New mounting technique opens the door to long-term imaging with SPIM [with video]

Photo damage and inappropriate mounting strategies limit the duration of in vivo imaging in conventional microscopy techniques. Combining light sheet microscopy with optimized mounting methods allows physiological imaging over days.

HFSP Career Development Award holder Jan Huisken and colleagues
authored on Tue, 30 October 2012

Light sheet microscopy (also known as selective plane illumination microscopy (SPIM)) has become a popular tool for studying developmental processes in living organisms. Due to the gentle illumination with a thin sheet of light, photo toxicity is dramatically reduced compared to conventional methods such as confocal microscopy, making SPIM ideal for imaging embryos over long periods of time with high spatial and temporal resolution. However, up to now the success of light sheet microscopy has been challenged by the lack of physiological mounting techniques adequate for experiments that last many hours or days.

Commonly, in SPIM, just as in many other microscopy techniques, the specimen has been embedded in agarose, which provides enough stiffness to hold the sample precisely in place. However, time lapse imaging of embryos embedded in a rigid scaffold, such as agarose, leads to severe growth restrictions and developmental defects. Therefore, to exploit the full potential of SPIM, we have developed new mounting strategies optimized to provide immobilization and growth at the same time.

We have evaluated the development of zebrafish embryos in different mounting media and tested their suitability for SPIM. We found that mounting the sample in low concentrations of agarose or methylcellulose in refractive-index-matched fluorinated ethylene propylene (FEP) tubes ensures both immobility and growth of the embryo as well as optimal image quality from all sides. Our paper provides mounting protocols for the first days of zebrafish development for light sheet microscopy and other long-term imaging techniques.

Movie 1: Developing zebrafish embryo with vascular marker Tg(kdrl:GFP) from 24-96 hpf.
The embryo was mounted in 0.1% agarose with 200 mg/l Tricaine in an FEP enclosure coated with 3% methylcellulose. Stacks were recorded every 10 minutes with SPIM in two channels, transmission illumination (bottom) and 488-nm excitation (top). Scale bar: 300 µm.

We have shown that dedicated sample mounting techniques are a key component in the application of current and future developments in microscopy. Our results will also be applicable to other multi-angle imaging techniques, such as tomography (e.g. OPT).

Movie 2:Developing zebrafish embryo with ubiquitous nuclei marker Tg(H2A:GFP) from 8-20 hpf.
The embryo was left in the chorion and mounted in an FEP tube filled with fish medium. The inner diameter of the tube is slightly smaller than the chorion, such that the chorion and by extension the embryo is held in place. Additionally, the embryo was imaged from 2 different angles simultaneously (compare left and right).

Text by Michaela Mickoleit


Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope.  Kaufmann, A., Mickoleit, M., Weber, M. & Huisken.  J. Development 139, 3242–3247 (2012).

PubMed link