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A new technique for the acceleration of iterative image restoration algorithms is proposed. The method is based on the principles of vector extrapolation and does not require the minimization of a cost function. The algorithm is derived and its performance illustrated with Richardson-Lucy (R-L) and maximum entropy (ME) deconvolution algorithms and the Gerchberg-Saxton magnitude and phase retrieval algorithms. Considerable reduction in restoration times is achieved with little image distortion or computational overhead per iteration. The speedup achieved is shown to increase with the number of iterations performed and is easily adapted to suit different algorithms. An example R-L restoration achieves an average speedup of 40 times after 250 iterations and an ME method 20 times after only 50 iterations. An expression for estimating the acceleration factor is derived and confirmed experimentally. Comparisons with other acceleration techniques in the literature reveal significant improvements in speed and stability.

3D deconvolution microscopy is a combination of optical and computational techniques that are used to maximize the observed resolution and signal from a biological specimen. Mathematical models are used to predict the distribution of out‐of‐focus light caused by the inherent optical limitations of the instrument, which can then be compensated for using computer algorithms. This unit will review the theory of image formation and characteristics of the point spread function (PSF) based on the instrument modality and objective lens parameters. A variety of commonly used deblurring and deconvolution methods are described, and their applications to sample datasets are illustrated to show the performance of each algorithm. Steps for setting up the image acquisition to acquire data suitable for deconvolution are described, and the challenge of maximizing signal levels while minimizing light exposure addressed. Deconvolution examples from widefield epi‐fluorescence and laser scanning confocal are shown, and suitability for other modalities discussed. Curr. Protoc. Cytom. 52:12.19.1‐12.19.20. © 2010 by John Wiley & Sons, Inc.

The article describes combined orientation-independent (OI-) DIC and polarization microscope and its biological applications. Several conventional DIC images were recorded with the specimen oriented in different directions followed by digital alignment and processing of the images. Then the obtained images are used for computation of the phase gradient magnitude and azimuth distribution, and, further, the phase image. The OI-DIC images were obtained using optics having numerical aperture 1.4, thus achieving a level of resolution never before achieved with any phase contrast or interference microscope. The combined system yields two complementary phase images of thin optical sections of the specimen: distribution of refractive index and distribution of birefringence due to anisotropy of the cell structure. For instance, in a live dividing cell, the OI-DIC image clearly shows the detailed shape of the chromosomes while the polarization image quantitatively depicts the distribution of birefringent microtubules in the spindle, both without any need for staining or other modifications of the cell. We present pseudo-color combined images of a crane-fly spermatocyte at diakinesis and metaphase of meiosis I. Those images provide clear evidence that the proposed technique can reveal fine architecture and molecular organization in live cells without perturbation associated with staining or fluorescent labeling.

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