Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope

Abstract

SUMMARYLight 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).

Developing a Novel Embryo–Larval Zebrafish Xenograft Assay to Prioritize Human Glioblastoma Therapeutics

“…This concentration of anesthetic was selected based on Kaufmann et al for complete immobilization of zebrafish for several hours. 27 Anesthetized xenografts were transferred to a 96-well imaging plate (Greiner BioOne, Monroe, NC) at one fish per well by hand and embedded in 0.1 mL of 0.8% (w/v) low-melt agarose (Promega, Madison, WI) containing 200 mg/L Tricaine. Before agarose solidified, xenograft heads were centered, dorsal side down, at the bottom of the well ( Fig.…”

Animal models of medullary thyroid cancer: state of the art and view to the future

“…In addition to the conventional applications in epifluorescence, confocal and multiphoton microscopy, zebrafish has recently resulted a valid platform for selective plane illumination microscopy (SPIM) (Kaufmann et al 2012), a technique that achieves excellent resolution at high penetration depth in translucent samples. The most prominent advantages of SPIM is the high acquisition speed and the reduced photo-toxicity, making this technique particularly suitable for long-term timelapse experiments in live samples (Huisken & Stainier 2009).…”

Unleashing Optics and Optoacoustics for Developmental Biology

“…As examples of specimens being imaged with high quality using light sheet microscopy, we should point out invertebrates such as D. melanogaster [48], C. elegans [58], and T. castaneum [59], and vertebrates such as zebrafish [47,60] (for very useful information regarding zebrafish preparation for laser sheet imaging see [61]). Of particular importance is the work by Ichikawa et al where light sheet microscopy was applied to a developing mouse embryo, given the complexity of keeping a mouse embryo under controlled CO 2 and temperature conditions [62].…”