Measurement of specimen‐induced aberrations of biological samples using phase stepping interferometry

Schwertner, Michael; Booth, Martin J.; Neil, Mark A. A.; Wilson, Tony

doi:10.1111/j.1365-2818.2004.01267.x

Cited by 75 publications

(47 citation statements)

References 16 publications

(19 reference statements)

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“…For example, one of the most widely applied microscopy techniques for in vivo imaging, two-photon fluorescence microscopy, often uses water-dipping objectives. Because biological samples are comprised of structures (i.e., proteins, nuclear acids, and lipids) with refractive indices different from that of water, they induce optical aberrations to the incoming excitation wave and result in an enlarged focal spot within the sample and a concomitant deterioration of signal and resolution (2,3). As a result, the resolution and contrast of optical microscopes is compromised in vivo, especially deep in tissue.…”

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Characterization and adaptive optical correction of aberrations during in vivo imaging in the mouse cortex

Satō

Betzig

2011

Proc. Natl. Acad. Sci. U.S.A.

183

158

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The signal and resolution during in vivo imaging of the mouse brain is limited by sample-induced optical aberrations. We find that, although the optical aberrations can vary across the sample and increase in magnitude with depth, they remain stable for hours. As a result, two-photon adaptive optics can recover diffraction-limited performance to depths of 450 μm and improve imaging quality over fields of view of hundreds of microns. Adaptive optical correction yielded fivefold signal enhancement for small neuronal structures and a threefold increase in axial resolution. The corrections allowed us to detect smaller neuronal structures at greater contrast and also improve the signal-to-noise ratio during functional Ca 2 imaging in single neurons.T he ability to visualize biological systems in vivo has been a major attraction of optical microscopy, because studying biological systems as they evolve in their natural, physiological state provides relevant information that in vitro preparations often do not allow (1). However, for conventional optical microscopes to achieve their optimal, diffraction-limited resolution, the specimen needs to have identical optical properties to those of the immersion media for which the microscope objective is designed. For example, one of the most widely applied microscopy techniques for in vivo imaging, two-photon fluorescence microscopy, often uses water-dipping objectives. Because biological samples are comprised of structures (i.e., proteins, nuclear acids, and lipids) with refractive indices different from that of water, they induce optical aberrations to the incoming excitation wave and result in an enlarged focal spot within the sample and a concomitant deterioration of signal and resolution (2, 3). As a result, the resolution and contrast of optical microscopes is compromised in vivo, especially deep in tissue.Many questions related to how the brain processes information on both the neuronal circuit level and the cell biological level can be addressed by observing the morphology and activity of neurons inside a living and, preferably, awake and behaving mouse (1). In a typical experiment, an area of the skull is surgically removed and replaced with a cover glass to provide optical access to the underlying structure of interest (4). For imaging during behavior, the cover glass is often attached to an optically transparent plug embedded in the skull to improve mechanical stability and to prevent the skull from growing back and blocking the optical access (5, 6) (Fig. 1A). Before the excitation light of a two-photon microscope reaches the desired focal plane inside the brain, it has to traverse first the cranial window and then the brain tissue, both with optical properties different from water. Thus, they both impart optical aberrations on the excitation light, which leads to a distorted focus, even at the surface of the brain.These sample-induced aberrations can be corrected with adaptive optics (AO) to recover diffraction-limited resolution. In AO, a wavefront-shaping device m...

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mentioning

confidence: 99%

Characterization and adaptive optical correction of aberrations during in vivo imaging in the mouse cortex

Satō

Betzig

2011

Proc. Natl. Acad. Sci. U.S.A.

183

158

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“…With practical specimens, the induced aberrations generally consist of combinations of aberration modes (Schwertner et al, 2004). The effects of multiple aberration modes on confocal/two-photon imaging of the four structures were also modelled.…”

Section: Confocal and Two-photon Fluorescence Microscopesmentioning

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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“…Both versions are listed. For more information on Zernike polynomials see Noll (1976), Born & Wolf (1983) and Schwertner et al (2004). …”

Section: Appendix: Zernike Polynomialsmentioning

confidence: 99%

“…A mouse oocyte cell and an optical fibre are modelled and the simulations are compared with experimental results. The interferometer set-up we used for the direct measurements of the specimen-induced aberrations is described in Schwertner et al (2004).…”

Section: Introductionmentioning

confidence: 99%

Simulation of specimen‐induced aberrations for objects with spherical and cylindrical symmetry

Schwertner

Booth

Wilson

2004

Journal of Microscopy

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SummaryWavefront aberrations caused by the refractive index structure of the specimen are known to compromise signal intensity and three-dimensional resolution in confocal and multiphoton microscopy. However, adaptive optics can measure and correct specimen-induced aberrations. For the design of an adaptive optics system, information on the type and amount of the aberration is required. We have previously described an interferometric set-up capable of measuring specimen-induced aberrations and a method for the extraction of the Zernike mode content. In this paper we have modelled specimen-induced aberrations caused by spherical and cylindrical objects using a ray tracing method. The Zernike mode content of the wavefronts was then extracted from the simulated wavefronts and compared with experimental results. Aberrations for a simple model of an oocyte cell consisting of two spherical regions and for a model of a well-characterized optical fibre are calculated. This simple model gave Zernike mode data that are in good agreement with experimental results.

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Measurement of specimen‐induced aberrations of biological samples using phase stepping interferometry

Cited by 75 publications

References 16 publications

Characterization and adaptive optical correction of aberrations during in vivo imaging in the mouse cortex

Characterization and adaptive optical correction of aberrations during in vivo imaging in the mouse cortex

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

Simulation of specimen‐induced aberrations for objects with spherical and cylindrical symmetry

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