Extended depth-of-field fluorescence microscope for stable imaging during random positioning machine running

The random positioning machine (RPM) is used to simulate microgravity for biodynamical study via stochastic movements, during which real-time imaging in vivo is highly desired. However, the jitter induced by RPM rotating complicates stable imaging of live samples. In this paper, we develop an extended depth-of-field (DOF) fluorescence microscope (EDOFM) for stable imaging during the operation of RPM. By interposing a 5 mm thick glass plate between the objective lens and the sample, we purposely induce spherical aberration to extend the DOF. We also integrate a low-numerical-aperture (NA) light stop to mitigate the reduction of imaging clarity caused by spherical aberration. Our design extends the DOF of the EDOFM by over 15-fold compared to conventional wide-field setups, verified by both ZEMAX simulations and practical experiments. We assessed our EDOFM system with transgenic zebrafish larva in vivo for blood cell tracking, under both stationary and rotating conditions of the RPM. With the EDOFM, we captured more signals during RPM operation, demonstrating its superior imaging stability during RPM-induced jitter. We expect broad applications of the EDOFM system in the study of microgravity.