Frequency comb ptychoscopy.

Multiheterodyne techniques using frequency combs-radiation sources whose lines are perfectly evenly-spaced-have revolutionized science. By beating sources with the many lines of a comb, their spectra are recovered. Even so, these approaches are fundamentally limited to probing coherent sources, such as lasers. They are unable to measure most spectra that occur in nature. Here we present frequency comb ptychoscopy, a technique that allows for the spectrum of any complex broadband source to be retrieved using a comb. In this approach, the spectrum is reconstructed by unfolding the simultaneous beating of a source with each comb line. We demonstrate this both theoretically and experimentally, at microwave frequencies. This approach can reconstruct the spectrum of nearly any complex source to high resolution, and the speed, resolution, and generality of this technique will allow chip-scale frequency combs to have an impact in a wide swath of new applications, such as remote sensing and passive spectral imaging.

Generalized stepwise optical saturation enables super-resolution fluorescence lifetime imaging microscopy.

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. Express9, 1613 (2018)]. 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 M , 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.

Saturation-compensated measurements for fluorescence lifetime imaging microscopy.

Fluorophore saturation is the key factor limiting the speed and excitation range of fluorescence lifetime imaging microscopy (FLIM). For example, fluorophore saturation causes incorrect lifetime measurements when using conventional frequency-domain FLIM at high excitation powers. In this Letter, we present an analytical theoretical description of this error and present a method for compensating for this error in order to extract correct lifetime measurements in the limit of fluorophore saturation. We perform a series of simulations and experiments to validate our methods. The simulations and experiments show a 13.2× and a 2.6× increase in excitation range, respectively. The presented method is based on algorithms that can be easily applied to existing FLIM setups.