In Vivo Light-Sheet Imaging of Senescence Reporter Activity in a Transparent Killifish

Aging is associated with progressive accumulation of senescent cells, which contribute to tissue dysfunction and organismal decline. Conventional approaches for assessing cellular senescence, such as histological or immunofluorescence analyses of fixed tissue sections and flow cytometry, require tissue collection, thereby precluding longitudinal in vivo studies. To enable the analysis of cellular senescence in a living vertebrate model, we have previously generated a cdkn1a (p21)-driven GFP reporter line that was established in the transparent klara background of Nothobranchius furzeri. Here, we describe a protocol for in vivo light-sheet microscopy of the reporter activity as readout for senescence-associated cell cycle arrest with single-cell resolution. The procedure involves anesthesia and mounting of fish for stable positioning within the imaging chamber, with particular attention to animal welfare considerations. It further includes the acquisition of three-dimensional image stacks and subsequent image processing. The workflow allows monitoring of GFP-positive cells in intact living killifish at different developmental stages. Although imaging depth remains limited despite organismal transparency, this method provides high-resolution volumetric imaging with minimal phototoxicity and enables analysis of senescence dynamics in a short-lived vertebrate model. It is currently performed as a terminal procedure under approved ethical regulations, but longitudinal imaging would also be possible with additional ethical authorization. Key features • This protocol allows intravital imaging of fluorescent reporter activity in intact, anesthetized small teleost fish using light-sheet microscopy. • Two complementary mounting strategies enable stable imaging across different developmental stages. • A customized mounting support is rapidly prepared using UV-curable adhesive that fits directly into the standard Lightsheet Z.1 sample holder (Zeiss). • Monitoring of vital parameters enables continuous assessment of the animal's physiological state during imaging and early detection of stress or compromised well-being.