915868.fig.003a
(a)
915868.fig.003b
(b)
915868.fig.003c
(c)
915868.fig.003d
(d)
915868.fig.003e
(e)
915868.fig.003f
(f)
Figure 3: Direct in vivo measurement of tissue neutrophil lifespan. Neutrophils were photoconverted using the PhotoKinesis device on the Laser Confocal Imaging System (Perkin Elmer Inc.). 40% 405 nm laser power for 120 cycles was used to photoconvert 3 individual tissue neutrophils in each of 32 fish at 3 dpf. The images shown above were selected as examples of larvae with a range of photoconverted neutrophils visible. Images were selected from an image series covering 0–48 hours after photoconversion. Images are Z-stacks taken using a 2x objective. (a) Larvae with no photoconverted neutrophils visible. (b) Larva with 3 photoconverted neutrophils present in the tail region. (c) Larva with 2 photoconverted neutrophils present in the tail region. (d) Larva with one photoconverted neutrophil on the dorsal aspect. (e) The number of photoconverted neutrophils was counted from Z-stack images taken at 0, 24, and 48 hours after photoconversion using a 2x objective lens. 160 larvae were used in total over 5 experimental repeats. The data were fitted with a linear regression, with error bars indicating standard error mean (SEM). (f) The number of photoconverted neutrophils was counted from Z-stack images taken at 0, 24, and 48 hours after photoconversion using a 2x objective lens. 30% and 60% laser power was repeated twice, and the 40% laser 5 times resulting in a total of 288 larvae being included in the analysis. 𝑃 < 0 . 0 5 for the difference between remaining neutrophils identified at 24 hours for 60% laser power and 40% and 30% laser power (2-way ANOVA). There is no significant difference in the percentage neutrophils remaining at 48 hours after photoconversion. Mean ± SEM is shown.