Kaufmann, A., Mickoleit, M., Weber, M., and Huisken, J. (2012) Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope. Development (Cambridge, England). 139(17):3242-3247.
Light sheet microscopy techniques, such as selective plane illumination microscopy (SPIM), are ideally suited for time-lapse
imaging of developmental processes lasting several hours to a few days. The success of this promising technology has mainly
been limited by the lack of suitable techniques for mounting fragile samples. Embedding zebrafish embryos in agarose, which
is common in conventional confocal microscopy, has resulted in severe growth defects and unreliable results. In this study,
we systematically quantified the viability and mobility of zebrafish embryos mounted under more suitable conditions. We found
that tubes made of fluorinated ethylene propylene (FEP) filled with low concentrations of agarose or methylcellulose provided
an optimal balance between sufficient confinement of the living embryo in a physiological environment over 3 days and optical
clarity suitable for fluorescence imaging. We also compared the effect of different concentrations of Tricaine on the development
of zebrafish and provide guidelines for its optimal use depending on the application. Our results will make light sheet microscopy
techniques applicable to more fields of developmental biology, in particular the multiview long-term imaging of zebrafish
embryos and other small organisms. Furthermore, the refinement of sample preparation for in toto and in vivo imaging will
promote other emerging optical imaging techniques, such as optical projection tomography (OPT).