Fig. 5.

CARS images compared with conventional fluorescent lipid dyes on fixed eggs. (A-D) DIC (using a 1.27 NA water objective and a 1.4 NA oil condenser), (E-H) false-coloured CARS images at wavenumber 2850 cm⁻¹ and (I-L) TPF xy images, accompanied by (M-P) false-coloured overlays, of GV and MII eggs stained with BODIPY (n=23 and 30, respectively) or LipidTox green neutral lipid stain (n=7 and 20, respectively). All images are maximum intensity projections of stacks with 0.5 µm z-steps. 0.1×0.1 µm pixel size; 0.01 ms pixel dwell time; ∼12 mW (∼9 mW) pump (Stokes) power at the sample. CARS and TPF signal intensities are given in photoelectrons per second (ph.e⁻/s). Scale bars: 10 µm. (Q-T) Scatterplots of pixel-coordinate correlation between CARS (x-axis) and TPF images (y-axis), and the mean Pearson's correlation coefficients of all investigated eggs, to show the degree of reliability of these dyes. A colocalisation is apparent with a Pearson's coefficient of >0.5, but is only considered significant if >0.95 as in accordance with normal 95% confidence limits. Data from ≥4 trials, using 1-3 mice each.