Abstract
High resolution microscopy is essential for advanced study of biological structures and accurate diagnosis of medical diseases. The spatial resolution of conventional microscopes is light diffraction limited. Structured illumination has been extensively explored to break the diffraction limit in wide field light microscopy. However, deployable application of the structured illumination in scanning laser microscopy is challenging due to the complexity of the illumination system and possible phase errors in sequential illumination patterns required for super-resolution reconstruction. We report here a super-resolution scanning laser imaging system which employs virtually structured detection (VSD) to break the diffraction limit. Without the complexity of structured illumination, VSD provides an easy, low-cost and phase-artifact free strategy to achieve super-resolution in scanning laser microscopy.
Highlights
High resolution imaging is essential for biomedical study and disease evaluation
The spatial resolution of conventional imaging systems is constrained by light diffraction, which precludes the observation of fine structures of biological specimens
The stimulated emission depletion (STED) laser can deactivate the fluorophores in the periphery of the excitation laser focus, allowing only the fluorescence from the subdiffraction-limited center to contribute to super-resolution recording [1, 2]
Summary
High resolution imaging is essential for biomedical study and disease evaluation. the spatial resolution of conventional imaging systems is constrained by light diffraction, which precludes the observation of fine structures of biological specimens. The STED laser can deactivate the fluorophores in the periphery of the excitation laser focus, allowing only the fluorescence from the subdiffraction-limited center to contribute to super-resolution recording [1, 2]. The extremely intensive laser exposure limits its applications for live cell imaging of biological systems, such as delicate and fragile retina. STORM [3], PALM [4], or fluorescence PALM (FPALM) [5] can achieve super-resolution by mapping localizations of individual molecules with photo-switchable fluorescence probes. Single molecule localization based imaging approach has been demonstrated for live cell imaging [6,7,8], the imaging speed is limited due to the requirement of acquiring multiple sub-images for superresolution reconstruction. Its application for high temporal resolution monitoring of live systems is still challenging
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