McCully ME (1964) Polychromatic staining of plant cell walls by toluidine blue O. Protoplasma 59:368–373CrossRefGoogle Scholar
  50. Pérez-Rangel M, Quiroz-Figueroa FR, González-Castañeda J, Valdez-Vazquez I (2015) Microscopic analysis of wheat straw cell wall degradation by microbial consortia for hydrogen production. Int J Hydrogen Energ 1:151–160CrossRefGoogle Scholar
  51. Reddy N, Yang Y (2005) Structure and properties of high quality natural cellulose fibers from cornstalks. Polymer 46:5494–5500CrossRefGoogle Scholar
  52. Rose HH (2009) Historical aspects of aberration correction. J Electron Microsc 58:77–85CrossRefGoogle Scholar
  53. Ruska E (1987) The development of the electron microscope and of electron microscopy. Biosci Rep 7:607–629CrossRefGoogle Scholar
  54. Sant´Anna C, de Souza W (2012) Microscopy as a tool to follow deconstruction of lignocellulosic biomass. In: Méndez-Vilas A (ed) Current microscopy contributions to advances in science and technology. Formatex, Badajoz, pp 639–645Google Scholar
  55. Sarkar P, Bosneaga E, Auer M (2009) Plant cell walls throughout evolution: towards a molecular understanding of their design principles. J Exp Bot 60:3615–3635CrossRefGoogle Scholar
  56. Schuldt A (2009) Seeing the wood for the trees. Nat Cell Biol 11:S12–S13Google Scholar
  57. Singh S, Simmons BA, Vogel KP (2009) Visualization of biomass solubilization and cellulose regeneration during ionic liquid pretreatment of switchgrass. Biotechnol Bioeng 104:68–75CrossRefGoogle Scholar
  58. Smith CL (2011) Basic confocal microscopy. Curr Protoc Neurosci 56:2.2.1–2.2.18Google Scholar
  59. Somleva MN, Snell KD, Beaulieu JJ, Peoples OP, Garrison BR, Patterson NA (2008) Production of polyhydroxybutyrate in switchgrass, a value-added co-product in an important lignocellulosic biomass crop. Plant Biotechnol J 6:663–678CrossRefGoogle Scholar
  60. Sun L, Li C, Xue Z, Simmons BA, Singh S (2013) Unveiling high-resolution, tissue specific dynamic changes in corn stover during ionic liquid pretreatment. RSC Adv 3:2017–2027CrossRefGoogle Scholar
  61. Tan HT, Lee KT, Mohamed AR (2011) Pretreatment of lignocellulosic palm biomass using a solvent-ionic liquid [BMIM] Cl for glucose recovery: an optimisation study using response surface methodology. Carbohydr Polym 83:1862–1868CrossRefGoogle Scholar
  62. Travis AJ, Murison SD, Perry P, Chesson A (1997) Measurement of cell wall volume using confocal microscopy and its application to studies of forage degradation. Ann Bot 80:1–11CrossRefGoogle Scholar
  63. Ushiki T, Kawabata K (2008) Scanning probe microscopy in biological research. In: Bharat B, Harald F, Masahiko T (eds) Applied scanning probe methods X. Springer, Berlin, pp 285–308CrossRefGoogle Scholar
  64. Wang C, Yadavalli VK (2014) Investigating biomolecular recognition at the cell surface using atomic force microscopy. Micron 60:5–17CrossRefGoogle Scholar
  65. Wang J, Quirk A, Lipkowski J, Dutcher JR, Clarke AJ (2013) Direct in situ observation of synergism between cellulolytic enzymes during the biodegradation of crystalline cellulose fibers. Langmuir 29:14997–15005CrossRefGoogle Scholar
  66. Wei H, Donohoe BS, Vinzant TB, Ciesielski PN, Wang E, Gedvilas LM, Zeng Y, Johnson DK, Ding SY, Himmel ME, Tucker MP (2011) Elucidating the role of ferrous ion cocatalyst inenhancing dilute acid pretreatment of lignocellulosic biomass. Biotechnol Biofuels 4:48CrossRefGoogle Scholar
  67. Williams DB, Carter CB (2009) The transmission electron microscope. Transmission electron microscopy. Springer, New York, NYCrossRefGoogle Scholar
  68. Wilson SM, Bacic A (2012) Preparation of plant cells for transmission electron microscopy to optimize immunogold labeling of carbohydrate and protein epitopes. Nat Protoc 7:1716–1727CrossRefGoogle Scholar
  69. Winey M, Meehl JB, O’Toole ET, Giddings TH (2014) Conventional transmission electron microscopy. Mol Biol Cell 25:319–323CrossRefGoogle Scholar
  70. Zhou W, Apkarian R, Wang ZL, Joy D (2006) Fundamentals of scanning electron microscopy (SEM). In: Zhou W, Wang ZL (eds) Scanning microscopy for nanotechnology. Springer, New York, NYGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  1. 1.Unidad Académica Juriquilla, Instituto de IngenieríaUniversidad Nacional Autónoma de MéxicoQuerétaroMexico
  2. 2.Laboratorio de Fitomejoramiento Molecular, Instituto Politécnico NacionalCentro Interdisciplinario de Investigación para el Desarrollo Integral Regional Unidad Sinaloa (CIIDIR-IPN Unidad Sinaloa)GuasaveMexico

Personalised recommendations