bio sphere
Peering into the depths of embryos E
SPIM has limitations. SPIM’s resolution movies show that medaka and fruit fly rnst Stelzer, Jan Huisken, and colis not yet as good as it could be, say ex(Drosophila melanogaster) embryos releagues at the European Molecular perts. Hardin says that although the main alive and develop normally over Biology Laboratory (Germany) have current resolution is fine for the larger several days in the agarose cylinder. developed an inexpensive microscopy medaka embryos, it is not optimal for The muscles of the medaka embryos method that allows researchers to see imaging small fruit fly and nematode that the researchers chose to examine deep inside living embryos (Science embryos. “But this is just 2004, 305, 1007–1009). (a) (b) the first generation of the Called selective plane ildevice, so I fully expect lumination microscopy that this will get better (SPIM), the new technique over time,” he says. Mountpenetrates more deeply ing samples in agarose also into large embryos than could be a limitation in conventional methods, some cases. “If your emsuch as multiphoton and bryo is changing shape confocal laser scanning during the course of filmmicroscopy. ing in such a way that the SPIM uses a focused A multiview reconstruction of (a) a medaka embryo head and (b) a medaka agarose becomes a mesheet of laser light to illumiheart cut open virtually by computer. (Adapted with permission. Copyright chanical constraint to nate a single slice through a 2004 American Association for the Advancement of Science.) morphogenesis, that would fluorescently labeled sample. were labeled with green fluorescent pro- be a problem,” says Hardin. Emitted light is detected by an objective Stelzer plans to improve the technique tein. Sections of the fish were viewed at that is perpendicular to the light sheet. and combine it with other microscopy a resolution of ~6 µm and at a depth of The sample can be imaged, rotated, and methods, such as fluorescence recovery 500 µm. Multiview reconstruction can imaged again from the opposite side in after photobleaching or fluorescence resextend the depth that SPIM can image an optional multiview reconstruction onance energy transfer. “The nice thing to ~3–5 mm, says Huisken. procedure, which merges the individual is that we have an excellent signal-toDepth is the big advantage of SPIM, data sets into a single high-resolution noise ratio that is really a dramatic differsay experts. According to Pawley, al3-D image. ence in comparison to other technolothough multiphoton microscopy can James Pawley at the University of Wisgies, and we have a relatively high speed consin says, “The light sheet is a very clev- also illuminate and image a single sec[of detection],” says Stelzer. These proption of an embryo, it cannot penetrate er idea.” He notes that SPIM is based erties make SPIM an ideal partner for as far as SPIM. “In addition, because on the same principles as the slit lamps other visualization methods. [SPIM] is a parallel image technique, it used by ophthalmologists, although Because SPIM provides information can image each plane quite quickly, and SPIM works on a much smaller scale. at both the organism and cellular levels, this is important when imaging living After crafting the SPIM instrument Stelzer says SPIM may help scientists embryos,” says Pawley. Jeff Hardin at from common, off-the-shelf parts, Stelsee things in ways they never dreamed the University of Wisconsin says, “I zer and Huisken struck up a collaborapossible. “SPIM should be regarded as a think [SPIM] is going to be really good tion with developmental biologists at tool that can be used for true 3-D cell for looking at big embryos, and by big, their institution who were studying biology,” he says. “I really think that I mean things that are on the order of a medaka fish (Oryzias latipes). Samples [looking at] flat specimens, such as fimillimeter.” Hardin was particularly imwere mounted in a 0.5% agarose cylinbroblasts on coverslips, is not really the pressed with the images the researchers der. “The agarose just acts as a scaffold kind of cell biology people should do acquired of medaka hearts and says this that spatially fixes the sample so that it because in our body, the cells are simply “would have been really hard with any doesn’t move anymore, and we can not flat; they are 3-D objects.” a other technique in living embryos.” move it through the light sheet,” says —Katie Cottingham Like any new analytical technique, Huisken. He adds that time-lapse
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