Before injecting a new drug into pregnant animals or spraying a new pesticide on or near insects, researchers must know how the chemical might affect the organisms. Many chemicals can cause an increase in cell death in developing embryos, which is sometimes detrimental to the development of a fetus. Researchers from the Division of Reproductive Toxicology at the US Environmental Protection Agency in Research Triangle Park, N.C., have discovered a way to look deep within fetal tissue using confocal microscopy to see how various chemicals affect growth and development. Robert M. Zucker, reporting the division’s research in the November issue of Cytometry, explained that the goal was to look both at the morphology of developing mammalian embryos and insect larvae and at the amount of apoptotic cell death occurring within embryos. These two parameters would help the researchers to understand the toxic effect of a chemical on the environment and on the health of a fetus. The image at left reveals the regions of apoptosis in a 10-day-old mouse’s future ear and the one at right shows the back of a mosquito larva head. Confocal microscopy can be used to look at morphological changes in organisms. However, because of their thickness, imaging whole insect or mammalian embryos without physically slicing them into thin sections has proved difficult because of the optical limitations of the microscope. Therefore, researchers have traditionally been using a light microscope in bright-field mode with Nile blue to observe embryo apoptosis. Although this technique can image apoptotic cell death, it cannot show detailed morphological changes within the embryo in the way that confocal microscopy can. The Nile blue staining pattern on live tissue changes or erodes with time because the dye is incompatible with the tissue and cannot be fixed. Now Zucker has found that, with careful specimen preparation combined with different fluorescence dyes, his team could obtain high-quality images of apoptosis in 500-μm-thick embryo tissue using confocal microscopy. To observe fluorescent dyes deep within embryos under a confocal microscope, the dye and tissue should be fixed (preserved) so that they can withstand an alcohol dehydration procedure followed by tissue clearing — which involves adding a solution with a similar refractive index to the embryo to make the tissue nearly transparent. Through careful preparation of tissue samples, researchers discovered that they could view developing embryos using confocal microscopy. The left confocal image reveals the apoptotic regions between the digits of a rat forelimb, and the middle one shows cell death during muscle development in a mouse eye. On the right, the front of a mosquito larva head is pictured. Red and green regions indicate selective responses to fluorescent dyes and reveal the mosquito’s morphological structures. Yellow is the collocalization of the two fluorescent emissions. Zucker found that LysoTracker Red, a paraformaldehyde fixable dye from Invitrogen/Molecular Probes of Eugene, Ore., could handle the fixation/clearing procedure. The fluorescent fixable dye proved more stable than most classical dyes (i.e., Nile blue) that are used with bright-field microscopy for indicating cell death. The red fluorescent dye allowed the researchers to observe the regions of the tissue that were acidic, which indicated high lysosomal activity and phagocytosis — indicators of apoptotic activity. After staining the tissue with the red fluorescent dye, the researchers fixed the tissue with glutaraldehyde and paraformaldehyde, which provided an increased amount of green fluorescence. This made it possible for the researchers to observe background morphological features of the embryo in a green wavelength range. To better observe tissue structures using confocal microscopy, the tissue and suspending medium must have similar refractive indices. The refractive index of tissue is usually about 1.55, whereas it is about 1.3 for water — meaning that a laser could not penetrate the tissue because of the barrier caused by the water in the tissue. The researchers, therefore, dehydrated the tissue with methanol. They removed the methanol from the tissue using a benzyl alcohol/benzyl benzoate solution from Sigma-Aldrich Inc. of St. Louis. Zucker explained that this was the critical step in the procedure that allowed them to observe 500 μm of tissue. The refractive index of the solution was very similar to the refractive index of the embryo, providing an almost transparent embryo. This reduced some of the artifacts contained in the light scattering. The clearing technique enabled them to view the tissue under a confocal microscope with considerably more laser penetration without fluorescent signal attenuation. In a departure from usual confocal microscopy methods, the researchers used a low-power lens to view the tissue on their Leica laser-scanning confocal inverted microscope. Through trial and error, they discovered that a 10x objective with a 0.4 NA enabled the most fluorescent light transmission in the tissue with the least amount of signal attenuation and degradation. Higher-powered lenses, such as a 63x lens, caused too much light scatter/absorption, allowing observation of only about 100 μm into the tissue with a very small field of view, Zucker said. An argon-krypton laser with three emitting wavelengths from Melles Griot of Irvine, Calif., allowed them to excite the red fluorescent dye with a 568-nm line and the glutaraldehyde-paraformaldehyde fixative fluorescence with a 488-nm line. Apoptosis, which appeared as bright red, clearly stood out on the green background, which revealed the morphological structures. The combination of both fluorescent signals allowed the team to detect the location of cell death in fetal limbs and embryos. Similar confocal microscopy procedures were used to observe tissue morphology in insects. Zucker believes that the technique should be very helpful for researchers who want to know how pesticides might affect the normal development of insects and for those who need to investigate how a chemical might affect a mammalian system.