Single Cell Ablation in the Zebrafish Kidney using SPIM and a Bessel Beam

Abstract

Selective plane illumination microscopy (SPIM) uses low‐powered laser light to rapidly and precisely capture high‐resolution images of cells within live organisms, revealing fine details of the cell structure. By combining a bespoke SPIM/Bessel beam in combination with appropriate fluorescent cell‐specific reporters, individual cells can be located and targeted for ablation via apoptosis.Incorporation of fluorescent reporters such as LifeAct‐red fluorescent protein (RFP) or the photosensitizing protein, KillerRed (KR), into the genome of the optically clear zebrafish larvae, allows specifically labelled cells to be visualized and/or optically ablated deep within developing organs and in real‐time. We have used this technology to visualize and target a) cells of the renin lineage (CoRLs)1 and b) podocytes, within the live Zebrafish kidney. Transgenic fish expressing KR under the control of the renin and podocin promoters were established and used independently for imaging/ablation experiments. Using a Bessel beam in combination with SPIM to precisely activate KR allows relatively low laser power to be employed resulting in 100% viability of the fish following laser treatment. Low energy laser ablation allows the cell injury/ablation to be imaged and assessed over an extensive time period (hours).Support or Funding InformationBHF Center of Research Excellence Award; Kidney Research UK award; EPSRC/MRC DT (Optima) AwardThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.