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Super-resolution optical fluctuation imaging (SOFI) 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 as well as the sampling can be increased up to n-fold in all three spatial dimensions. In this study, we extend the cumulant analysis into the spectral domain and propose a novel multicolor super-resolution scheme. The simultaneous acquisition of two spectral channels followed by spectral cross-cumulant analysis and unmixing increase 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 multiple experiments with different cellular structures, fluorophores and filter sets. Based on an eigenvalue/ vector analysis we propose a scheme for an optimized spectral filter choice. Overall, our methodology provides a novel route for easy-to-implement multicolor sub-diffraction imaging using standard microscopes while conserving the spatial super-resolution property. This makes simultaneous multiplexed super-resolution fluorescence imaging widely accessible to the life science community interested to probe colocalization between two or more molecular species., Comment: main: 21 pages & 4 figures, supplementary 20 pages & 16 figures

Super-resolution fluorescence microscopy provides unprecedented insight into cellular and subcellular structures. However, going "beyond the diffraction barrier" comes at a price since most far-field super-resolution imaging techniques trade temporal for spatial super-resolution. We propose the combination of a novel label-free white light quantitative phase tomography with fluorescence imaging to provide high-speed imaging and spatial super-resolution. The non-iterative phase reconstruction relies on the acquisition of single images at each z-location and thus enables straightforward 3D phase imaging using a classical microscope. We realized multi-plane imaging using a customized prism for the simultaneous acquisition of 8 planes. This allowed us to not only image live cells in 3D at up to 200 Hz, but also to integrate fluorescence super-resolution optical fluctuation imaging within the same optical instrument. This 4D microscope platform unifies the sensitivity and high temporal resolution of phase tomography with the specificity and high spatial resolution of fluorescence imaging.

A straightforward method to achieve super-resolution consists of taking an image sequence of stochastically blinking emitters using a standard wide-field fluorescence microscope. Densely packed single molecules can be distinguished sequentially in time using high-precision localization algorithms (e.g., PALM and STORM) or by analyzing the statistics of the temporal fluctuations (SOFI). In a face-to-face comparison of the two post-processing algorithms, we show that localization-based super-resolution can deliver higher resolution enhancements but imposes significant constraints on the blinking behavior of the probes, which limits its applicability for live-cell imaging. SOFI, on the other hand, works more consistently over different photo-switching kinetics and also delivers information about the specific blinking statistics. Its suitability for low SNR acquisition reveals SOFI's potential as a high-speed super-resolution imaging technique.

In the version of this Article originally published, there were some errors in equations in Fig. 1a; the details are shown in the correction notice. In the Acknowledgments, grant number ‘686271’ should have read ‘686271/SEFRI 16.0047’. These errors have now been corrected online.

Super-resolution optical fluctuation imaging (SOFI) provides an elegant way of overcoming the diffraction limit in all three spatial dimensions by computing higher-order cumulants of image sequences of blinking fluorophores acquired with a classical widefield microscope. Previously, three-dimensional (3D) SOFI has been demonstrated by sequential imaging of multiple depth positions. Here we introduce a multiplexed imaging scheme for the simultaneous acquisition of multiple focal planes. Using 3D cross-cumulants, we show that the depth sampling can be increased. The simultaneous acquisition of multiple focal planes significantly reduces the acquisition time and thus the photobleaching. We demonstrate multiplane 3D SOFI by imaging fluorescently labelled cells over an imaged volume of up to 65 × 65 × 3.5 μm3 without depth scanning. In particular, we image the 3D network of mitochondria in fixed C2C12 cells immunostained with Alexa 647 fluorophores and the 3D vimentin structure in living Hela cells expressing the fluorescent protein Dreiklang., Super-resolution optical fluctuation imaging provides 3D images of biological specimens via blinking fluorophores. Geissbuehler et al. present a multiplexed version of this method that captures images at multiple focal planes simultaneously, reducing the acquisition time compared with standard approaches.

The use of fluorescent light is of fundamental importance in the natural and life sciences as it offers the unique possibility for tackling quantitative measurement problems in three dimensions with high specificity and at various spatial resolution scales ranging from several micrometers to a few tens of nanometers. Typically, fluorescence-based metrology and imaging techniques use the intensity of light waves, which is easily measured by detectors. However, the phase of a fluorescent light wave contains important information about the wave; yet, it has been largely unexplored. In this chapter, we will introduce the concept of fluorescence self-interference to allow the measurement of the phase information of fluorescent light waves. In general, fluorescence self-interferometry is a unique form of low-coherence self-referencing optical interferometry that utilizes fluorophores as a light source of low-temporal coherence. Fluorescence self-interferometry opens up new avenues for the development of optical microscopes based on the phase, rather than on the intensity solely, of fluorescent waves as will be described here. In particular, we will present several experimental realizations of fluorescence phase microscopy and discuss their measurement capabilities and limitations for metrology and imaging applications at the mesoscopic (1–10 μm) to nanoscopic (

We present a novel concept for optical spectroscopy called nonlinear correlation spectroscopy (NLCS). NLCS analyses coherent field fluctuations of the second and third harmonic light generated by diffusing nanoparticles. Particles based on noncentrosymmetric nonlinear materials such as KNbO(3) show a strong second as well as third harmonic response. The method and the theory are introduced and experimental NLCS results in fetal calf serum are presented showing the promising selectivity of this technique for measurement in complex biological environments.

The diffraction patterns of fixed fluorophores are characteristic of the orientation of the molecules' underlying dipole. Fluorescence localization microscopy techniques such as PALM and STORM achieve super-resolution by sequentially imaging sparse subsets of fluorophores, which are localized by means of Gaussian-based localization. This approach is based on the assumption of isotropic emitters, where the diffraction pattern corresponds to a section of the point spread function. Applied to fixed fluorophores, it can lead to an estimation bias in the range of 5-20nm. We introduce a method for the joint estimation of position and orientation of single fluorophores, based on an accurate image formation model expressed as a 3-D steerable filter. We demonstrate experimental estimation accuracies of 5 nm for position and 2 degrees for orientation.

We present a combination of self-interference microscopy with lateral super-resolution microscopy and introduce a novel approach for localizing a single nano-emitter to within a few nanometers in all three dimensions over a large axial range. We demonstrate nanometer displacements of quantum dots placed on top of polymer bilayers that undergo swelling when changing from an air to a water environment, achieving standard deviations below 10 nm for axial and lateral localization.

A classical technique to monitor dynamical processes at the molecular level is fluorescence correlation spectroscopy (FCS). FCS requires fluorescent labels that are typically limited by photobleaching and saturation. We present a new method that uses noble-metal nanoparticles instead of fluorophores: optical coherence correlation spectroscopy (OCCS). OCCS is a correlation spectroscopy technique based on dark-field optical coherence microscopy, a Fourier domain optical coherence tomography technique. In OCCS, several sampling volumes are measured simultaneously with high detection sensitivity. OCCS measures the time correlation function of the light back-scattered by the nanoparticles. Using a mode-locked Ti:Sapphire laser (780nm central wavelength) we performed first experiments with different nanoparticles down to 30nm in diameter. We present experimental results and a preliminary model to fit the correlation curves and extract the particles’ concentrations and diffusion coefficients. The experimental determination of the diffusion times of gold nanoparticles using this model is presented, showing the potential of our method. In the near future, we aim at investigating smaller gold nanoparticles that interfere less with the biological phenomena under study.