Characterizing specimen induced aberrations for high NA adaptive optical microscopy

Schwertner, Michael; Booth, Martin J.; Wilson, Tony

doi:10.1364/opex.12.006540

Cited by 139 publications

(92 citation statements)

References 28 publications

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“…One should therefore expect reduced performance both in terms of transmission and aberrations. Furthermore, aberrations are introduced by the specimens themselves, particularly by thick specimens to which HGM is often applied [5]. As the harmonic generation is nonlinearly dependent on the excitation intensity, these factors could have a significant effect on the imaging performance.…”

Section: Introductionmentioning

confidence: 99%

“…In early-stage mouse embryos, THG images reveal cellular structure and sub-cellular features, whereas SHG has shown mitotic spindles and the zona pellucida. It has also been shown that large aberrations are induced when focussing through embryos [5]. When combined with the system aberrations arising from the nonoptimal objective lenses, the effects can cause a significant reduction in signal level and resolution.…”

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Adaptive harmonic generation microscopy of mammalian embryos

et al. 2009

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Adaptive optics is implemented in a harmonic generation microscope using a wavefront sensorless correction scheme. Both the second-and third-harmonic intensity signals are used as the optimization metric. Aberration correction is performed to compensate both system-and specimen-induced aberrations by using an efficient optimization routine based upon Zernike polynomial modes. Images of live mouse embryos show an improved signal level and resolution.Harmonic generation microscopy (HGM) was proposed as a method for label-free imaging of biological specimens [1]. Using the nonlinear optical properties of the tissue, third-and second-harmonic generation (THG, SHG) are produced at the focus of a short-pulsed laser beam, revealing cellular structure with three-dimensional resolution. Implementations of combined SHG and THG imaging have often used excitation wavelengths around 1200 nm [2][3][4]. This choice was influenced by the relatively low absorption of this wavelength by biological tissues and the convenient placement of the SHG and THG around 600 and 400 nm, respectively, permitting the use of standard optics and detectors. A disadvantage of this excitation wavelength is that high-numerical-aperture objectives required for such imaging are not typically specified for this wavelength. One should therefore expect reduced performance both in terms of transmission and aberrations. Furthermore, aberrations are introduced by the specimens themselves, particularly by thick specimens to which HGM is often applied [5]. As the harmonic generation is nonlinearly dependent on the excitation intensity, these factors could have a significant effect on the imaging performance.One application area where HGM has shown promise is in imaging for developmental biology [3,2,4]. In early-stage mouse embryos, THG images reveal cellular structure and sub-cellular features, whereas SHG has shown mitotic spindles and the zona pellucida. It has also been shown that large aberrations are induced when focussing through embryos [5]. When combined with the system aberrations arising from the nonoptimal objective lenses, the effects can cause a significant reduction in signal level and resolution. Adaptive optics has been used to compensate for aberrations in various microscopes [6]. In this Letter we demonstrate the use of adaptive optics for the correction of system and specimen-induced aberrations in the HGM of live mouse embryos. OCIS codes: 180.0180, 180.4315, 110.1080, 170.3880, 180.6900, 190.4160. A schematic of the adaptive microscope is shown in Fig. 1. The chromium forsterite laser (Mavericks, Del Mar Photonics) emits 65 fs pulses at a repetition rate of 100 MHz, center wavelength λ=1235 nm and output power around 200 mW. The expanded beam was steered by two-axis galvanometer mirrors that are imaged onto a deformable membrane mirror (MIRAO 52-e, Imagine Eyes). The deformable mirror (DM) was imaged onto the pupil plane of the microscope objective. The illumination power in the focus was approximately 30 mW. The harmonic emiss...

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Section: Introductionmentioning

confidence: 99%

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Adaptive harmonic generation microscopy of mammalian embryos

et al. 2009

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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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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.

184

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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“…It is assumed that the wavefront aberration can be represented by a finite low-order Zernike aberrations [13,26,27], then it is possible that the DM can generate these low-order Zernike modes efficiently and Δφ x (ξ , η) can be neglected. As a result, Eq.…”

Section: Modeling Of the Wfsless Ao Systemmentioning

confidence: 99%

Model-based aberration correction in a closed-loop wavefront-sensor-less adaptive optics system

et al. 2010

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Abstract:In many scientific and medical applications, such as laser systems and microscopes, wavefront-sensor-less (WFSless) adaptive optics (AO) systems are used to improve the laser beam quality or the image resolution by correcting the wavefront aberration in the optical path. The lack of direct wavefront measurement in WFSless AO systems imposes a challenge to achieve efficient aberration correction. This paper presents an aberration correction approach for WFSlss AO systems based on the model of the WFSless AO system and a small number of intensity measurements, where the model is identified from the input-output data of the WFSless AO system by black-box identification. This approach is validated in an experimental setup with 20 static aberrations having Kolmogorov spatial distributions. By correcting N = 9 Zernike modes (N is the number of aberration modes), an intensity improvement from 49% of the maximum value to 89% has been achieved in average based on N + 5 = 14 intensity measurements. With the worst initial intensity, an improvement from 17% of the maximum value to 86% has been achieved based on N + 4 = 13 intensity measurements.

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Characterizing specimen induced aberrations for high NA adaptive optical microscopy

Cited by 139 publications

References 28 publications

Adaptive harmonic generation microscopy of mammalian embryos

Adaptive harmonic generation microscopy of mammalian embryos

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

Model-based aberration correction in a closed-loop wavefront-sensor-less adaptive optics system

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