Wide-field fluorescence microscopy is an essential tool in modern cell biology. Unfortunately the image quality of fluorescence microscopes is often significantly degraded due to aberrations that occur under normal imaging conditions. In this article, we examine the use of adaptive optics technology to dynamically correct these problems to achieve close to ideal diffraction limited performance. Simultaneously, this technology also allows ultra-rapid focusing without having to move either the stage or the objective lens. We perform optical simulations to demonstrate the degree of correction that can be achieved.
"The principle of MCAO, though well understood in the astronomy community, seems to be less appreciated in the microscopy community . A few reports have discussed various benefits of MCAO in the context of microscopy  , though these have relied on numerical simulation only. MCAO has also been used in retinal imaging applications  and in benchtop experiments designed to simulate astronomical imaging . "
[Show abstract][Hide abstract] ABSTRACT: The imaging performance of an optical microscope can be degraded by
sample-induced aberrations. A general strategy to undo the effect of these
aberrations is to apply wavefront correction with a deformable mirror (DM). In
most cases, the DM is placed conjugate to the microscope pupil, called pupil
adaptive optics (AO). When the aberrations are spatially variant, an
alternative configuration involves placing the DM conjugate to the main source
of aberrations, called conjugate AO. We provide theoretical and experimental
comparison of both configurations for the simplified case where spatially
variant aberrations are produced by a well defined phase screen. We pay
particular attention to the resulting correction field of view (FOV). Conjugate
AO is found to provide a significant FOV advantage. While this result is well
known in the astronomy community, our goal here is to recast it specifically
for the optical microscopy community.
"Such WF can be corrected in the excitation beam , in the collected signal beam or in both. For example, in wide field techniques such as PALM or STORM, only the correction of the collected signal is important in order to maintain the contrast of the acquired images . In confocal microscopy, both the excitation and collected beams need to be compensated to improve the intensity of the reconstructed images [10,11]. "
[Show abstract][Hide abstract] ABSTRACT: We demonstrate that sample induced aberrations can be measured in a nonlinear microscope. This uses the fact that two-photon excited fluorescence naturally produces a localized point source inside the sample: the nonlinear guide-star (NL-GS). The wavefront emitted from the NL-GS can then be recorded using a Shack-Hartmann sensor. Compensation of the recorded sample aberrations is performed by the deformable mirror in a single-step. This technique is applied to fixed and in vivo biological samples, showing, in some cases, more than one order of magnitude improvement in the total collected signal intensity.
"corrected for a single depth. Researchers have proposed numerous ways to reduce aberrations, including introducing additional optics (akin to the cover slip corrector found on some objectives), changing the index of refraction of the immersion medium, or employing computer-controlled deformable mirrors (Albert et al., 2000; Booth et al., 2002; Sherman et al., 2002; Kam et al., 2007; Wright, 2007). However, these techniques offer only a limited ability to correct for aberrations that vary spatially. "
[Show abstract][Hide abstract] ABSTRACT: By inserting a microlens array at the intermediate image plane of an optical microscope, one can record four-dimensional light fields of biological specimens in a single snapshot. Unlike a conventional photograph, light fields permit manipulation of viewpoint and focus after the snapshot has been taken, subject to the resolution of the camera and the diffraction limit of the optical system. By inserting a second microlens array and video projector into the microscope's illumination path, one can control the incident light field falling on the specimen in a similar way. In this paper, we describe a prototype system we have built that implements these ideas, and we demonstrate two applications for it: simulating exotic microscope illumination modalities and correcting for optical aberrations digitally.
Journal of Microscopy 09/2009; 235(2):144-62. DOI:10.1111/j.1365-2818.2009.03195.x · 2.33 Impact Factor
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