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Structured illumination microscopy (SIM) is a powerful technique for obtaining super-resolved fluorescence maps of samples, but it is very sensitive to aberrations or misalignments affecting the excitation patterns. Here, we present a reconstruction algorithm that is able to process SIM data even if the illuminations are strongly distorted. The approach is an extension of the recent blind-SIM technique, which reconstructs simultaneously the sample and the excitation patterns without a priori information on the latter. Our algorithm was checked on synthetic and experimental data using distorted and nondistorted illuminations. The reconstructions were similar to that obtained by up-to-date SIM methods when the illuminations were periodic and remained artifact-free when the illuminations were strongly distorted.

We report the implementation and exploitation of fluorescence polarization measurements, in the form of anisotropy fluorescence lifetime imaging microscopy (rFLIM) and energy migration Förster resonance energy transfer (emFRET) modalities, for wide-field, confocal laser-scanning microscopy and flow cytometry of cells. These methods permit the assessment of rotational motion, association and proximity of cellular proteins in vivo. They are particularly applicable to probes generated by fusions of visible fluorescence proteins, as exemplified by studies of the erbB receptor tyrosine kinases involved in growth-factor-mediated signal transduction.

During the last years, measurements considerably beyond the conventional “Abbe-Limit” of optical resolution in far field light microscopy were realized by several light microscopical approaches. Point spread function (PSF) engineering, spectral precision distance microscopy (SPDM), and related methods were used to demonstrate the feasibility of such measurements. SPDM allows the measurement of position and multiple distances between point-like fluorescent objects of different spectral signatures far below the optical resolution criterion as defined by the full width at half maximum of the PSF. Here, we report a software method to obtain online visualization of light distribution in the lateral and axial direction of any object detected in a spatially modulated illumination (SMI) microscope. This strongly facilitates routine application of SMI microscopy. The software was developed using Microsoft Visual C++ running on Windows NT. Furthermore, some aspects of the theoretical limits of the SPDM method were studied by virtual microscopy. For the case of SMI microscopy the precision of axial distance measurements was studied, taking into account photon statistics and image analysis procedures. The results indicate that even under low fluorescence intensity conditions typical for biological structure research, precise distance measurements in the nanometer range can be determined, and that axial distances in the order of 40 nm are detectable with such precision. © 2001 Society of Photo-Optical Instrumentation Engineers.