Enhancing the Axial Resolution in Far-field Light Microscopy: Two-photon 4Pi Confocal Fluorescence Microscopy

Hell, Stefan W.; Lindek, Steffen; Stelzer, Ernst H. K.

doi:10.1080/09500349414550701

Cited by 81 publications

(45 citation statements)

References 7 publications

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“…This limit, as proposed by Abbe, is approximately half the wavelength of the excitation light [31]. The axial resolution is approximately twice as long as the lateral resolution, or approximately equal to the wavelength of the excitation light [32]. This axial confinement of the excitation spot lends two-photon laser scanning microscopy the same optical sectioning ability as confocal microscopy [25].…”

Section: Two-photon Laser Scanning Microscopymentioning

confidence: 98%

Two-photon in vivo imaging of cells

Christensen

Nedergaard

2011

Pediatr Nephrol

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In vivo imaging of cells gives a glimpse into the world of biology in a natural setting unparalleled by any other venue. Two-photon imaging of fluorescently labeled cells has become the standard to obtain high-resolution, dynamic images of living specimens with great specificity. This review focuses on providing the reader with a short history of, and impetus behind, two-photon imaging, its working mechanics, and emerging technologies related to biological multiphoton imaging.

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Section: Two-photon Laser Scanning Microscopymentioning

confidence: 98%

Two-photon in vivo imaging of cells

Christensen

Nedergaard

2011

Pediatr Nephrol

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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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“…17 This approach when used in combination with multiphoton excitation reduces the presence of axial side lobes and provides a higher contrast of the interference pattern. 18 Combining confocalisation and multiphoton excitation in this way is known as multiphoton 4Pi confocal microscopy. In addition it was found that the utilisation of specific imaging algorithms i.e.…”

Section: The Use Of Linear Processesmentioning

confidence: 99%

Beyond the diffraction limit: far-field fluorescence imaging with ultrahigh resolution

Rice

2007

Mol. BioSyst.

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Fluorescence microscopy is an important and extensively utilised tool for imaging biological systems. However, the image resolution that can be obtained has a limit as defined through the laws of diffraction. Demand for improved resolution has stimulated research into developing methods to image beyond the diffraction limit based on far-field fluorescence microscopy techniques. Rapid progress is being made in this area of science with methods emerging that enable fluorescence imaging in the far-field to possess a resolution well beyond the diffraction limit. This review outlines developments in far-field fluorescence methods which enable ultrahigh resolution imaging and application of these techniques to biology. Future possible trends and directions in far-field fluorescence imaging with ultrahigh resolution are also outlined.

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Enhancing the Axial Resolution in Far-field Light Microscopy: Two-photon 4Pi Confocal Fluorescence Microscopy

Cited by 81 publications

References 7 publications

Two-photon in vivo imaging of cells

Two-photon in vivo imaging of cells

Imaging properties of high aperture multiphoton fluorescence scanning optical microscopes

Beyond the diffraction limit: far-field fluorescence imaging with ultrahigh resolution

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