Larval and adult zebrafish were anaesthetised and mounted in 1% low-gelling agarose (Sigma). Live imaging was performed on a ZEISS Lightsheet Z.1 system with a 40x W Plan Apochromat objective or a Leica TCS SP8 AOBS confocal laser scanning microscope with a 25x/0,95 W HC FLUOTAR objective with resonant scanner (frame intervals of 0.02-0.04 seconds). To image free particle movement in larvae, synthetic EVs containing a Cy5 conjugated microRNA (cel-miR-39-3p; not present in zebrafish 38 ) were microinjected into the pericardial space. Adult hearts were dissected, fixed in 4% Paraformaldehyde and embedded and imaged as above with the conventional confocal scanner. Images were processed using Fiji 39 (link), IMARIS (version 9.5.0, Oxford Instruments, United Kingdom) and Huygens Professional (version 16.10, Scientific Volume Imaging, The Netherlands). Deconvolved images are noted in the figure legends. For manual counts of EVs, all analysis was blinded and positive events were counted from 1-minute videos of the peripheral circulation.
Lightsheet z 1 system
Manufactured by Leica
The Lightsheet Z.1 system is a high-performance light sheet fluorescence microscope designed for fast and gentle imaging of large specimens. It utilizes a thin sheet of light to selectively illuminate the sample, enabling optical sectioning and reducing phototoxicity. The system is optimized for long-term, live-cell imaging and comprehensive 3D visualization of intact samples.
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2 protocols using lightsheet z 1 system
1
Live Imaging of Zebrafish Extracellular Vesicles
2
Visualizing Extracellular Vesicles in Zebrafish
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Larval and adult zebrafish were anesthetized and mounted in 1% low-gelling agarose (Sigma). Live imaging was performed on a ZEISS Lightsheet Z.1 system with a 40× W Plan Apochromat objective or a Leica TCS SP8 AOBS confocal laser scanning microscope with a 25×/0.95 W HC FLUOTAR objective with resonant scanner (frame intervals of 0.02–0.04 seconds). To image free particle movement in larvae, synthetic EVs containing a Cy5 conjugated microRNA (cel-miR-39-3p; not present in zebrafish38 ) were microinjected into the pericardial space. Adult hearts were dissected, fixed in 4% paraformaldehyde, and embedded and imaged as above with the conventional confocal scanner. Images were processed using Fiji,39 (link) IMARIS (version 9.5.0, Oxford Instruments, United Kingdom), and Huygens Professional (version 16.10, Scientific Volume Imaging, The Netherlands). Deconvolved images are noted in the figure legends. For manual counts of EVs, all analysis was blinded and positive events were counted from 1-minute videos of the peripheral circulation.
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