Single particle tracking methods have been used to dissect the viral infection process step-by-step and elucidate new quantitative understanding of the interactions and dynamics between virus and host. The process begins when a virus diffusing rapidly through solution impacts and binds to the cell. The disparate spatial scales between the virus and a host cell make capturing these moments of transition between fast, 3D viral diffusion and cell-bound interaction an impossible imaging challenge that no single microscope is built to task for. Instead, tradeoffs are required between imaging the narrow field of view required to target-lock a solution-phase virus or imaging the larger cellular environment, temporally limited to confined viral dynamics inside cells after binding. In this work, we introduce the 3D Tracking and Imaging, (3D-TrIm) microscope which merges the best of both worlds; allowing for large-scale, 3D multiphoton volumes to be simultaneously acquired and co-localized with single-particle trajectories obtained via active feedback tracking capable of target-locking freely-diffusive virus-scale particles. This feat is accomplished by the integration of 3D-Dynamic Photon Localization Tracking (3D-DyPLoT), and the 3D Fast Acquisition Scan via z-Translating Raster (3D-FASTR) point-scan imaging pattern. Both microscopes utilize dynamic 3D laser scan patterns to survey two disparate fields of view, coupled through the same objective lens and piezoelectric stage which serves as the reference spatial coordinate system and allows point-scanned multiphoton voxels to be rapidly localized without motion smearing. The ability to quickly and simultaneously image surrounding 3D cellular regions during tracking can provide new insight into the nature of viral activities spanning the range of spatiotemporal regimes in the infection process. Such activities can yield new insight into the critical moments of the process missed by only having access to single spatiotemporal imaging regimes and modalities.
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