The effects of spherical aberration on multiphoton fluorescence excitation microscopy

Multiphoton fluorescence excitation microscopy is almost invariably conducted with samples whose refractive index differ from that of the objective immersion medium, conditions that cause spherical aberration. Due to the quadratic nature of multiphoton fluorescence excitation, spherical aberration is expected to profoundly affect the depth dependence of fluorescence excitation. In order to determine the effect of refractive index mismatch in multiphoton fluorescence excitation microscopy, we measured signal attenuation, photobleaching rates and resolution degradation with depth in homogeneous samples with minimal light scattering and absorption over a range of refractive indices. These studies demonstrate that signal levels and resolution both rapidly decline with depth into refractive index mismatched samples. Analyses of photobleaching rates indicate that the preponderance of signal attenuation with depth results from decreased rates of fluorescence excitation, even in a system with a descanned emission collection pathway. Similar results were obtained in analyses of fluorescence microspheres embedded in rat kidney tissue, demonstrating that spherical aberration is an important limiting factor in multiphoton fluorescence excitation microscopy of biological samples.     

Blind deconvolution of 3D fluorescence microscopy using depth‐variant asymmetric PSF

The 3D wide‐field fluorescence microscopy suffers from depth‐variant asymmetric blur. The depth‐variance and axial asymmetry are due to refractive index mismatch between the immersion and the specimen layer. The radial asymmetry is due to lens imperfections and local refractive index inhomogeneities in the specimen. To obtain the PSF that has these characteristics, there were PSF premeasurement trials. However, they are useless since imaging conditions such as camera position and refractive index of the specimen are changed between the premeasurement and actual imaging. In this article, we focus on removing unknown depth‐variant asymmetric blur in such an optical system under the assumption of refractive index homogeneities in the specimen. We propose finding few parameters in the mathematical PSF model from observed images in which the PSF model has a depth‐variant asymmetric shape. After generating an initial PSF from the analysis of intensities in the observed image, the parameters are estimated based on a maximum likelihood estimator. Using the estimated PSF, we implement an accelerated GEM algorithm for image deconvolution. Deconvolution result shows the superiority of our algorithm in terms of accuracy, which quantitatively evaluated by FWHM, relative contrast, standard deviation values of intensity peaks and FWHM. Microsc. Res. Tech. 79:480–494, 2016. © 2016 Wiley Periodicals, Inc.     

3D deconvolution of spherically aberrated images using commercial software

Refractive index mismatch between the specimen and the objective immersion oil results in spherical aberration, which causes distortion and spreading of the point spread function, as well as incorrect readings of the axial coordinates. These effects have to be taken into account when performing three-dimensional restoration of wide-field fluorescence images. By using objects with well-defined geometry (fluorescently stained Escherichia coli or actin filaments) separated from a cover slip by a layer of oil with known refractive index, we investigated the accuracy of three-dimensional shape restoration by the commercial programs Huygens and Autoquant. Aberration correction available in the software dramatically reduced the axial blur compared to deconvolution that ignored the refractive index mismatch. At the same time, it failed to completely recover the cylindrical symmetry of bacteria or of actin fibres, which showed up to a three to five times larger width along the optical axis compared to the lateral plane. The quality of restoration was only moderately sensitive to the exact values of the specimen refractive index but in some cases improved significantly by assuming a reduced NA of the objective. Because image restoration depends on the knowledge of the vertical scale, we also performed detailed measurements of the axial scaling factor and concluded (in agreement with some previous authors) that scaling is adequately described by the simple paraxial formula, even when high-NA oil-immersion objectives are used.     

An open‐source deconvolution software package for 3‐D quantitative fluorescence microscopy imaging

Deconvolution techniques have been widely used for restoring the 3‐D quantitative information of an unknown specimen observed using a wide‐field fluorescence microscope. Deconv , an open‐source deconvolution software package, was developed for 3‐D quantitative fluorescence microscopy imaging and was released under the GNU Public License. Deconv provides numerical routines for simulation of a 3‐D point spread function and deconvolution routines implemented three constrained iterative deconvolution algorithms: one based on a Poisson noise model and two others based on a Gaussian noise model. These algorithms are presented and evaluated using synthetic images and experimentally obtained microscope images, and the use of the library is explained. Deconv allows users to assess the utility of these deconvolution algorithms and to determine which are suited for a particular imaging application. The design of Deconv makes it easy for deconvolution capabilities to be incorporated into existing imaging applications.     

An application of spatial deconvolution to a capillary-based high-pressure chamber for fluorescence microscopy imaging

Capillary‐based high‐pressure chambers for which the wall serves as both the optical window and mechanical support have been reported for fluorescence microscopy imaging. Although capillary chambers are straightforward and economical to construct, the curved capillary wall introduces image aberrations. The significance of these aberrations in imaging sub‐cellular‐dimension objects has yet to be assessed. Using a capillary chamber that is routinely pressurized to between 20 and 30 MPa, a pressure range suitable for studying a wide variety of cellular processes, we demonstrate sub‐cellular‐dimension spatial resolution in the imaging of fluorescent micro‐spheres. Objectives with a range of numerical apertures (0.5–1.3) and working distances (0.1–7.4 mm) are considered. We show that spatial (or point‐spread function, PSF) deconvolution improves image contrast in capillary‐based images by comparing deconvolution results with those obtained from slide‐mounted controls. Furthermore, similar deconvolution results between a measured PSF and a calculated, flat‐geometry PSF indicate that the capillary wall is optically flat on cellular length scales. Results here facilitate the application of contemporary techniques in fluorescence microscopy to high‐pressure imaging fields.     

The point-spread function of a confocal microscope: its measurement and use in deconvolution of 3-D data

We have measured the point-spread function (PSF) for an MRC-500 confocal scanning laser microscope using subresolution fluorescent beads. PSFs were measured for two lenses of high numerical aperture—the Zeiss plan-neofluar 63 × water immersion and Leitz plan-apo 63 × oil immersion—at three different sizes of the confocal detector aperture. The measured PSFs are fairly symmetrical, both radially and axially. In particular there is considerably less axial asymmetry than has been demonstrated in measurements of conventional (non-confocal) PSFs. Measurements of the peak width at half-maximum peak height for the minimum detector aperture gave approximately 0·23 and 0·8 μm for the radial and axial resolution respectively (4·6 and 15·9 in dimensionless optical units). This increased to 0·38 and 1·5 μm (7·5 and 29·8 in dimensionless units) for the largest detector aperture examined. The resulting optical transfer functions (OTFs) were used in an iterative, constrained deconvolution procedure to process three-dimensional confocal data sets from a biological specimen—pea root cells labelled in situ with a fluorescent probe to ribosomal genes. The deconvolution significantly improved the clarity and contrast of the data. Furthermore, the loss in resolution produced by increasing the size of the detector aperture could be restored by the deconvolution procedure. Therefore for many biological specimens which are only weakly fluorescent it may be preferable to open the detector aperture to increase the strength of the detected signal, and thus the signal-to-noise ratio, and then to restore the resolution by deconvolution.     

Artefacts in restored images due to intensity loss in three-dimensional fluorescence microscopy

Computational algorithms for three-dimensional deconvolution have proven successful in reducing blurring and improving the resolution of fluorescence microscopic images. However, discrepancies between the imaging conditions and the models on which such deconvolution algorithms are based may lead to artefacts and/or distortions in the images restored by application of the algorithms. In this paper, artefacts associated with a decrease of fluorescence intensity with time or slice in three-dimensional wide-field images are demonstrated using simulated images. Loss of intensity, whether due to photobleaching or other factors, leads to artefacts in the form of bands or stripes in the restored images. An empirical method for correcting the intensity losses in wide-field images has been implemented and used to correct biological images. This method is based on fitting a decreasing function to the slice intensity curve computed by summing all pixel values in each slice. The fitted curve is then used for the calculation of correction factors for each slice.     

The role of specimen-induced spherical aberration in confocal microscopy

We present an overview of recent theories for describing specimen-induced spherical aberration in confocal microscopy. One of these theories is used to compute numerically the role of spherical aberration in general confocal, and especially in biological confocal, microscopy for a variety of three-layer specimen structures. In particular, we study the effect of specimen-induced spherical aberration on the maximum value of the overall confocal point spread function, the accompanying focal shift and the size of the optical probe in both fluorescence and brightfield confocal microscopy.