The role of specimen‐induced spherical aberration in confocal microscopy

Török, Péter; Hewlett, S. J.; Варга, П.

doi:10.1046/j.1365-2818.1997.2440802.x

Cited by 60 publications

(32 citation statements)

References 20 publications

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“…This causes the light rays to be focused at multiple levels within the sample to create images that suffer from reduced intensity and degraded resolution. Spherical aberration increases commensurate with depth of focus relative to the coverglass and becomes more significant with high NA objectives (Carlsson, 1991, Hell et al 1993, Torok et al, 1997, Egner and Hell, 2006. Backscatter of excitation and fluorescent emissions at these interfaces additionally contributes to loss of signal intensity and contrast.…”

Section: Discussionmentioning

confidence: 99%

Three-dimensional imaging of the intact mouse cochlea by fluorescent laser scanning confocal microscopy

MacDonald

Rubel

2008

Hearing Research

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The complex anatomy of the mammalian cochlea is most readily understood by representation in three-dimensions. However, the cochlea is often sectioned to minimize the effects of its anatomic complexity and optical properties on image acquisition by light microscopy. We have found that optical aberrations present in the decalcified cochlea can be greatly reduced by dehydration through graded ethanols followed by clearing with a mixture of 5 parts methyl salicylate and 3 parts benzyl benzoate (MSBB). Clearing the cochlea with MSBB enables acquisition of high-resolution images with multiple fluorescent labels, through the full volume of the cochlea by laser scanning confocal microscopy. The resulting images are readily applicable to three-dimensional morphometric analysis and volumetric visualizations. This method promises to be particularly useful for three-dimensional characterization of anatomy, innervation and expression of genes or proteins in the many new animal models of hearing and balance generated by genetic manipulation. Furthermore, the MSBB is compatible with most non-protein fluorophores used for histological labeling, and may be removed with traditional transitional solvents to allow subsequent epoxy embedding for sectioning.

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Section: Discussionmentioning

confidence: 99%

Three-dimensional imaging of the intact mouse cochlea by fluorescent laser scanning confocal microscopy

MacDonald

Rubel

2008

Hearing Research

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“…A few studies have been done on the role of the specimen-induced degradation of the performance. 6,7 Here we consider the effect of a refractive-index-mismatched specimen on the performance of the reflecting optical system by evaluating the rms wave front error on the surface of the spherical mirror ͑Fig. 2͒.…”

Section: Improvement Of Detected Intensity In Confocal Microscopy By mentioning

confidence: 99%

“…However, the optical probe is distorted as the probe depth increases. 6 This imposes a real limitation on the possible thickness of the specimen. As can be seen from the discussion above, the allowable thickness of the specimen is limited, but not by the reflecting optical system, when autofocusing is used.…”

Section: Improvement Of Detected Intensity In Confocal Microscopy By mentioning

confidence: 99%

Improvement of detected intensity in confocal microscopy by using reflecting optical system

Kang

Seo

Gweon

2004

Review of Scientific Instruments

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In confocal microscopy, the level of the detected intensity is low, which reduces the contrast of the images. We propose a reflecting optical system to improve the detected intensity. The result of the optimal design is presented and the performance of the resulting reflecting optical system is evaluated. A numerical simulation of the effect of the reflecting optical system on the total system performance is also presented. A standard specimen is imaged to show the benefits of the proposed reflecting optical system. The results in this Note demonstrate a 37% increase of the detected intensity and better contrast of the image.

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“…Due to the rotation of the capillary in micro axial tomography, the microscopical objects can be imaged in varying depths of focus. Since an increasing amount of monochromatic aberrations with increasing depth of focus was observed both practically and theoretically [12,18], an effect on the chromatic aberration had to be expected.…”

Section: Micro Axial Tomography (Mat)mentioning

confidence: 99%

<title>Correlation of chromatic shifts and focal depth in spectral precision distance microscopy measured by microaxial tomography</title>

et al. 1999

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Confocal laser scanning fluorescence microscopy is presently being used widely in biomedical research. A severe limitation for its use is its often still insufficient resolution. In situ measurements in 3D conserved human cell nuclei showed that distance measurements between fluorescent targets located in the interior of such objects are limited to a resolution regime of appr. O.3 tm in lateral and appr. tm in axial direction. A technique to overcome these restrictions is the recently developed Spectral Precision Distance Microscopy (SPM). This approach allows the determination of distances between targets which cany different spectral signatures with high precision. In situ measurements revealed that the SPM approach allows the determination of distances in 3D intact cell nuclei with a "Resolution Equivalent" better than 50 nm. Here we present an improved chromatic shift calibration method for Spectral Precision Distance Microscopy. Furthermore, micro axial tomography allows the tilting of objects perpendicular to the optical axis; thus two objects can always be tilted in such a way that they can be recorded in the same focal plane. Therefore the error in distance determination is minimized. Here we present some preliminary data for the applicability of spectral precision distance microscopy (SPM) to micro axial microscopy.

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The role of specimen‐induced spherical aberration in confocal microscopy

Cited by 60 publications

References 20 publications

Three-dimensional imaging of the intact mouse cochlea by fluorescent laser scanning confocal microscopy

Three-dimensional imaging of the intact mouse cochlea by fluorescent laser scanning confocal microscopy

Improvement of detected intensity in confocal microscopy by using reflecting optical system

<title>Correlation of chromatic shifts and focal depth in spectral precision distance microscopy measured by microaxial tomography</title>

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