bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/045187

Light sheet microscopy for everyone? Experience of building an OpenSPIM to study flatworm development

Johannes Girstmair·Anne Zakrzewski·François Lapraz·Mette Handberg-Thorsager·Pavel Tomancak·Peter Gabriel Pitrone·Fraser Simpson·Maximilian J Telford
View original source ↗Export metadata ↓

Abstract

BACKGROUNDSelective plane illumination microscopy (SPIM a type of light-sheet microscopy) involves focusing a thin sheet of laser light through a specimen at right angles to the objective lens. As only the thin section of the specimen at the focal plane of the lens is illuminated, out of focus light is naturally absent and toxicity due to light (phototoxicity) is greatly reduced enabling longer term live imaging. OpenSPIM is an open access platform (Pitrone et al. 2013 and OpenSPIM.org) created to give new users step-by-step instructions on building a basic configuration of a SPIM microscope, which can in principle be adapted and upgraded to each laboratorys own requirements and budget. Here we describe our own experience with the process of designing, building, configuring and using an OpenSPIM for our research into the early development of the polyclad flatworm Maritigrella crozieri - a non-model animal.\n\nRESULTSOur OpenSPIM builds on the standard design with the addition of two colour laser illumination for simultaneous detection of two probes/molecules and dual sided illumination, which provides more even signal intensity across a specimen. Our OpenSPIM provides high resolution 3d images and time lapse recordings, and we demonstrate the use of two colour lasers and the benefits of two color dual-sided imaging. We used our microscope to study the development of the embryo of the polyclad flatworm Maritigrella crozieri. The capabilities of our microscope are demonstrated by our ability to record the stereotypical spiral cleavage pattern of Maritigrella with high-speed multi-view time lapse imaging. 3D and 4D (3D + time) reconstruction of early development from these data is possible using image registration and deconvolution tools provided as part of the open source Fiji platform. We discuss our findings on the pros and cons of a self built microscope.\n\nCONCLUSIONSWe conclude that home-built microscopes, such as an OpenSPIM, together with the available open source software, such as MicroManager and Fiji, make SPIM accessible to anyone interested in having continuous access to their own light-sheet microscope. However, building an OpenSPIM is not without challenges and an open access microscope is a worthwhile, if significant, investment of time and money. Multi-view 4D microscopy is more challenging than we had expected. We hope that our gained experience during this project will help future OpenSPIM users with similar ambitions.

Source connections

Explore related subjects

Developmental BiologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Johannes Girstmair, Anne Zakrzewski, François Lapraz, Mette Handberg-Thorsager, Pavel Tomancak, Peter Gabriel Pitrone, Fraser Simpson, Maximilian J Telford. 2016-03-23. Light sheet microscopy for everyone? Experience of building an OpenSPIM to study flatworm development. https://doi.org/10.1101/045187

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Neural tracking of stressed syllables in Dutch nursery rhymes relates to vocabulary outcomes in a large, longitudinal sample

Speech consists of regularities at different timescales. Already during infancy, neural electrophysiological activity aligns to these rhythms. The degree to which infants exhibit neural tracking of speech can be linked to their language development. In this study, we examined how the neural tracking of sung speech develops across age, from infancy to early childhood, and across different frequency bands (i.e., at the stress, syllabic, and phonemic rates), and whether neural tracking at each frequency and age predicts childrens language outcomes. We included 2565 children of the longitudinal YOUth cohort. Children listened to Dutch sung nursery rhymes while EEG was recorded at three measurement waves. After preprocessing the data, we included 955 children at 5 months, 1048 children at 10 months, and 795 children at 2-4 years. The final sample consisted of 750 children who also completed a receptive vocabulary test at 2-4 years. Children from 5 months onwards showed significant neural tracking of stressed syllables, syllables, and phonemes, measured with speech-brain coherence (SBC). Unexpectedly, there were no developmental changes in SBC across different frequency bands from infancy to early childhood. As expected, children with larger receptive vocabularies showed increased SBC in the stressed syllable rate. These findings suggest that stronger tracking of stressed syllables is related to individual differences in language ability.

Developmental Biology↗
bioRxiv2026

FGF Signaling Potentiates Müller Glia for Mammalian Retinal Regeneration

Muller glia possess latent regenerative potential that could be harnessed to restore retinal neurons lost to injury or disease. Although fibroblast growth factor (FGF) signaling is upregulated following retinal damage, its role in mammalian retinal regeneration remains unclear. Here, we investigated the function of FGF signaling in Muller glial reprogramming using genetic, pharmacological, and single-cell transcriptomic approaches. Activation of FGF signaling alone was insufficient to induce Muller glial proliferation in the mouse retina. However, conditional deletion of FGFR1/2 in Muller glia abolished regenerative responses induced by multiple independent pathways, demonstrating that FGF signaling is essential for regenerative competence. Mechanistically, loss of FGF signaling impaired sustained ERK/MAPK activation following injury, while MEK/ERK inhibition phenocopied the regenerative defect. Conversely, constitutive MEK activation induced limited Muller glial proliferation in the absence of injury. Although STAT3/5 inhibition synergized with Activin-A to promote robust proliferation and neurogenic gene expression, it failed to rescue regeneration in FGF-deficient Muller glia. Single-cell RNA sequencing revealed that FGF signaling suppresses multiple anti-regenerative programs, including Hes1, S1pr1, p27, NF-{kappa}B, and p300/CBP activity. Together, these findings identify FGF signaling as a critical permissive regulator of mammalian retinal regeneration that potentiates Muller glia through sustained ERK activation and suppression of transcriptional barriers to regeneration.

Developmental Biology↗
bioRxiv2026

Doublesex- mediated regulation of insulin signaling drives sex-specific body growth

Most animals develop sex-biased body sizes driven by sexually divergent plasticity in nutrient-dependent growth. Prior work in Drosophila, implicated the sex-determination gene transformer (tra) in regulation of sex differences in body size. However, tra does not widely mediate sex determination across insects. Thus, the unifying molecular pathway governing female-biased sexual size dimorphism (SSD) remains poorly established in non-drosophilid insects. The rice stem borer Chilo suppressalis, a devastating lepidopteran crop pest, exhibits a robust SSD, offering an ideal system to dissect underlying mechanisms. We report that female-specific splice forms of the sex-determining gene doublesex (Csdsx) are master regulators of female-biased growth. Disruption of the female-specific Csdsx exon 3 via CRISPR knockout or RNA interference drastically reduces female body size and completely erases the SSD. Csilp2, encoding a key insulin-like peptide (ILP2), is selectively upregulated in late-instar female larvae, and female CsDsx proteins directly bind and activate the Csilp2 promoter to boost transcription. Loss-of-function of Csilp2 eliminates the SSD by suppressing female somatic overgrowth. Our results identify a novel regulatory cascade: female-specific Dsx directly stimulates insulin signaling via Csilp2, bridging core sex-determination circuitry and nutrient-dependent body growth control. SignificanceSexual size dimorphism (SSD), widespread across insects with larger females, strongly shapes reproductive fitness. Yet reports on the molecular connection between sex determination and dimorphic growth are scarce. Using a major agricultural pest, the rice stem borer with a prominent female-biased SSD, we show that female-specific Doublesex (dsx) splice variants directly activate the promoter of Insulin-like peptide 2 in late larvae to trigger increased growth. Knocking out either gene in females abolishes the SSD. Our findings thus establish dsx as a novel link connecting sex-determination, insulin signaling and nutrient-dependent body growth.

Developmental Biology↗