Abstract
The FLUMIAS (Fluorescence-Microscopic Analyses System for Life-Cell-Imaging in Space) confocal laser spinning disk fluorescence microscope represents a new imaging capability for live cell imaging experiments on suborbital ballistic rocket missions. During the second pioneer mission of this microscope system on the TEXUS-54 suborbital rocket flight, we developed and performed a live imaging experiment with primary human macrophages. We simultaneously imaged four different cellular structures (nucleus, cytoplasm, lysosomes, actin cytoskeleton) by using four different live cell dyes (Nuclear Violet, Calcein, LysoBrite, SiR-actin) and laser wavelengths (405, 488, 561, and 642 nm), and investigated the cellular morphology in microgravity (10−4 to 10−5 g) over a period of about six minutes compared to 1 g controls. For live imaging of the cytoskeleton during spaceflight, we combined confocal laser microscopy with the SiR-actin probe, a fluorogenic silicon-rhodamine (SiR) conjugated jasplakinolide probe that binds to F-actin and displays minimal toxicity. We determined changes in 3D cell volume and surface, nuclear volume and in the actin cytoskeleton, which responded rapidly to the microgravity environment with a significant reduction of SiR-actin fluorescence after 4–19 s microgravity, and adapted subsequently until 126–151 s microgravity. We conclude that microgravity induces geometric cellular changes and rapid response and adaptation of the potential gravity-transducing cytoskeleton in primary human macrophages.
Highlights
The monocyte-macrophage system (MMS) belongs to the first line of immune defense, acts as a crucial effector system for attacking and killing bacteria and routinely clears more than one billion apoptotic cells from almost all tissues of the organism [1,2], thereby maintaining homeostasis of the human immune and tissue systems
During the CELLBOX-PRIME International Space Station (ISS) experiment, we recently demonstrated, that 11 days in microgravity resulted neither in quantitative nor structural changes of the actin and vimentin cytoskeleton in primary human macrophages when compared to 1 g controls [23]
Four different fluorescence colors can be used, since the microscope is equipped with four lasers with the wavelengths 405, 488, 561, and 642 nm
Summary
The monocyte-macrophage system (MMS) belongs to the first line of immune defense, acts as a crucial effector system for attacking and killing bacteria and routinely clears more than one billion apoptotic cells from almost all tissues of the organism [1,2], thereby maintaining homeostasis of the human immune and tissue systems. Only relatively simple optical systems existed for life sciences, which consisted of a simple camera to record smaller organisms, such as cichlid fish or daphnia [39,40], light microscopy to record bright field images, or epifluorescence systems for fluorescence-labelled cells [41,42,43,44] These systems did not allow high-resolution imaging of subcellular structures in living cells. A recent live imaging study with the FLUMIAS microscope reported disturbances of actin bundles in the cytoplasm of human thyroid carcinoma cells immediately after the onset of microgravity in a parabolic flight and suborbital ballistic rocket experiments, using Lifeact-GFP transfection [11], but without providing quantitative data. We were able to observe for the first time, and live in primary human macrophages, rapid changes in the volume and surface of cell nuclei and cells in response to microgravity and rapid response and adaptation of the actin-cytoskeleton
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