Influence of refractive-index mismatch in high-resolution three-dimensional confocal microscopy

Diaspro, Alberto; Federici, Federico; Robello, Mauro

doi:10.1364/ao.41.000685

Cited by 108 publications

(114 citation statements)

References 20 publications

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“…This has been measured using subresolution beads either embedded in optical cement [10] or fixed to a tilted surface [28]. However, both of these measurements use beads placed at random depths to measure this effect.…”

Section: Discussionmentioning

confidence: 99%

A Quantitative Study of the Quality of Deconvolved Wide-field Microscopy Images as Function of Empirical Three-dimensional Point Spread Functions

Adur

Vicente

Zamboni

et al. 2011

Journal of the Optical Society of Korea

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In this work, for the first time, the quality of restoration in wide-field microscopy images after deconvolution was analyzed as a function of different Point Spread Functions using one deconvolution method, on a specimen of known size and on a biological specimen. The empirical Point Spread Function determination can significantly depend on the numerical aperture, refractive index of the embedding medium, refractive index of the immersion oil and cover slip thickness. The influence of all of these factors is shown in the same article and using the same microscope. We have found that the best deconvolution results are obtained when the empirical PSF utilized is obtained under the same conditions as the specimen. We also demonstrated that it is very important to quantitatively check the process' outcome using several quality indicators: Full-Width at Half-Maximum, Contrast-to-Noise Ratio, Signal-to-Noise Ratio and a Tenengrad-based function. We detected a significant improvement when using an indicator to measure the focus of the whole stack. Therefore, to qualitatively determinate the best deconvolved image between different conditions, one approach that we are pursuing is to use Tenengrad-based function indicators in images obtained using a wide-field microscope.

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Section: Discussionmentioning

confidence: 99%

A Quantitative Study of the Quality of Deconvolved Wide-field Microscopy Images as Function of Empirical Three-dimensional Point Spread Functions

Adur

Vicente

Zamboni

et al. 2011

Journal of the Optical Society of Korea

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show abstract

“…This distortion is manifested as a simple elongation resulting from the unequal movement of focal plane and objective (Diaspro et al, 2002).…”

Section: Discussionmentioning

confidence: 99%

“…2) (Diaspro et al, 2002). Because each of these aberrations shares a common source, it is difficult and often impossible to accurately represent the axial dimensions in the 3D solid model through computational or optical methods alone.…”

Section: Discussionmentioning

confidence: 99%

High-resolution solid modeling of biological samples imaged with 3D fluorescence microscopy

Ferko

Patterson

Butler

2006

Microsc. Res. Tech.

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Optical-sectioning, digital fluorescence microscopy provides images representing temporally-and spatially-resolved molecular-scale details of the substructures of living cells. To render such images into solid models for further computational analyses, we have developed an integrated system of image acquisition, processing, and rendering, which includes a new empirical technique to correct for axial distortions inherent in fluorescence microscopy due to refractive index mismatches between microscope objective immersion medium, coverslip glass, and water. This system takes advantage of the capabilities of ultra-high numerical aperture objectives (e.g. total internal reflection fluorescence microscopy) and enables faithful three-dimensional rendering of living cells into solid models amenable to further computational analysis. An example of solid modeling of bovine aortic endothelial cells and their nuclei is presented. Since many cellular level events are temporally and spatially confined, such integrated image acquisition, processing, rendering, and computational analysis, will enable, in silico, the generation of new computational models for cell mechanics and signaling.

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“…These aberrations limit the ability of microscopes to image deep within thick specimens (Diaspro et al, 2002), although recent developments in adaptive optics for aberration correction have led to improvements (Booth, 2007;Girkin et al, 2009;Ji et al, 2010). The effects of aberrations on microscope images include reduction in resolution, blurring and reduction of intensity.…”

Section: Introductionmentioning

confidence: 99%

Effects of aberrations and specimen structure in conventional, confocal and two‐photon fluorescence microscopy

Simmonds

Wilson

Booth

2011

Journal of Microscopy

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SummarySpecimen-induced aberrations cause a reduction in signal levels and resolution in fluorescence microscopy. Aberrations also affect the image contrast achieved by these microscopes. We model the effects of aberrations on the fluorescence signals acquired from different specimen structures, such as pointlike, linear, planar and volume structures, when imaged by conventional, confocal and two-photon microscopes. From this we derive the image contrast obtained when observing combinations of such structures. We show that the effect of aberrations on the visibility of fine features depends upon the specimen morphology and that the contrast is less significantly affected in microscopes exhibiting optical sectioning. For example, we show that point objects become indistinguishable from background fluorescence in the presence of aberrations, particularly when imaged in a conventional fluorescence microscope. This demonstrates the significant advantage of using confocal or two-photon microscopes over conventional instruments when aberrations are present.

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Influence of refractive-index mismatch in high-resolution three-dimensional confocal microscopy

Cited by 108 publications

References 20 publications

A Quantitative Study of the Quality of Deconvolved Wide-field Microscopy Images as Function of Empirical Three-dimensional Point Spread Functions

A Quantitative Study of the Quality of Deconvolved Wide-field Microscopy Images as Function of Empirical Three-dimensional Point Spread Functions

High-resolution solid modeling of biological samples imaged with 3D fluorescence microscopy

Effects of aberrations and specimen structure in conventional, confocal and two‐photon fluorescence microscopy

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