Electromagnetic description of image formation in confocal fluorescence microscopy

Visser, Taco D.; Wiersma, Sjoerd H.

doi:10.1364/josaa.11.000599

Cited by 20 publications

(16 citation statements)

References 21 publications

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“…by using electromagnetic diffraction theory based on the actual instrument configuration. [21,[25][26][27][28] Details of the calculation procedure are presented in the Supporting Information. We then used the calculated observation volume profile in the PCH model to fit the experimental PCH.…”

Section: Deviation Of the 3 Dg Approximationmentioning

confidence: 99%

Photon Counting Histogram: One‐Photon Excitation

2004

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The photon counting histogram (PCH) analysis is a fluorescence fluctuation method that is able to characterize the brightness and concentration of different fluorescent species present in a liquid sample. We find that the PCH model using a three-dimensional Gaussian observation volume profile is inadequate for fitting experimental data obtained from a confocal setup with one-photon excitation. We propose an imoroved model, which is based on the correction to the observation volume profile for the out-of-focus emission. We demonstrate that this model is able to resolve different species present under a wide range of conditions. Attention is given to how this model allows the examination of the effects of different instrumental setups on the resolvability.

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Section: Deviation Of the 3 Dg Approximationmentioning

confidence: 99%

Photon Counting Histogram: One‐Photon Excitation

2004

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“…As a result, the image captured on the camera is a convolution of the illuminated platonic image with the PSF, and not simply the illuminated dye itself. While complicated, this PSF has been calculated exactly by many researchers for different geometries [26][27][28][29][30][31][32][33]. For microscope samples with a refractive index different from what the optical train is designed for, the PSF worsens with depth, becoming significantly broader and more aberrated.…”

Section: Resultsmentioning

confidence: 99%

Light Microscopy at Maximal Precision

et al. 2017

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Microscopy is the workhorse of the physical and life sciences, producing crisp images of everything from atoms to cells well beyond the capabilities of the human eye. However, the analysis of these images is frequently little more accurate than manual marking. Here, we revolutionize the analysis of microscopy images, extracting all the useful information theoretically contained in a complex microscope image. Using a generic, methodological approach, we extract the information by fitting experimental images with a detailed optical model of the microscope, a method we call parameter extraction from reconstructing images (PERI). As a proof of principle, we demonstrate this approach with a confocal image of colloidal spheres, improving measurements of particle positions and radii by 10-100 times over current methods and attaining the maximum possible accuracy. With this unprecedented accuracy, we measure nanometer-scale colloidal interactions in dense suspensions solely with light microscopy, a previously impossible feat. Our approach is generic and applicable to imaging methods from brightfield to electron microscopy, where we expect accuracies of 1 nm and 0.1 pm, respectively.

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“…The first is to consider the fluorescent emission as being randomly polarised. In previous publications (Van der Voort & Brakenhoff, 1990;Hell et al, 1994;Visser & Wiersma, 1994) this led to mathematical formulae in which the point spread function of the illumination and excitation optical paths were simply multiplied. The other, more rigorous, approach is to consider the mechanism of fluorescence emission as a polarisation dependent process.…”

Section: Multiphoton Fluorescence Imagingmentioning

confidence: 99%

Imaging properties of high aperture multiphoton fluorescence scanning optical microscopes

Higdon

Török

Wilson

1999

Journal of Microscopy

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SummaryA theory for multiphoton fluorescence imaging in high aperture scanning optical microscopes employing finite sized detectors is presented. The effect of polarisation of the fluorescent emission on the imaging properties of such microscopes is investigated. The lateral and axial resolutions are calculated for one-, two-and three-photon excitation of p-quaterphenyl for high and low aperture optical systems. Significant improvement in lateral resolution is found to be achieved by employing a confocal pinhole. This improvement increases with the order of the multiphoton process. Simultaneously, it is found that, when the size of the pinhole is reduced to achieve the best possible resolution, the signal-to-noise ratio is not degraded by more than 30%. The degree of optical sectioning achieved is found to improve dramatically with the use of confocal detection. For two-and three-photon excitation axial full width half-maximum improvement of 30% is predicted.

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Electromagnetic description of image formation in confocal fluorescence microscopy

Cited by 20 publications

References 21 publications

Photon Counting Histogram: One‐Photon Excitation

Photon Counting Histogram: One‐Photon Excitation

Light Microscopy at Maximal Precision

Imaging properties of high aperture multiphoton fluorescence scanning optical microscopes

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