Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

Max Planck Institute of Molecular Cell Biology and Genetics

The piRNA (PIWI-interacting RNA) pathway is a small RNA silencing system that acts in animal gonads and protects the genome against the deleterious influence of transposons. A major bottleneck in the field is the lack of comprehensive knowledge of the factors and molecular processes that constitute this pathway. We conducted an RNAi screen in Drosophila and identified ~50 genes that strongly impact the ovarian somatic piRNA pathway. Many identified genes fall into functional categories that indicate essential roles for mitochondrial metabolism, RNA export, the nuclear pore, transcription elongation, and chromatin regulation in the pathway. Follow-up studies on two factors demonstrate that components acting at distinct hierarchical levels of the pathway were identified. Finally, we define CG2183/Gasz as an essential primary piRNA biogenesis factor in somatic and germline cells. Based on the similarities between insect and vertebrate piRNA pathways, our results have far-reaching implications for the understanding of this conserved genome defense system.

The genetic makeup of the Drosophila piRNA pathway.

Light sheet microscopy is an emerging technique allowing comprehensive visualization of dynamic biological processes, at high spatial and temporal resolution without significant damage to the sample by the imaging process itself. It thus lends itself to time-lapse observation of fluorescently labeled molecular markers over long periods of time in a living specimen. In combination with sample rotation light sheet microscopy and in particular its selective plane illumination microscopy (SPIM) flavor, enables imaging of relatively large specimens, such as embryos of animal model organisms, in their entirety. The benefits of SPIM multiview imaging come to the cost of image data postprocessing necessary to deliver the final output that can be analyzed. Here, we provide a set of practical recipes that walk biologists through the complex processes of SPIM data registration, fusion, deconvolution, and time-lapse registration using publicly available open-source tools. We explain, in plain language, the basic principles behind SPIM image-processing algorithms that should enable users to make informed decisions during parameter tuning of the various processing steps applied to their own datasets. Importantly, the protocols presented here are applicable equally to processing of multiview SPIM data from the commercial Zeiss Lightsheet Z.1 microscope and from the open-access SPIM platforms such as OpenSPIM.

Open-source solutions for SPIMage processing.

To study the development and interactions of cells and tissues, multiple fluorescent markers need to be imaged efficiently in a single living organism. Instead of acquiring individual colours sequentially with filters, we created a platform based on line-scanning light sheet microscopy to record the entire spectrum for each pixel in a three-dimensional volume. We evaluated data sets with varying spectral sampling and determined the optimal channel width to be around 5 nm. With the help of these data sets, we show that our setup outperforms filter-based approaches with regard to image quality and discrimination of fluorophores. By spectral unmixing we resolved overlapping fluorophores with up to nanometre resolution and removed autofluorescence in zebrafish and fruit fly embryos.

Hyperspectral light sheet microscopy.

Selective Plane Illumination Microscopy (SPIM) allows to image developing organisms in 3D at unprecedented temporal resolution over long periods of time. The resulting massive amounts of raw image data requires extensive processing interactively via dedicated graphical user interface (GUI) applications. The consecutive processing steps can be easily automated and the individual time points can be processed independently, which lends itself to trivial parallelization on a high performance computing (HPC) cluster. Here, we introduce an automated workflow for processing large multiview, multichannel, multiillumination time-lapse SPIM data on a single workstation or in parallel on a HPC cluster. The pipeline relies on snakemake to resolve dependencies among consecutive processing steps and can be easily adapted to any cluster environment for processing SPIM data in a fraction of the time required to collect it.

An automated workflow for parallel processing of large multiview SPIM recordings.

The Drosophila genome contains >13000 protein-coding genes, the majority of which remain poorly investigated. Important reasons include the lack of antibodies or reporter constructs to visualise these proteins. Here, we present a genome-wide fosmid library of 10000 GFP-tagged clones, comprising tagged genes and most of their regulatory information. For 880 tagged proteins, we created transgenic lines, and for a total of 207 lines, we assessed protein expression and localisation in ovaries, embryos, pupae or adults by stainings and live imaging approaches. Importantly, we visualised many proteins at endogenous expression levels and found a large fraction of them localising to subcellular compartments. By applying genetic complementation tests, we estimate that about two-thirds of the tagged proteins are functional. Moreover, these tagged proteins enable interaction proteomics from developing pupae and adult flies. Taken together, this resource will boost systematic analysis of protein expression and localisation in various cellular and developmental contexts.

Christopher Schmied

Max Planck Institute of Molecular Cell Biology and Genetics

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development