Culturing murine embryonic organs: Pros, cons, tips and tricks

McClelland, Kathryn S.; Bowles, Josephine

doi:10.1016/j.diff.2016.01.008

Cited by 7 publications

(5 citation statements)

References 29 publications

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“…This has enabled researchers to image real-time biological phenomena such as flow dynamics in naturally optically transparent zebrafish embryonic hearts (Fei et al, 2016). Ex vivo organ culture methods have been used for decades in developmental biology (McClelland and Bowles, 2016). LSFM/SPIM of cultured murine or human embryonic genital tubercles could illuminate real-time external genital development in controlled environments rather than simply at single points in development.…”

Section: Discussionmentioning

confidence: 99%

Imaging the developing human external and internal urogenital organs with light sheet fluorescence microscopy

et al. 2020

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Technological advances in three-dimensional (3D) reconstruction techniques have previously enabled paradigm shifts in our understanding of human embryonic and fetal development. Light sheet fluorescence microscopy (LSFM) is a recently-developed technique that uses thin planes of light to optically section whole-mount cleared and immunolabeled biologic specimens. The advent of commercially-available light sheet microscopes has facilitated a new generation of research into protein localization and tissue dynamics at extremely high resolution. Our group has applied LSFM to study developing human fetal external genitalia, internal genitalia and kidneys. This review describes LSFM and presents our group's technique for preparing, clearing, immunostaining and imaging human fetal urogenital specimens. We then present light sheet images and videos of each element of the developing human urogenital system. To the extent of our knowledge, the work conducted by our laboratory represents the first description of a method for performing LSFM on the full human urogenital system during the embryonic and fetal periods.

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Section: Discussionmentioning

confidence: 99%

Imaging the developing human external and internal urogenital organs with light sheet fluorescence microscopy

et al. 2020

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“…(d) Finally, the low levels of energy to which specimens are exposed in light sheet microscopy enable non-invasive imaging of live organisms or tissues (Fei et al, 2016). Ex vivo tissue and organ culture methods are well-established in developmental biology (McClelland and Bowles, 2016). The Light sheet Z.1 device has chambers equipped for specimen incubation and gas exchange.…”

Section: Discussionmentioning

confidence: 99%

Three-dimensional imaging of the developing human fetal urogenital-genital tract: Indifferent stage to male and female differentiation

et al. 2018

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Recent studies in our lab have utilized three imaging techniques to visualize the developing human fetal urogenital tract in three dimensions: optical projection tomography, scanning electron microscopy and lightsheet fluorescence microscopy. We have applied these technologies to examine changes in morphology and differential gene expression in developing human external genital specimens from the ambisexual stage (<9 weeks fetal age) to well-differentiated male and female organs (>13 weeks fetal age). This work outlines the history and function of each of these three imaging modalities, our methods to prepare specimens for each and the novel findings we have produced thus far. We believe the images in this paper of human fetal urogenital organs produced using lightsheet fluorescence microscopy are the first published to date.

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“…Agarose can easily be melted to a liquid and then solidified by cooling to room temperature. This approach is favored for samples such as whole organs or whole organisms (McClelland & Bowles, 2016; McClelland, Ng, & Bowles, 2016; Swoger, Pampaloni, & Stelzer, 2014). The embedding material should not be excessively heated to the point where it will damage a living specimen.…”

Section: Arrangements For Holding the Samplementioning

confidence: 99%

“…(2016) have respectively published protocols for studying gastrulation and the mouse intestine with the lightsheet microscope. Nevertheless, the eventual size of the mouse embryo and the fact that it cannot be immobilized for lengthy periods (Xavier da Silveira Dos Santos & Liberali, 2019) is a challenge peculiar to mouse developmental biology, particularly when imaging adult mice or complete organs (McClelland & Bowles, 2016). Phillipp Keller at the Janelia Research Campus has had some success (McDole et al., 2018; Strack, 2018) in doing this by using a custom sample chamber for longitudinal embryo culturing and automated software to keep the growing embryo in focus.…”

Section: Background Informationmentioning

confidence: 99%

Lightsheet Microscopy

et al. 2022

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In this paper, we review lightsheet (selective plane illumination) microscopy for mouse developmental biologists. There are different means of forming the illumination sheet, and we discuss these. We explain how we introduced the lightsheet microscope economically into our core facility and present our results on fixed and living samples. We also describe methods of clearing fixed samples for three-dimensional imaging and discuss the various means of preparing samples with particular reference to mouse cilia, adipose spheroids, and cochleae.

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Culturing murine embryonic organs: Pros, cons, tips and tricks

Cited by 7 publications

References 29 publications

Imaging the developing human external and internal urogenital organs with light sheet fluorescence microscopy

Imaging the developing human external and internal urogenital organs with light sheet fluorescence microscopy

Three-dimensional imaging of the developing human fetal urogenital-genital tract: Indifferent stage to male and female differentiation

Lightsheet Microscopy

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