Abstract
We present a novel super-resolution fluorescence lifetime microscopy technique called generalized stepwise optical saturation (GSOS) that generalizes and extends the concept of the recently demonstrated stepwise optical saturation (SOS) super-resolution microscopy [Biomed. Opt. Express 9, 1613 (2018)29675306]. The theoretical basis of GSOS is developed based on exploring the dynamics of a two-level fluorophore model and using perturbation theory. We show that although both SOS and GSOS utilize the linear combination of M raw images to increase the imaging resolution by a factor of , SOS is a special and the simplest case of GSOS. The super-resolution capability is demonstrated with theoretical analysis and numerical simulations for GSOS with sinusoidal and pulse-train modulations. Using GSOS with pulse-train modulation, super-resolution and fluorescence lifetime imaging microscopy (FLIM) images can be obtained simultaneously. The super-resolution FLIM capability is experimentally demonstrated with a cell sample on a custom-built two-photon frequency-domain (FD) FLIM system based on radio frequency analog signal processing. To our knowledge, this is the first implementation of super-resolution imaging in FD-FLIM.
Highlights
Both stepwise optical saturation (SOS) and generalized stepwise optical saturation (GSOS) can be implemented on existing fluorescence intensity or√FD-Fluorescence lifetime imaging microscopy (FLIM) microscopes
As discussed in [8], the SOS image generated from more than two steps of raw images is generally unacceptable for its signal-to-noise ratio (SNR) is at least two orders of magnitude lower than a conventional image; the same problem exists in GSOS microscopy
The SNR performance of GSOS could be even worse than SOS, since GSOS often relies on the harmonics qk of a modulated fluorescence signal, which are generally weaker and noisier than the intensity of an unmodulated fluorescence signal used in SOS
Summary
Super-resolution fluorescence microscopy techniques have enabled a dramatic development in modern biology by being able to discern fluorescent features that are smaller than the diffraction limit [1,2,3,4,5,6,7,8,9] and can be achieved using a variety of techniques such as stimulated emission depletion (STED) microscopy [2], photoactivated localization microscopy (PALM) [3], stochastic optical reconstruction microscopy (STORM) [4], structured illumination microscopy (SIM) [5], saturated excitation (SAX) microscopy [6, 7], and the recently demonstrated stepwise optical saturation (SOS) microscopy [8, 9]. Super-resolution and FLIM techniques can be combined into an integrated system, which is attractive for its capability to measure the microenvironment in living tissues with a subdiffractive resolution. We present a novel microscopy technique, termed generalized stepwise optical saturation (GSOS), that is able to generate super-resolution lifetime images using FD-FLIM approaches. We show with theory and simulations that both sinusoidal and pulse-train signals can be used as the excitation source in GSOS microscopy to generate super-resolution images, where pulse-train modulation is preferred for it can greatly simplify the lifetime measurement process in practice. We experimentally demonstrate the super-resolution FLIM capability enabled by GSOS with pulse-train modulation on a custom-built two-photon FD-FLIM system based on a low-cost, high-performance homodyne detection method using radio frequency (RF) analog signal processing
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