Benchmarking scanning mechanisms for high-fidelity 3D imaging in fluorescence microscopy

Three-dimensional imaging is vital in biological research for investigating intracellular dynamics and protein–protein interactions. Modern fluorescence microscopy typically achieves volumetric imaging by acquiring stacks of two-dimensional images at varying depths, often through sample scanning or objective scanning, translating the specimen or objective using a piezo stage. Recent approaches, such as remote focusing or laser scanning, enable volumetric acquisition without physically moving either the sample or the objective within the imaging space. In this study, we benchmark scanning strategies by performing a comparative analysis of two widely used 3D imaging approaches: sample scanning, which requires mechanical translation of the specimen, and laser scanning, which achieves volumetric imaging without motion in the sample space. Although sample scanning remains broadly adopted, our experiments demonstrate that it introduces mechanical instability, exhibiting cumulative drift of approximately 11 μm over a 116 μm scan range in 572 steps across 100 time points. In contrast, laser scanning, by keeping the sample and optics stationary at the sample space, demonstrates superior stability and is better suited for longitudinal imaging. We validate our findings through time-lapse imaging of a calibration slide, 200 nm fluorescent microspheres, and fixed human breast adenocarcinoma cells, highlighting the advantages of laser scanning for high-fidelity 3D imaging.