Abstract
We present a characterization of chemically treated cells using atomic force microscopy (AFM) which can observe changes in morphology and elasticity of cells. Since AFM has the significant advantage that it does not require fixation of samples, the method is simple and can capture various properties of living cells. In this study, corneal epithelial and endothelial cells were examined. The topography images of the corneal cells without glutaraldehyde (GA) fixation were successfully obtained. The images showed a natural three-dimensional shape of these cells, which scanning electron microscope (SEM) images could not provide. The AFM images of GA-fixed cells were taken and compared with a SEM image reported in the literature. Our results show that longer time for GA fixation makes the surface of the corneal endothelial tissue stiffer. Also, longer treatment results in relatively large structural variation in samples. Combined with conventional histochemical methods, this approach helps us gain an overall understanding of the influence of such chemical treatment.
This is a preview of subscription content, access via your institution.


References
- 1.
Colton RJ, Baselt DR, Dufrêne YF, Green J-BD, Lee GU (1997) Scanning probe microscopy. Curr Opin Chem Biol 1:370–377
- 2.
Hansma HG, Pietrasanta L (1998) Atomic force microscopy and other scanning probe microscopies. Curr Opin Chem Biol 2:579–584
- 3.
Binnig G, Quate CF, Gerber C (1986) Atomic force microscope. Phys Rev Lett 56:930–933
- 4.
Bart ZR, Hammond MA, Wallace JM (2014) Multi-scale analysis of bone chemistry, morphology and mechanics in the oim model of osteogenesis imperfecta. Connect Tissue Res 55:4–8
- 5.
Jena BP, Hörner JKH (2006) Force microscopy—application in biology and medicine: WILEY-LISS
- 6.
Chicoine L, Webster P (1998) Effect of microwave irradiation on antibody labeling efficiency when applied to ultrathin cryosections through fixed biological material. Microsc Res Tech 42:24–32
- 7.
Bohrmann B, Kellenberger E (2001) Cryosubstitution of frozen biological specimens in electron microscopy: use and application as an alternative to chemical fixation. Micron 32:11–19
- 8.
Hannouche D, Raould A, Nizard RS, Sedel L, Petite H (2007) Embedding of bone samples in methylmethacrylate: a suitable method for tracking LacZ mesenchymal stem cells in skeletal tissues. J Histochem Cytochem 55:255–262
- 9.
Troiano NW, Ciovacco WA, Kacena MA (2009) The effects of fixation and dehydration on the histological quality of undecalcified murine bone specimens embedded in methylmethacrylate. J Histotechnol 32:27–31
- 10.
Shibata-Seki T, Masai J, Tagawa T, Sorin T, Kondo S (1996) In-situ atomic force microscopy study of lipid vesicles adsorbed on a substrate. Thin Solid Films 273:297–303
- 11.
Butt HJ (1992) Measuring local surface-charge densities in electrolyte-solutions with a scanning force microscope. Biophys J 63:578–582
- 12.
Dokukin ME, Guz NV, Sokolov I (2013) Quantitative study of the elastic modulus of loosely attached cells in AFM indentation experiments. Biophys J 104:2123–2131
- 13.
Horimizu M, Kawase T, Tanaka T, Okuda K, Nagata M et al (2013) Biomechanical evaluation by AFM of cultured human cell-multilayered periosteal sheets. Micron 48:1–10
- 14.
A-Hassan E, Heinz WF, Antonik MD, D'Costa NP, Nageswaran S et al (1998) Relative microelastic mapping of living cells by atomic force microscopy. Biophys J 74:1564–1578
- 15.
Heu C, Berquand A, Elie-Caille C, Nicod L (2012) Glyphosate-induced stiffening of HaCaT keratinocytes, a peak force tapping study on living cells. J Struct Biol 178:1–7
- 16.
Spedden E, White JD, Naumova EN, Kaplan DL, Staii C (2012) Elasticity maps of living neurons measured by combined fluorescence and atomic force microscopy. Biophys J 103:868–877
- 17.
Pittenger B, Erina N, Su C (2011) Quantitative mechanical properties mapping at the nanoscale with PeakForceQNM. Bruker Application Note #128
- 18.
Pittenger B, Slade A, Berquand A, Milani P, Boudaoud A, Hamant O (2013) Toward quantitative nonmechanical measurements on live cells with PeakForce QNM. Bruker Application Note #141
- 19.
Derjaguin BV, Muller VM, Toporov YP (1975) Effect of contact deformations on adhesion of particles. J Colloid Interface Sci 53:314–326
- 20.
Kadonosono K, Ito N, Yazama F, Nishide T, Sugita M et al (1998) Effect of intracameral anesthesia on the corneal endothelium. J Cataract Refract Surg 24:1377–1381
- 21.
Nichols B, Dawson CR, Togni B (1983) Surface-features of the conjunctiva and cornea. Invest Ophthalmol Vis Sci 24:570–576
- 22.
Liu BY, Zhang GM, Li XL, Chen H (2012) Effect of glutaraldehyde fixation on bacterial cells observed by atomic force microscopy. Scanning 34:6–11
- 23.
Okuda K, Urabe I, Yamada Y, Okada H (1991) Reaction of glutaraldehyde with amino and thiol compounds. J Ferment Bioeng 71:100–105
- 24.
Kuznetsova NP, Mishaeva RN, Gudkin LR, Panarin EF (2013) Reactions of glutaraldehyde with dipolar ions of amino acids and proteins. Russ Chem Bull 62:918–927
- 25.
Kawahara J, Ohmori T, Ohkubo T, Hattori S, Kawamura M (1992) The structure of glutaraldehyde in aqueous-solution determined by ultraviolet-absorption and light-scattering. Anal Biochem 201:94–98
Acknowledgments
We would like to thank Dainichiseika Color & Chemicals Mfg. Co., Ltd. for financial support through Dainichiseika-Donated Chair of Research Division for Innovative Biomaterials in Tokyo Institute of Technology. This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 23592611.
Author information
Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Shibata-Seki, T., Tajima, K., Takahashi, H. et al. AFM characterization of chemically treated corneal cells. Anal Bioanal Chem 407, 2631–2635 (2015). https://doi.org/10.1007/s00216-015-8473-0
Received:
Revised:
Accepted:
Published:
Issue Date:
Keywords
- Atomic force microscopy
- Corneal epithelial and endothelial cells
- Mechanical property
- Young's modulus
- Glutaraldehyde