Focus variation due to near infrared laser in a confocal microscope

Abstract

Focus precision and stability is crucial in confocal microscopy not only for image sharpness but also to avoid radiometric fluctuations that can wrongly be interpreted as variations of the fluorescence intensity in the sample. Here we report a focus variation provoked by a continuous wave laser of 810-nm wavelength introduced along the optical path of an inverted confocal microscope with an oil immersion X60 objective. When the laser is turned on or off, the focus position drifts toward lower or high values of the vertical coordinate z, respectively. The maximum drift observed was 2.25 µm for a laser power of 40 mW at the sample and over a 600-s exposure time. The temporal evolution of the focus position is well fitted by exponential curves that mimic temperature variations due to a heat source. Our analysis strongly suggests that the focus drift is due to heating of the immersion oil.