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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Nat Protoc. Author manuscript; available in PMC 2010 September 10.
Published in final edited form as:
Nat Protoc. 2009; 4(10): 1397–1412.
Published online 2009 September 10. doi: 10.1038/nprot.2009.130

Figure 2

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Optical properties of mesoderm in early Drosophila and zebrafish embryos. Using brightfield microscopy, wild-type Drosophila embryos (a) at stage 8 (s8) appear darker than klarsicht mutants (b), which is due to the different light scattering properties of the cells. The experimental quantification of these optical properties is performed as explained in Box 1. Each fluorescent imaging dataset is analyzed by plotting G(z) (see Box 1 for its definition) and fitting the experimental data using a linear regression (c). This analysis allows estimation of the scattering mean free path lsex of the excitation light from embryonic tissues at different stages or from different species (d). The graph (d) shows that klarsicht Drosophila embryos (red) exhibit lsex ~ 76 μm, which is 20 μm larger than wild-type embryos at the same stage (blue). The error bars represent the standard deviation of the lsex estimations for N=8 embryos. Previous studies show that similar measurements performed in Drosophila blastoderm cells at stage 5 (s5) and in Zebrafish mesoderm cells at bud stage (10hpf, hours post fertilization) result in lsex two and three times larger, respectively11, 17 (dark and light gray in d, respectively). These measures are used to plot the typical 2PEF signal decay in depth (e) as explained in Box 1. This graph displays the loss of fluorescence signal when imaging deeper inside an embryonic tissue and permits comparison of the expected signal loss observed in tissues with different optical properties. It shows that the difference in optical properties between wild-type Drosophila (blue curve) and klarsicht (red curve) s8 embryos is significant, and results in the ability to obtain twice the fluorescent signal at 80 μm within klarsicht embryos. 80 μm is the position of the deepest mesoderm cells in this embryo. It also shows that the signal is 3 times higher in wild-type Drosophila at s5, and 5 times higher in zebrafish embryos under similar imaging conditions (dark and light gray curves at 80 μm, respectively). Scale bar in (a) indicates 50μm. wt: wild-type

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