High-precision fluorescence modulation microscopy using a Bessel beam for artifact-suppressed super-resolution imaging.

Fluorescence emission difference (FED) microscopy suffers from imperfect matching between solid and hollow excitation point spread functions (PSFs), causing subtraction-induced artifacts. Here, we introduce high-precision fluorescence modulation microscopy (HPFM) based on a first-order Bessel beam. The Bessel beam's smaller dark core and tunable annular structure enable superior PSF alignment, effectively suppressing subtraction-induced artifacts. HPFM achieves a lateral resolution of λ/4 to λ/6, outperforming FED and confocal microscopy in both fluorescent bead and fixed-cell imaging. Moreover, its low excitation power allows prolonged live-cell imaging with reduced photobleaching and phototoxicity, enabling clear visualization of subcellular dynamics. These capabilities make HPFM a robust and practical super-resolution tool for challenging biological applications.