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Precision and stability in confocal microscopy are vital for maintaining image clarity and preventing misinterpretation of radiometric fluctuations as changes in fluorescence intensity within the sample. This study discusses a focus variation induced by an 810-nm continuous wave laser integrated into the optical path of an inverted confocal microscope with a 60x oil immersion objective. Activation or deactivation of the laser leads to a drift in the focus position towards lower or higher values of the vertical coordinate z, respectively. The maximum observed drift is 2.25 μm, occurring with a 40 mW laser power at the sample over a 600-second exposure time. The temporal behavior of the focus position follows exponential curves resembling temperature fluctuations associated with a heat source. Our analysis strongly indicates that the focus drift is attributable to the heating of the immersion oil.
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Remy Avila, Melissa Matrecitos-Avila, Reinher Pimentel-Domínguez, Elisa Tamariz, Pablo Loza-Alvarez, Edén J. Parra-Fuentes, "Near infrared laser provoques focus variation in a confocal microscope," Proc. SPIE PC13006, Biomedical Spectroscopy, Microscopy, and Imaging III, PC130060B (20 June 2024); https://doi.org/10.1117/12.3022743