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Diapausing embryos of invertebrates represent investments in future populations. Thus, these embryos must be capable of withstanding a variety of environmental assaults. Consequently, their eggshells should be adapted to resist injuries from predators, sediments, or excessive shrinkage if desiccated. To date, there have been no direct nanomechanical measurements of the eggshells of most diapausing invertebrates. Here, we used three approaches to understand how eggshells of two rotifers, a freshwater species (Brachionus calyciflorus) and a brackish water species (B. plicatilis), tolerate harsh conditions: (1) atomic force microscopy to measure elasticity and hardness; (2) transmission electron microscopy to study ultrastructure; (3) scanning electron microscopy to examine surface features. We compare these values to measurements of brine shrimp (Artemia salina) cysts and mosquito (Aedes aegypti) overwintering eggs. Our results revealed that rotifer eggshells are structurally similar and have comparable nanomechanical values. While rotifer eggshells had lower Young’s moduli (ca. 13–16 MPa) and hardness values (1.84–1.85x10-2 GPa) than eggshells of Artemia and Aedes, eggshells of all species were relatively elastic and not particularly resistant to deformation. Pliancy of shells that form egg banks (i.e., Artemia, Brachionus) may be an adaptation to resist cracking under the physical forces of buried sediments. Though there are no obvious relationship between eggshell thickness, ultrastructure, ornamentation, or nanomechanical values in rotifer eggshells, we hypothesize that eggshell chemistry may play an important role in determining elasticity and hardness. Future studies should consider an integrative approach to understand importance of eggshell structure, chemistry, and mechanics in protecting diapausing embryos. 

Imaging sub-diffraction dynamics of neural nanostructures involved in behaviors such as learning and memory in a freely moving animal is not possible with existing techniques. Here, we present a solution in the form of a two-photon (2P), fiber-coupled, stimulated emission depletion microscope and demonstrate its capabilities by acquiring super-resolution imaging of mammalian cells. A polarization-maintaining fiber is used to transport both the 2P excitation light (915 nm) and the donut-shaped depletion beam (592 nm), which is constructed by adding two temporally incoherent and orthogonally polarized Hermite–Gaussian fiber modes. The fiber output is insensitive to bending or temperature changes and is the first demonstration toward deep tissue super-resolution imaging in awake behaving animals. 

The DyMIN method reduces photobleaching, a problem in STED microscopy. Labs implementing custom-built STED microscopes would greatly benefit from DyMIN capabilities. We present an inexpensive, open-source version utilizing an FPGA and multiplexer. 

We demonstrate a two photon (2P) fiber STED microscope in which the excitation and STED light are delivered to the sample in polarization maintaining (PM) fiber.