Spectral cross-cumulants for multicolor super-resolved SOFI imaging.

Super-resolution optical fluctuation imaging provides a resolution beyond the diffraction limit by analysing stochastic fluorescence fluctuations with higher-order statistics. Using nth order spatio-temporal cross-cumulants the spatial resolution and the sampling can be increased up to n-fold in all spatial dimensions. In this study, we extend the cumulant analysis into the spectral domain and propose a multicolor super-resolution scheme. The simultaneous acquisition of two spectral channels followed by spectral cross-cumulant analysis and unmixing increases the spectral sampling. The number of discriminable fluorophore species is thus not limited to the number of physical detection channels. Using two color channels, we demonstrate spectral unmixing of three fluorophore species in simulations and experiments in fixed and live cells. Based on an eigenvalue/vector analysis, we propose a scheme for an optimized spectral filter choice. Overall, our methodology provides a route for easy-to-implement multicolor sub-diffraction imaging using standard microscopes while conserving the spatial super-resolution property.

Parameter-free image resolution estimation based on decorrelation analysis.

Super-resolution microscopy opened diverse new avenues of research by overcoming the resolution limit imposed by diffraction. Exploitation of the fluorescent emission of individual fluorophores made it possible to reveal structures beyond the diffraction limit. To accurately determine the resolution achieved during imaging is challenging with existing metrics. Here, we propose a method for assessing the resolution of individual super-resolved images based on image partial phase autocorrelation. The algorithm is model-free and does not require any user-defined parameters. We demonstrate its performance on a wide variety of imaging modalities, including diffraction-limited techniques. Finally, we show how our method can be used to optimize image acquisition and post-processing in super-resolution microscopy.

Photons in - numbers out: perspectives in quantitative fluorescence microscopy for in situ protein counting.

The full understanding of cellular functions requires information about protein numbers for various biomolecular assemblies and their dynamics, which can be partly accessed by super-resolution fluorescence microscopy. Yet, many protein assemblies and cellular structures remain below the accessible resolution on the order of tens of nanometers thereby evading direct observation of processes, like self-association or oligomerization, that are crucial for many cellular functions. Over the recent years, several approaches have been developed addressing concentrations and copy numbers of biomolecules in cellular samples for specific applications. This has been achieved by new labeling strategies and improved sample preparation as well as advancements in super-resolution and single-molecule fluorescence microscopy. So far, none of the methods has reached a level of general and versatile usability due to individual advantages and limitations. In this article, important requirements of an ideal quantitative microscopy approach of general usability are outlined and discussed in the context of existing methods including sample preparation and labeling quality which are essential for the robustness and reliability of the methods and future applications in cell biology.

Time-resolved molecule counting by photon statistics across the visible spectrum.

In the past few years quantification of fluorescently labeled (bio-) molecules has become of increasing importance and several approaches have been developed to address this task. Counting by photon statistics measures the distribution of multiple photon detection events that carry information about the number and brightness of independently emitting fluorophores. The method enables absolute and non-destructive quantification, with the quality of estimates critically depending on the ability to accurately measure said photon statistics. Here, we present a combination of simulations and experiments that relate fundamental properties of fluorophores, i.e. their molecular brightness and photostability, to important experimental conditions, i.e. excitation power and acquisition time. Thereby, experimental settings and analysis parameters can be quantitatively evaluated, making counting by photon statistics a robust method for absolute counting of the number of emitters in a diffraction limited observation volume. We show that the time-resolution of counting varies with the fluorophore brightness and can be as fast as 10-100 ms. At the same time, the range of suitable fluorophores can be easily assessed. We evaluated the brightness and photostability of 16 organic dyes across the visible spectrum, providing information crucial for a range of single-molecule spectroscopy applications. This opens up exciting possibilities to analyze absolute stoichiometries in dynamic multi-component complexes.