Abstract
Atomic force microscopy is used for characterization of biological samples, individual molecules, molecular interaction and their roles in friction and adhesion at molecular and cellular levels. The current paper aims to summarize the atomic force microscopy, its working principle, the usage of it in characterization of bone and latest developments appeared. The proposed workflow for the AFM is developed based on current observations based on articles studied in current paper and their results, their limitations and remained gaps to be solved, as well as there are also additional steps shown for educational purposes. Potential bias in elastic modulus and boundary condition errors, variability of values and lack of standardization plus possible bias sources, biases in algorithm are the limitations and problems needed to be still solved. Therefore, a combined effort of specialists in biomechanics, tissue engineering and biomaterials is necessary to be conducted further.
References
Andany, S. H., Hlawacek, G., Hummel, S., Brillard, C., Kangül, M., & Fantner, G. E. (2020). An atomic force microscope integrated with a helium ion microscope for correlative nanoscale characterization. Beilstein Journal of Nanotechnology, 11, 1272–1279. https://doi.org/10.3762/bjnano.11.111
Arnold, K. M., Sicard, D., Tschumperlin, D. J., & Westendorf, J. J. (2023). Atomic force microscopy micro-indentation methods for determining the elastic modulus of murine articular cartilage. Sensors, 23(4), Article 1835. https://doi.org/10.3390/s23041835
Băncilă, I. -C. (2025). Mini-review: Experimental approaches for the biomechanical testing of bone. International Journal of Emerging Engineering & Technology, 4(1), 11–20. https://doi.org/10.57041/bnc97632
Băncilă, I. -C. (2026). Nanoindentation: A comprehensive review with a proposed standardized workflow. Advances in Mechanical and Materials Engineering, 43, 79–94. https://doi.org/10.7862/rm.2026.6
Bouxsein, M. L., Boyd, S. K., Christiansen, B. A., Guldberg, R. E., Jepsen, K. J., & Müller, R. (2010). Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. Journal of Bone and Mineral Research, 25(7), 1468–1486. https://doi.org/10.1002/jbmr.141
Brent, M. B. (2024). Imaging, dynamic histomorphometry, and mechanical testing in preclinical bone research. Osteology, 4(3), 120–131. https://doi.org/10.3390/osteology4030010
Charrier, A., Lefèvre, E., Pithioux, M., & Esteve, F. (2013). Determination of mechanical properties of cortical bone using AFM under dry and immersed conditions. Computer Methods in Biomechanics and Biomedical Engineering, 16, 102–103. https://doi.org/10.1080/10255842.2013.815974
Choi, J. W., & Kim, J. J. (2025). A computational approach to investigate the structural behavior of bone scaffold-implanted proximal femur in routine clinical resolution. International Journal for Numerical Methods in Biomedical Engineering, 41(7), Article e70072. https://doi.org/10.1002/cnm.70072
Eaton, P., & West, P. (2010a). AFM Image Artefacts. In P. Eaton, P. West (Eds.)., Atomic force microscopy (pp. 121-138). Oxford University Press. https://doi.org/10.1093/acprof:oso/9780199570454.003.0006
Eaton, P., & West, P. (2010b). Atomic force microscopy. Oxford University Press. https://doi.org/10.1093/acprof:oso/9780199570454.001.0001
Fadhil, K., & Amin, B. K. (2025). Effect of bleaching on the surface roughness of resin composites evaluated by atomic force microscopy (AFM). Dentistry Journal, 13(10), Article 470. https://doi.org/10.3390/dj13100470
Fukuda, S., & Ando, T. (2021). Faster high-speed atomic force microscopy for imaging of biomolecular processes. Review of Scientific Instruments, 92(3), Article 033705. https://doi.org/10.1063/5.0032948
Herruzo, E. T., Asakawa, H., Fukuma, T., & Garcia, R. (2016). Sub-nanometer resolution imaging with amplitude-modulation atomic force microscopy in liquid. JoVE Journal. https://dx.doi.org/10.3791/54924
Hutter, J. L., & Bechhoefer, J. (1993). Calibration of atomic‐force microscope tips. Review of Scientific Instruments, 64(7), 1868–1873. https://doi.org/10.1063/1.1143970
Kontomaris, S. V., Psychogios, I., Stergiopoulos, M., Malamou, A., & Stylianou, A. (2025). Quantitative criteria for the validity of the elastic half-space assumption in AFM nanoindentation. Next Materials, 9, Article 101180. https://doi.org/10.1016/j.nxmate.2025.101180
Ma, C., & Zhou, F. (2025). Surface and subsurface mechanical testing at the nanoscale: A review on ultrasonic atomic force microscopy. Langmuir, 41(24), 15203–15220. https://doi.org/10.1021/acs.langmuir.5c01456
Melters, D. P., Neuman, K. C., & Dalal, Y. (2025). Single-molecule studies with AFM and high-speed AFM: From nucleosomes to chromosomes. In J. M. Walker (Ed.), Methods in Molecular Biology (pp. 485–508). Humana Press. https://doi.org/10.1007/978-1-0716-4750-9_29
Millan-Solsona, R., Brown, S. R., Zhang, L., Madugula, S. S., Zhao, H. H., Dumerer, B., Bible, A. N., Lavrik, N. V., Vasudevan, R. K., Biswas, A., Morrell-Falvey, J. L., Retterer, S., Checa, M., & Collins, L. (2025). Analysis of biofilm assembly by large area automated AFM. npj Biofilms and Microbiomes, 11(1), Article 75. https://doi.org/10.1038/s41522-025-00704-y
Morita, S., Giessibl, F. J., Meyer, E., & Wiesendanger, R. (Eds.). (2015). Noncontact atomic force microscopy: Volume 3. Springer International Publishing. https://doi.org/10.1007/978-3-319-15588-3
Morris, V. J., Kirby, A. R., & Gunning, P. A. (2009). Atomic force microscopy for biologists (2nd ed.). Imperial College Press.
Rana, M. S., Pota, H. R., & Petersen, I.R. (2017). Improvement in the imaging performance of atomic force microscopy: A survey. IEEE Transactions on Automation Science and Engineering, 14(2), 1265-1285. https://doi.org/10.1109/TASE.2016.2538319
Sader, J. E., Chon, J. W. M., & Mulvaney, P. (1999). Calibration of rectangular atomic force microscope cantilevers. Review of Scientific Instruments, 70(10), 3967–3969. https://doi.org/10.1063/1.1150021
Scarano, E., Arvidsson, E. K., Roos, A. K., Holmgren, E., Borgani, R., Tholén, M. O., & Haviland D. B. (2025). Low-temperature AFM with a microwave cavity optomechanical transducer. Beilstein Journal of Nanotechnology, 16, 1873–1882. https://doi.org/10.3762/bjnano.16.130
Schitter, G., Fantner, G., Kindt, J. H., & Thurner, P. J. (2005). On recent developments for high-speed atomic force microscopy. In Proceedings of the International Conference on Advanced Intelligent Mechatronics. IEEE.
Schitter, G., Fantner, G. E., Thurner, P. J., Adams, J., & Hansma, P. K. (2006). Design and characterization of a novel scanner for high-speed atomic force microscopy. In Proceedings of the 4th IFAC Symposium on Mechatronic Systems (pp. 819–824).
Sumbul, F., Hassanpour, N., Rodriguez-Ramos, J., & Rico, F. (2020). One-step calibration of AFM in liquid. Frontiers in Physics, 8, Article 301. https://doi.org/10.3389/fphy.2020.00301
Vuillemot, R., Pellequer, J. L., & Grudinin, S. (2025). Deciphering conformational dynamics in AFM data using fast nonlinear NMA and FFT-based search with AFMFit. Communications Biology, 8, Article 1381. https://doi.org/10.1038/s42003-025-08365-5
Wen, C.-Y., Wu, C.-B., Tang, B., Pan, H., Hu, Y., & Chiu K. Y. (2012). Collagen fibril stiffening in osteoarthritic cartilage of human beings revealed by atomic force microscopy. Osteoarthritis and Cartilage, 20(8), 916–922. https://doi.org/10.1016/j.joca.2012.04.018
Zeng, Y., Chen, Y., Wu, T., & Han, G. (2025). Adaptive compressive sensing imaging in AFM based on target block detection. Microscopy Research and Technique, 88(12), 3282–3309. https://doi.org/10.1002/jemt.70021
Zhou, Y., & Du, J. (2022). Atomic force microscopy (AFM) and its applications to bone-related research. Progress in Biophysics and Molecular Biology, 176, 52–66. https://doi.org/10.1016/j.pbiomolbio.2022.10.002

