Comparison between SOFI and STORM

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

Detection efficiency in total internal reflection fluorescence microscopy

We present a rapid and flexible framework for the accurate calculation of the detection efficiency of fluorescence emission in isotropic media as well as in the vicinity of dielectric or metallic interfaces. The framework accounts for the dipole characteristics of the emitted fluorescence and yields the absolute detection efficiency by taking into account the total power radiated by the fluorophore. This analysis proved to be useful for quantitative measurements, i.e. the fluorescence detection at a glass–water interface for total internal reflection fluorescence microscopy in an epi- and a trans-illumination configuration

Image formation and tomogram reconstruction in optical coherence microscopy

In this work we present a model for image formation in optical coherence microscopy. In the spectral domain detection, each wavenumber has a specific coherent transfer function that samples a different part of the object's spatial frequency spectrum. The reconstruction of the tomogram is usually accurate only in a short depth of field. Using numerical simulations based on the developed model, we identified two distinct mechanisms that influence the signal of out-of-focus sample information. Besides the lateral blurring induced through defocusing, an additional axial envelope contributing equally to the signal degradation was found. (C) 2010 Optical Society of Americ

Multiplane 3D superresolution optical fluctuation imaging

By switching fluorophores on and off in either a deterministic or a stochastic manner, superresolution microscopy has enabled the imaging of biological structures at resolutions well beyond the diffraction limit. Superresolution 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 conventional widefield microscope. So far, three-dimensional (3D) SOFI has only been demonstrated by sequential imaging of multiple depth positions. Here we introduce a versatile imaging scheme which allows for the simultaneous acquisition of multiple focal planes. Using 3D cross-cumulants, we show that the depth sampling can be increased. Consequently, the simultaneous acquisition of multiple focal planes reduces the acquisition time and hence the photo-bleaching of fluorescent markers. We demonstrate multiplane 3D SOFI by imaging the mitochondria network in fixed C2C12 cells over a total volume of $65\times65\times3.5 \mu\textrm{m}^3$ without depth scanning.Comment: 7 pages, 3 figure

Visible spectrum extended-focus optical coherence microscopy for label-free sub-cellular tomography

We present a novel extended-focus optical coherence microscope (OCM) attaining 0.7 {\mu}m axial and 0.4 {\mu}m lateral resolution maintained over a depth of 40 {\mu}m, while preserving the advantages of Fourier domain OCM. Our method uses an ultra-broad spectrum from a super- continuum laser source. As the spectrum spans from near-infrared to visible wavelengths (240 nm in bandwidth), we call the method visOCM. The combination of such a broad spectrum with a high-NA objective creates an almost isotropic 3D submicron resolution. We analyze the imaging performance of visOCM on microbead samples and demonstrate its image quality on cell cultures and ex-vivo mouse brain tissue.Comment: 15 pages, 7 figure

Quantifying protein densities on cell membranes using super-resolution optical fluctuation imaging

Surface molecules, distributed in diverse patterns and clusters on cell membranes, influence vital functions of living cells. It is therefore important to understand their molecular surface organisation under different physiological and pathological conditions. Here, we present a model-free, quantitative method to determine the distribution of cell surface molecules based on TIRF illumination and super-resolution optical fluctuation imaging (SOFI). This SOFI-based approach is robust towards single emitter multiple-blinking events, high labelling densities and high blinking rates. In SOFI, the molecular density is not based on counting events, but results as an intrinsic property due to the correlation of the intensity fluctuations. The effectiveness and robustness of the method was validated using simulated data, as well as experimental data investigating the impact of palmitoylation on CD4 protein nanoscale distribution in the plasma membrane of resting T cells.Comment: 9 pages, 3 figures plus Supplementary Informatio

Complementarity of PALM and SOFI for super-resolution live cell imaging of focal adhesions

Live cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenging task for super-resolution microscopy. We have addressed this important issue by combining photo-activated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed cell focal adhesion images, we investigated the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework was used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualized the dynamics of focal adhesions, and revealed local mean velocities around 190 nm per minute. The complementarity of PALM and SOFI was assessed in detail with a methodology that integrates a quantitative resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as the fluorophore density and the photo-activation and photo-switching rates

Continuous wavelet transform ridge extraction for spectral interferometry imaging

The combination of wavelength multiplexing and spectral interferometry allows for the encoding of multidimensional information and its transmission over a mono-dimensional channel; for example, measurements of a surface's topography acquired through a monomode fiber in a small endoscope. The local depth of the imaged object is encoded in the local spatial frequency of the signal measured at the output of the fiber-decoder system. We propose a procedure to retrieve the depth-map by determining the signal's instantaneous frequency. First, we compute its continuous, complex-valued, wavelet transform (CWT). The frequency signature at every position is contained in the resulting scalogram. We then extract the ridge of maximal response by use of a dynamic programming algorithm thus directly recovering the object's topography. We present results that validate this procedure based on both simulated and experimental data