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.

Here we report the successful first operation of FLUMIAS-DEA, a miniaturized high-resolution 3D fluorescence microscope on the International Space Station (ISS) by imaging two scientific samples in a temperature-constant system, one sample with fixed cells and one sample with living human cells. The FLUMIAS-DEA microscope combines features of a high-resolution 3D fluorescence microscope based on structured illumination microscope (SIM) technology with hardware designs to meet the requirements of a space instrument. We successfully demonstrated that the FLUMIAS technology was able to acquire, transmit, and store high-resolution 3D fluorescence images from fixed and living cells, allowing quantitative and dynamic analysis of subcellular structures, e.g., the cytoskeleton. The capability of real-time analysis methods on ISS will dramatically extend our knowledge about the dynamics of cellular reactions and adaptations to the space environment, which is not only an option, but a requirement of evidence-based medical risk assessment, monitoring and countermeasure development for exploration class missions.

Of all 3D-super resolution techniques, structured illumination microscopy (SIM) provides the best compromise with respect to resolution, signal-to-noise ratio (S/N), speed and cell viability. Its ability to achieve double resolution in all three dimensions enables resolving 3D-volumes almost 10× smaller than with a normal light microscope. Its major drawback is noise contained in the out-of-focus-signal, which—unlike the out-of-focus signal itself—cannot be removed mathematically. The resulting “noise-pollution” grows bigger the more light is removed, thus rendering thicker biological samples unsuitable for SIM. By using a slit confocal pattern, we employ optical means to suppress out-of-focus light before its noise can spoil SIM mathematics. This not only increases tissue penetration considerably, but also provides a better S/N performance and an improved confocality. The SIM pattern we employ is no line grid, but a two-dimensional hexagonal structure, which makes pattern rotation between image acquisitions obsolete and thus simplifies image acquisition and yields more robust fit parameters for SIM.

Human inducible pluripotent stem cells (hiPSCs) hold a large potential for disease modeling. hiPSC-derived human astrocyte and neuronal cultures permit investigations of neural signaling pathways with subcellular resolution. Combinatorial cultures, and three-dimensional (3-D) embryonic bodies enlarge the scope of investigations to multi-cellular phenomena. A the highest level of complexity, brain organoids that – in many aspects – recapitulate anatomical and functional features of the developing brain permit the study of developmental and morphological aspects of human disease. An ideal microscope for 3-D tissue imaging at these different scales would combine features from both confocal laser-scanning and light-sheet microscopes: a micrometric optical sectioning capacity and sub-micrometric spatial resolution, a large field of view and high frame rate, and a low degree of invasiveness, i.e., ideally, a better photon efficiency than that of a confocal microscope. In the present work, we describe such an instrument that belongs to the class of two-photon (2P) light-sheet microsocpes. Its particularity is that – unlike existing two- or three-lens designs – it is using a single, low-magnification, high-numerical aperture objective for the generation and scanning of a virtual light sheet. The microscope builds on a modified Nipkow-Petran spinning-disk scheme for achieving wide-field excitation. However, unlike the common Yokogawa design that uses a tandem disk, our concept combines micro lenses, dichroic mirrors and detection pinholes on a single disk. This design, advantageous for 2P excitation circumvents problems arising with the tandem disk from the large wavelength-difference between the infrared excitation light and visible fluorescence. 2P fluorescence excited in by the light sheet is collected by the same objective and imaged onto a fast sCMOS camera. We demonstrate three-dimensional imaging of TO-PRO3-stained embryonic bodies and of brain organoids, under control conditions and after rapid (partial) transparisation with triethanolamine and /ormamide (RTF) and compare the performance of our instrument to that of a confocal microscope having a similar numerical aperture. 2P-virtual light-sheet microscopy permits one order of magnitude faster imaging, affords less photobleaching and permits better depth penetration than a confocal microscope with similar spatial resolution.