Optimized extended depth of focus simulated analysis for confocal microscopy.

In imaging systems and especially in confocal microscopy systems there is a trade-off between the lateral resolution and the obtained depth of focus. The use of complex pupils to improve the lateral resolution by engineering the point spread function is a common approach; however the lateral improvement reduces the effective depth of focus and therefore the fluorescence efficiency. In this work we analytically develop an optimized approach for obtaining a complex pupil with an extended depth of focus. The proposed solution is numerically applied and tested in designing an improved focal depth in confocal microscope configuration.

Second harmonic imaging of chloroplasts using the two-photon laser scanning microscope.

BACKGROUND: Second harmonic information reveals information about the structure of spatially oriented structures with an asymmetry. We study the second harmonic images of the grana and intergranal parts of chloroplasts in the leaves of the shade moss Plagiomnium affine. RESULTS: A two-photon microscope with blocking filters to suppress fluorescence generated both forward and backward second harmonic illumination to be captured. In the starch free chloroplasts of the single cell layered laminae strong second harmonic generation (SHG) from the granal regions was emitted. Upon illumination the chloroplasts changed their orientation affecting the SHG signal. Chloroplast is reoriented upon illumination. CONCLUSIONS: SHG signals were successfully obtained from the grana without any possible confusion from SHG starch grains due to their absence.

Super-resolution via iterative phase retrieval for blurred and saturated biological images.

One of the most fascinating problems addressed today is retrieving high-resolution data of blurred images obtained from biological objects. In most cases the research relays either on a priory knowledge of the image nature or a large number of images (either of the same object or of different objects obtained by the same imaging setup). If saturation is added to the blurring, most algorithms fail to sharpen the image and in some cases researchers decline to use such images as an input. In this work a single captured blurred and saturated image is given with no a priori knowledge except of the fact that the primary blurring is due to defocused imaging setup. The authors suggest a novel three-stage approach for retrieving higher resolution data from the intensity distribution of the blurred and saturated image. The core of the process is the phase retrieval algorithm suggested by Gerchberg and Saxton in 1972. The new method is explained in details and the algorithm is tested numerically and experimentally on several images to show the improvement in the sharpness of the spatial details.

Coherent super-resolution microscopy via laterally structured illumination.

Theory describing a super-resolution microscopy experiment using temporally and spatially coherent structured illumination was developed, and used to derive a method for processing experimental data. Numerical simulations were performed to verify that the method can, in principle, produce super-resolved images that are exactly equivalent to an image processed by a system with a much larger aperture (that is, the correct weighting between different regions of the image spectrum is maintained). The process was then demonstrated experimentally, showing a factor of two improvement in resolution over a diffraction-limited, coherently illuminated, microscope.

Resolution enhancement by subtraction of confocal signals taken at different pinhole sizes.

Subtractive imaging in confocal fluorescence light microscopy is based on the subtraction of a suitably weighted widefield image from a confocal image. An approximation to a widefield image can be obtained by detection with an opened confocal pinhole. The subtraction of images enhances the resolution in-plane as well as along the optic axis. Due to the linearity of the approach, the effect of subtractive imaging in Fourier-space corresponds to a reduction of low spatial frequency contributions leading to a relative enhancement of the high frequencies. Along the direction of the optic axis this also results in an improved sectioning. Image processing can achieve a similar effect. However, a 3D volume dataset must be acquired and processed, yielding a result essentially identical to subtractive imaging but superior in signal-to-noise ratio. The latter can be increased further with the technique of weighted averaging in Fourier-space. A comparison of 2D and 3D experimental data analysed with subtractive imaging, the equivalent Fourier-space processing of the confocal data only, and Fourier-space weighted averaging is presented.

Bizonoplast, a unique chloroplast in the epidermal cells of microphylls in the shade plant Selaginella erythropus (Selaginellaceae).

Study of the unique leaf anatomy and chloroplast structure in shade-adapted plants will aid our understanding of how plants use light efficiently in low light environments. Unusual chloroplasts in terms of size and thylakoid membrane stacking have been described previously in several deep-shade plants. In this study, a single giant cup-shaped chloroplast, termed a bizonoplast, was found in the abaxial epidermal cells of the dorsal microphylls and the adaxial epidermal cells of the ventral microphylls in the deep-shade spike moss Selaginella erythropus. Bizonoplasts are dimorphic in ultrastructure: the upper zone is occupied by numerous layers of 2-4 stacked thylakoid membranes while the lower zone contains both unstacked stromal thylakoids and thylakoid lamellae stacked in normal grana structure oriented in different directions. In contrast, other cell types in the microphylls contain chloroplasts with typical structure. This unique chloroplast has not been reported from any other species. The enlargement of epidermal cells into funnel-shaped, photosynthetic cells coupled with specific localization of a large bizonoplast in the lower part of the cells and differential modification in ultrastructure within the chloroplast may allow the plant to better adapt to low light. Further experiments are required to determine whether this shade-adapted organism derives any evolutionary or ecophysiological fitness from these unique chloroplasts.