Here we describe a relatively inexpensive and easy method to produce high quality images that reveal fine topological details of vertebrate embryonic structures. Electron Microscopy (SEM). The fluorescent nuclear staining may be performed with a variety of cell permeable nuclear dyes enabling the technique to be performed with multiple standard microscope/illumination or confocal/laser CI-1011 systems. The method may be used to document morphology of embryos of a variety of organisms as well as individual organs and tissues. Nuclear stain imaging imposes minimal impact on embryonic specimens enabling imaged specimens to be utilized for additional assays. frog embryos that have been bleached with H202(Wallingford 2010 The efficacy of the technique may be limited in embryos at older stages of development as the skin matures and begins to form a barrier with reduced permeability which in the case of mouse embryos occurs at embryonic day 16.5 (E16.5) (Hardman et al. 1998 For mouse embryos we find that nuclear stain penetration is successful in whole mount specimens through E15.5 (Fig 2D). For zebrafish and chick we have found nuclear staining to be effective until at least day 5 and day 9 respectively. High quality whole mount nuclear stained embryo images may be achieved by staining with a variety of nuclear dyes (Table 1). The choice of dye depends primarily on the illumination and filter CI-1011 options available for microscopy and imaging. DAPI or Hoechst dyes can be used for imaging on any fluorescent microscope or stereomicroscope that has fluorescence CI-1011 illumination and a UV filter or on a confocal microscope with a 405nm laser and a bandpass filter for emission of violet wavelength light. For confocal microscopes that have far-red laser/filter combinations far-red nuclear stains such as Draq5 (Cell Signaling Technology) or Red-Dot (Biotium) may Rabbit polyclonal to Sp2. be used. For our studies we have used membrane permeant dyes. Membrane impermeant dyes such as Ethidium bromide and Propidium iodide have been used by others. (Hu and Marcucio 2009 Zucker 2006 Zucker et al. 1999 Table 1 Examples of nuclear stains that can be used for Pseudo-SEM imaging of vertebrate embryos and embryonic tissues. With respect to mode of microscopy conventional fluorescent microscopy has advantages over confocal microscopy in terms of speed ease accessibility and modest digital file size. Conventional fluorescent microscopy can yield images that while not of confocal quality are distinctly superior to those obtained by brightfield microscopy (Fig. 1b Fig. 2 b d e Fig. 3 e). Also since many confocal systems are not set up for low CI-1011 magnification imaging conventional fluorescence stereomicroscopy is often the most appropriate choice for large specimens such as whole mouse embryos E12.5 or older or any specimen for which relatively low magnification is required. Confocal microscopy is superior to conventional microscopy in terms of apparent depth of field resolution and contrast which together yield images of exceptional clarity and exquisite morphological detail. The disadvantages of confocal microscopy are the lengthy time requirement to capture the needed z-stack CI-1011 of images and the large data storage size of the resulting multiple image files. For many embryo specimens excellent detail can be obtained using a 10× objective which depending on the optics of the microscope can allow a field of view in the X-Y axis sufficient to collect an entire E9.5 mouse embryo in a single frame. For larger specimens lower power objectives such as 5× or 2.5× may be used or multiple z-stack frames may be collected with a 10× objective and subsequently spliced into a single composite image. A key component in generating a good pseudo-SEM image CI-1011 from a confocal z-stack is properly defining the top and bottom optical slice position when setting up the parameters for collecting an image stack. The position of the top and bottom optical slice must be defined to avoid cropping the specimen in the z-axis (Fig. 4 a-b). In practice this can be accomplished by defining the first and last optical section according to the focal positions at the starting and ending limits of detection of nuclear staining. In addition when setting the confocal microscopy acquisition parameters it is important to keep sufficient overlap.