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Drosophila is an important model animal to study connectomics since its brain is complicated and small enough to be mapped by optical microscopy with single-cell resolution. Compared to other model animals, its genetic toolbox is more sophisticated, and a connectome map with single-cell resolution has been established, serving as an invaluable reference for functional connectome study. Two-photon microscopy (2PM) is now the most popular tool to study functional connectome by taking the advantages of low photobleaching, subcellular resolution and deep penetration depth. However, using GFP-labeling with excitation wavelength ~ 920-nm, the reported penetration depths in a living Drosophila brain are limited to ~ 100-μm, which are much smaller than that in living mouse or zebrafish brains. The underlying reason is air vessels, i.e., trachea, instead of blood vessels, are responsible for oxygen exchange in Drosophila brains. The trachea structures induce extraordinarily strong scattering and aberration since the air/tissue refractive index difference is much larger than blood/tissue. By expelling the air inside trachea, whole Drosophila brain can be penetrated by 2PM without difficulty. However, the Drosophila is not alive anymore. Here, three-photon microscopy based on a 1300-nm laser is demonstrated to penetrate a living Drosophila brain with single-cell resolution. The long wavelength intrinsically reduces scattering, when combined with normal dispersion of brain tissue, aberration from trachea/tissue interface is reduced to some extent. As a result, the penetration depth is improved more than twice using 1300-nm excitation. This technique is believed to significantly contribute on functional connectome studies in the future.
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