Recent advances in cell biology and genetic tools have allowed researchers to explore the architecture of developing organisms like never before. Model organisms, such as zebrafish, Drosophila, C. elegans and Xenopus, have emerged as powerful model systems that lend themselves particularly well to these sorts of quantitative investigations due to their rapid development and high optical clarity in embryonic and larval stages. In particular, live-cell imaging using fluorescent proteins has become an indispensable tool for developmental biology. However, due to their large size, these model organisms pose a unique challenge with respect to imaging. Here, I explore some of these challenges and how light-sheet fluorescence microscopy (LSFM) can overcome these challenges.
By Dr. Dane Maxfield, Bruker
Published September 1, 2020 | Est. Read Time: 5 Min.
Large multicellular organisms such as mouse blastocysts, Drosophila larva and zebrafish embryos, provide a significant imaging challenge for fluorescent microscopy. Image quality can suffer from several limiting factors, including light scattering and tissue penetration by light at physiological wavelengths, compromising the image quality at depth. Depending on the opacity and makeup of these tissues, visible light will only travel approximately 200µm into the sample, making it difficult to obtain a clear view of the portion of the sample farthest from the objective. This is also complicated by the fact that most high NA objectives have a working distance significantly smaller than the size of the embryo. For example, a 1.4 NA 60x objective (the standard choice on most confocal systems) has a working distance of approximately 100µm, making it impossible to visualize the organism in its entirety.
An additional complication of the large sample size of these model organisms is the sample mounting. With the limitations of traditional microscopes, users traditionally have to manually rotate or manipulate the sample so that the area of interest is closest to the coverslip in order to obtain a high-quality image. To image these samples on a traditional microscope, users typically sandwich the sample between two coverslips, causing topological deformations. As embryos develop, they can dramatically change in size and shape, and such mechanical restrictions can alter or stall the developmental process. Even further, samples like Arabidopsis and Maize, naturally develop vertically making it impossible to recreate native conditions on a standard confocal microscope.
When studying development and other rapid biological processes, researchers need to be able to capture images on physiologically relevant time scales in order to observe dynamic processes and perform various types of functional imaging. Traditional point scanning confocal techniques limit the user to a single image (1024x1024 pixels) at ~0.5-2Hz; and even the newest point scanning confocals can only acquire a similar region at ~15Hz. This presents a significant bottleneck for imaging dynamic biological processes that happen in 3D on rapid time scales, such as imaging a zebrafish heartbeat or functional imaging of Ca2+ transients.
A particular strength of these model organisms is the ability to study embryonic development in vivo through the use of long-term timelapse imaging. This allows researchers to visualize cells in their native 3D environment and translate these studies of cellular dynamics to the organismal level. However, imaging studies of these large and sensitive specimens have traditionally been plagued by photobleaching and phototoxicity. Conventional imaging techniques, such as laser-scanning and spinning disk confocal microscopy, excite fluorophores above and below the focal place of interest and create optical sections by way of a pinhole (or disk of pinholes) placed in a conjugate focal plane which function to physically block light from out-of-focus planes from making it to the detector. This means with every image acquired, every fluorophore in the path of the laser beam becomes excited, even if it does not contribute to the final image. When a fluorescent protein is excited, it has the potential to enter a triplet state and photobleach or react with molecular O2 and produce free radials that can damage the cell, leading to cell death; also known as phototoxicity. These effects become particularly apparent during long-term timelapse studies where researchers commonly will photobleach their protein of interest or the embryo will arrest or die due to repeated exposure of high intensity excitation light.
Light-sheet microscopy, although a relatively new technique in developmental biology, has proven invaluable for visualizing and quantitatively measuring dynamic processes in developing model organisms. The combination of flexible sample mounting, inherent 3D optical sectioning, high spatiotemporal resolution, and low phototoxicity and photobleaching make multi-view light-sheet the perfect solution for your developmental biology imaging needs. Contact us to learn more about our innovative light-sheet solutions tailored around your experiments.
Dr. Dane Maxfield, Bruker