Abstract

Optical microscopes are essential tools widely used in clinical and laboratory research. The quality of images depends on various software and hardware criteria, with the focus point being a key factor. Accurate focus, determined by movement along the vertical axis, is crucial for precisely observing microscopic details, particularly cellular structures. A properly focused image enhances research reliability and repeatability. This study developed an automation system integrated into a graphical user interface (GUI) to automatically determine the focus distance of optical microscopes using gradient and intensity functions. A motorized mechanical system controlled by an embedded computer moves the microscope stage. The GUI evaluates real-time images, which adjusts the system to the correct focus point. Calibration tests with a 0.01 mm spaced cover slip confirmed the system’s high accuracy and precision, enabling a reliable universal autofocus solution for optical microscopes.

References

[1] J. Mertz, Introduction to optical microscopy. Cambridge University Press, 2019.

[2] A. P. Dhawan, B. D’Alessandro, and X. Fu, “Optical imaging modalities for biomedical applications,” IEEE Rev. Biomed. Eng., vol. 3, pp. 69–92, 2010.

[3] F. L. Pedrotti, L. M. Pedrotti, and L. S. Pedrotti, Introduction to optics. Cambridge University Press, 2018.

[4] A. R. Carter, G. M. King, T. A. Ulrich, W. Halsey, D. Alchenberger, and T. T. Perkins, “Stabilization of an optical microscope to 0.1 nm in three dimensions,” Appl. Opt., vol. 46, no. 3, pp. 421–427, 2007.

[5] C. Maurer, A. Jesacher, S. Bernet, and M. Ritsch‐Marte, “What spatial light modulators can do for optical microscopy,” Laser Photon. Rev., vol. 5, no. 1, pp. 81–101, 2011.

[6] S. Perrin, H. Li, S. Lecler, and P. Montgomery, “Unconventional magnification behaviour in microsphere-assisted microscopy,” Opt. Laser Technol., vol. 114, pp. 40–43, 2019.

[7] M. A. Pala, “Holografik Görüntülerde Kenar Tabanlı Fraktal Özniteliklerin Hücre Canlılık Analizlerinde Başarısı,” vol. 2, no. 2, pp. 86–94, 2021.

[8] M. A. Pala and M. Z. Yıldız, “Improving cellular analysis throughput of lens-free holographic microscopy with circular Hough transform and convolutional neural networks,” Opt. Laser Technol., vol. 176, p. 110920, 2024.

[9] R. Hurtado-Pérez, C. Toxqui-Quitl, A. Padilla-Vivanco, J. F. Aguilar-Valdez, and G. Ortega-Mendoza, “Focus measure method based on the modulus of the gradient of the color planes for digital microscopy,” Opt. Eng., vol. 57, no. 02, p. 1, 2018, doi: 10.1117/1.oe.57.2.023106.

[10] H. Pinkard, Z. Phillips, A. Babakhani, D. A. Fletcher, and L. Waller, “Deep learning for single-shot autofocus microscopy,” Optica, vol. 6, no. 6, p. 794, Jun. 2019, doi: 10.1364/optica.6.000794.

[11] Z. Yan, G. Chen, W. Xu, C. Yang, and Y. Lu, “Study of an image autofocus method based on power threshold function wavelet reconstruction and a quality evaluation algorithm,” Appl. Opt., vol. 57, no. 33, pp. 9714–9721, 2018.

[12] M. A. Bueno-Ibarra, J. A´ lvarez-Borrego, L. Acho, and M. a C. na Cha´ vez-Sa´ nchez, “Fast autofocus algorithm for automated microscopes,” Opt. Eng., vol. 44, no. 6, p. 63601, 2005.