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Correlative light and electron microscopy techniques are powerful tools that allow the identification and analysis of fluorescently labeled cells and structures of interest by an electron microscope (EM).1–3 Nevertheless, employing the methods listed above for the identification of rare cell populations can be very time consuming.4 A recently described method combines imaging of a fluorescently labeled tissue before fixation, with laser etching at the region of interest after embryo fixation.5 While the region of interest in the zebrafish embryo could be better focused on, this technique does not assist the identification of rare cell populations or specific cellular compartments when adjacent morphological landmarks are not present. In this study, we present a procedure based on a recently described method6 that allows effective identification and EM analysis of a small group of cells within the embryo, without relying on distinct morphological markers (Fig. 1). We demonstrate the merit of this procedure using zebrafish primordial germ cells, a population of less than 25 cells within early embryos, which at that developmental stage (8–12 hours post fertilization [hpf]) consist of more than 20,000 cells.7,8 This method relies on preservation of the fluorescent protein signal after fixation, combined with two-photon laser marking within the tissue at defined locations relative to the position where the cells of interest reside.    FIG. 1.  Schematic workflow of the correlative light and electron microscopy using laser marking. (A) Zebrafish embryo fixed in 2% PFA + 0.2% glutaraldehyde in 0.1 M PHEM buffer for 10 min, deyolked and fixed for an additional 2 h ...

Correlative Light and Electron Microscopy of Rare Cell Populations in Zebrafish Embryos Using Laser Marks