TY  - JOUR
TI  - In Vivo Light-Sheet Imaging of Senescence Reporter Activity in a Transparent Killifish
AU  - Perner, Birgit 
AU  - Englert, Christoph 
VL  - 16
IS  - 14
PY  - 2026
DA  - 2026/07/20
SP  - e5762
C1  - Bio-protocol 2026;16:e5762
DO  - 10.21769/BioProtoc.5762
UR  - https://doi.org/10.21769/BioProtoc.5762
AB  - 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.
KW  - Light-sheet microscopy
KW  - Intravital imaging
KW  - Nothobranchius furzeri
KW  - Aging model
KW  - cdkn1a (p21) reporter
KW  - Transparent killifish
KW  - Cellular senescence
KW  - Fluorescence microscopy
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False