specimens such as embryos, confocal microscopy is essential to
visualize diffraction-limited objects. Wide-field fluorescence
microscopy, used in many studies of cultured cells, is inappro-
priate because of the overwhelming signal from out-of-focus
planes.- Voxel size: For most applications, diffraction-limited objects
are most effectively detected by our custom MATLAB software
in images obtained with pixels of 7676 nm and a z increment
of between 250 and 420 nm (seeNote 22). - Total section thickness: We typically collect image stacks span-
ning 10–20μminz(seeNote 23).
Fig. 1Single molecule mRNA detection in Drosophila embryos. (a)Low magnification image of an embryo
approximately 2 h after fertilization following the twelfth nuclear division cycle and labeled with probes
complementary to the genehunchback(hb). Embryo is oriented with anterior to the left.Green:hbexpression;
magenta: DAPI stain to highlight nuclei. (b) Single confocal slice through the nuclear layer illustrating nascent
transcription sites and single mRNAs. (c) Series of adjacent confocal slices surrounding a single mRNA
illustrating detection on multiple z planes.Scalebars:50μm(a), 5μm(b), 2μm(c)
136 Shawn C. Little and Thomas Gregor