Search

Summary: Structured‐illumination microscopy allows widefield fluorescence imaging with resolution beyond the classical diffraction limit. Its linear form extends resolution by a factor of two, and its nonlinear form by an in‐principle infinite factor, the effective resolution in practice being determined by noise. In this paper, we analyse the noise properties and achievable resolution of linear and nonlinear 1D and 2D patterned SIM from a frequency–space perspective. We develop an analytical theory for a general case of linear or nonlinear fluorescent imaging, and verify the analytical calculations with numerical simulation for a special case where nonlinearity is produced by photoswitching of fluorescent labels. We compare the performance of two alternative implementations, using either two‐dimensional (2D) illumination patterns or sequentially rotated one‐dimensional (ID) patterns. We show that 1D patterns are advantageous in the linear case, and that in the nonlinear case 2D patterns provide a slight signal‐to‐noise advantage under idealised conditions, but perform worse than 1D patterns in the presence of nonswitchable fluorescent background. Lay Description: Structured‐illumination microscopy (SIM) is a high‐resolution light microscopy technique that allows imaging of fluorescence at a resolution about twice the classical diffraction limit. There are various ways that the illumination can be structured, but it is not obvious how the choice of illumination pattern affects the final image quality, especially in view of the noise. We present a detailed performance analysis considering two illumination techniques: sequential illumination with line‐gratings that are shifted and rotated during image acquisition and two‐dimensional (2D) illumination structures requiring only shift operations. Our analysis is based on analytical theory, supported by simulations of images considering noise. We also extend our analysis to a nonlinear variant of SIM, with which enhanced resolution can be achieved, limited only by noise. This includes nonlinear SIM based on the light‐induced switching of the fluorescent molecules between a bright and a dark state. We find sequential illumination with line‐gratings to be advantageous in ordinary (linear) SIM, whereas 2D patterns provides a slight signal‐to‐noise advantage under idealised conditions in nonlinear SIM if there is no nonswitching background. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Microscopy is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

EphAs and ephrinAs are expressed in multiple areas of the developing brain in overlapping countergradients, notably in the retina and tectum. Here they are involved in targeting retinal axons to their correct topographic position in the tectum. We have used truncated versions of EphA3, single–amino acid point mutants of ephrinA5 and fluorescence resonance energy transfer technology to uncover a cis interaction between EphA3 and ephrinA5 that is independent of the established ligand-binding domain of EphA3. This cis interaction abolishes the induction of tyrosine phosphorylation of EphA3 and results in a loss of sensitivity of retinal axons to ephrinAs in trans. Our data suggest that formation of this complex transforms the uniform expression of EphAs in the nasal part of the retina into a gradient of functional EphAs and has a key role in controlling retinotectal mapping. [ABSTRACT FROM AUTHOR]

Copyright of Microscopy Research & Technique is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Summary By physical rotation of the sample, axial tomography enables the acquisition of otherwise inaccessible spatial information from an object. In combination with confocal microscopy, the method can fundamentally improve the effective three-dimensional (3D) resolution. In this report we present a novel method for high resolution reconstruction of confocal axial tomographic data. The method automatically determines the relative angles of rotation, aligns the data from different rotational views and reconstructs a single high resolution 3D dataset. The reconstruction makes use of a known point spread function and is based on an unconstrained maximum likelihood deconvolution performed simultaneously from multiple (in our case three) angular views. It was applied to simulated as well as to experimental confocal datasets. The gain in resolution was quantified and the effect of choice of overrelaxation factors on the speed of convergence was investigated. A clearly improved 3D resolution was obtained by axial tomography together with reconstruction as compared with reconstruction of confocal data from only a single angular view. [ABSTRACT FROM AUTHOR]

A programmable array microscope (PAM) incorporates a spatial light modulator (SLM) placed in the primary image plane of a widefield microscope, where it is used to define patterns of illumination and/or detection. We describe the characteristics of a special type of PAM collecting two images simultaneously. The conjugate image (Ic) is formed by light originating from the object plane and returning along the optical path of the illumination light. The non-conjugate image (Inc) receives light from only those regions of the SLM that are not used for illuminating the sample. The dual-signal PAM provides much more time-efficient excitation than the confocal laser scanning microscope (CLSM) and greater utilization of the available emission light. It has superior noise characteristics in comparison to single-sided instruments. The axial responses of the system under a variety of conditions were measured and the behaviour of the novel Inc image characterized. As in systems in which only Ic images are collected (Nipkow-disc microscopes, and previously characterized PAMs), the axial response to thin fluorescent films showed a sharpening of the axial response as the unit cell of the repetitive patterns decreased in size. The dual-signal PAM can be adapted to a wide range of data analysis and collection strategies. We investigated systematically the effects of patterns and unit cell dimensions on the axial response. Sufficiently sparse patterns lead to an Ic image formed by the superposition of the many parallel beams, each of which is equivalent to the single scanning spot of a CLSM. The sectioning capabilities of the system, as given by its axial responses, were similar for a given scan pattern and for processed pseudorandom sequence (PRS) scans with the same size of the unit cell. For the PRS scans, optical sectioning was achieved by a subtraction of an Inc image or, alternatively, a scaled widefield image from the Ic image. Based on the comparative noise levels of the two methods, the non-conjugate subtraction was significantly superior. A point spread function for Ic and Inc was simulated and properties of the optical transfer functions (OTFs) were compared. Simulations of the OTF in non-conjugate imaging did not suffer from the missing cone problem, enabling a high quality deconvolution of the non-conjugate side alone. We also investigated the properties of images obtained by subjecting the Ic and Inc data to a combined maximum likelihood deconvolution. [ABSTRACT FROM AUTHOR]

Fluorescent confocal laser scanning microscopy allows an improved imaging of microscopic objects in three dimensions. However, the resolution along the axial direction is three times worse than the resolution in lateral directions. A method to overcome this axial limitation is tilting the object under the microscope, in a way that the direction of the optical axis points into different directions relative to the sample. A new technique for a simultaneous reconstruction from a number of such axial tomographic confocal data sets was developed and used for high resolution reconstruction of 3D-data both from experimental and virtual microscopic data sets. The reconstructed images have a highly improved 3D resolution, which is comparable to the lateral resolution of a single deconvolved data set. Axial tomographic imaging in combination with simultaneous data reconstruction also opens the possibility for a more precise quantification of 3D data.The color images of this publication can be accessed from http://www.esacp.org/acp/2000/20-1/heintzmann.htm. At this web address an interactive 3D viewer is additionally provided for browsing the 3D data. This java applet displays three orthogonal slices of the data set which are dynamically updated by user mouse clicks or keystrokes. [ABSTRACT FROM AUTHOR]