Abstract
Super-resolution fluorescence microscopy is an important tool in biomedical research for its ability to discern features smaller than the diffraction limit. However, due to its difficult implementation and high cost, the super-resolution microscopy is not feasible in many applications. In this paper, we propose and demonstrate a saturation-based super-resolution fluorescence microscopy technique that can be easily implemented and requires neither additional hardware nor complex post-processing. The method is based on the principle of stepwise optical saturation (SOS), where M steps of raw fluorescence images are linearly combined to generate an image with a [Formula: see text]-fold increase in resolution compared with conventional diffraction-limited images. For example, linearly combining (scaling and subtracting) two images obtained at regular powers extends the resolution by a factor of 1.4 beyond the diffraction limit. The resolution improvement in SOS microscopy is theoretically infinite but practically is limited by the signal-to-noise ratio. We perform simulations and experimentally demonstrate super-resolution microscopy with both one-photon (confocal) and multiphoton excitation fluorescence. We show that with the multiphoton modality, the SOS microscopy can provide super-resolution imaging deep in scattering samples.
Highlights
Super-resolution fluorescence microscopy techniques, such as stimulated emission depletion (STED) microscopy [1,2] and its related reversible saturable optical fluorescence transitions (RESOLFT) microscopy [3], photoactivated localization microscopy (PALM) [4], stochastic optical reconstruction microscopy (STORM) [5], and structured illumination microscopy (SIM) [6,7], have enabled a dramatic development in modern biology by being able to discern fluorescent molecules or features that are closer together than the diffraction limit [8, 9]
Considering the shot noise and the propagation of errors in stepwise optical saturation (SOS) algorithms [28], the signal-to-noise ratio (SNR) of a two-step SOS image will be an order of magnitude lower than a conventional image, while for SOS images generated from more than two steps, the SNR will be at least two orders of magnitude lower
We have proposed and demonstrated a new kind of super-resolution fluorescence microscopy using the principle of SOS
Summary
Super-resolution fluorescence microscopy techniques, such as stimulated emission depletion (STED) microscopy [1,2] and its related reversible saturable optical fluorescence transitions (RESOLFT) microscopy [3], photoactivated localization microscopy (PALM) [4], stochastic optical reconstruction microscopy (STORM) [5], and structured illumination microscopy (SIM) [6,7], have enabled a dramatic development in modern biology by being able to discern fluorescent molecules or features that are closer together than the diffraction limit [8, 9]. The implementation of most super-resolution techniques is difficult and expensive, which hinders the universal application of super-resolution microscopy in many labs. To increase the imaging depth, some super-resolution methods have been combined with multiphoton microscopy (MPM) [11,12,13,14], an imaging technique widely used in biomedical research for its inherent 3D resolution, deep penetration, and minimal phototoxicity [15,16,17]. To reduce the complexity and cost of super-resolution microscopy, novel techniques such as saturated excitation (SAX) microscopy and its variants [13, 18,19,20,21] and fluorescence emission difference (FED) microscopy [22] have been developed, whose implementations are easier and cheaper compared to traditional super-resolution methods. A super-resolution technique with both deep penetration and easy implementation would be desirable and widely utilized
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