Imaging Calcium in Neurons

In the field of biomedical optics, optical scattering has traditionally limited the range of imaging within tissue to a depth of one millimetre. A recently developed class of wavefront-shaping techniques now aims to overcome this limit and achieve diffraction-limited control of light beyond one centimetre. By manipulating the spatial profile of an optical field before it enters a scattering medium, it is possible to create a micrometre-scale focal spot deep within tissue. To successfully operate in vivo, these wavefront-shaping techniques typically require feedback from within the biological sample. This Review summarizes recently developed 'guidestar' mechanisms that provide feedback for intra-tissue focusing. Potential applications of guidestar-assisted focusing include optogenetic control over neurons, targeted photodynamic therapy and deep tissue imaging.

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Guidestar-assisted wavefront-shaping methods for focusing light into biological tissue

X-ray sources such as free-electron lasers offer the potential to study matter at unprecedented spatial and temporal resolution. But that potential is limited by the poor quality of conventional X-ray optical elements. An in situ technique that corrects for wavefront aberrations and allows X-rays to be focused to a spot just 7 nm wide could provide a solution.

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Breaking the 10 nm barrier in hard-X-ray focusing

The ability to dynamically image features deep within living organisms, permitting real-time analysis of cellular structure and function, is important for biological science. This Review article discusses multiphoton microscopy capable of such analysis, along with technologies that are pushing the limits of phenomena that can be quantitatively imaged.

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Advances in multiphoton microscopy technology

The imaging properties of optical microscopes are often compromised by aberrations that reduce image resolution and contrast. Adaptive optics technology has been employed in various systems to correct these aberrations and restore performance. This has required various departures from the traditional adaptive optics schemes that are used in astronomy. This review discusses the sources of aberrations, their effects and their correction with adaptive optics, particularly in confocal and two-photon microscopes. Different methods of wavefront sensing, indirect aberration measurement and aberration correction devices are discussed. Applications of adaptive optics in the related areas of optical data storage, optical tweezers and micro/nanofabrication are also reviewed.

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Adaptive optics in microscopy

Summary We demonstrate adaptive aberration correction for depthinduced spherical aberration in a multiphoton scanning microscope with a micromachined deformable mirror. Correction was made using a genetic learning algorithm with two-photon fluorescence intensity feedback to determine the desired shape for an adaptive mirror. For a 40 × /0.6 NA long working distance objective, the axial scanning range was increased from 150 mm to 600 mm.

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Adaptive correction of depth-induced aberrations in multiphoton scanning microscopy using a deformable mirror

Off-axis aberrations in a beam-scanning multiphoton confocal microscope are corrected with a deformable mirror. The optimal mirror shape for each pixel is determined by a genetic learning algorithm, in which the second-harmonic or two-photon fluorescence signal from a reference sample is maximized. The speed of the convergence is improved by use of a Zernike polynomial basis for the deformable mirror shape. This adaptive optical correction scheme is implemented in an all-reflective system by use of extremely short (10-fs) optical pulses, and it is shown that the scanning area of an f:1 off-axis parabola can be increased by nine times with this technique.

Smart microscope: an adaptive optics learning system for aberration correction in multiphoton confocal microscopy.

Summary form only given. In normal operation with a dry or oil immersion microscope objective there is an index mismatch with a water-based biological sample. This index mismatch causes a depth dependant spherical aberration that severely limits the range that a sample can be imaged along the optical axis. We have previously demonstrated the use of a membrane deformable mirror to correct for off-axis aberrations in a confocal microscopy system. In this study we apply a genetic learning algorithm in conjunction with third harmonic generation to determine the appropriate shape of the deformable mirror to correct for aberrations in longitudinal depth scanning in a confocal microscope.

L. Sherman

Papers

Adaptive correction of depth-induced aberrations in multiphoton scanning microscopy using a deformable mirror

Smart microscope: an adaptive optics learning system for aberration correction in multiphoton confocal microscopy.

Adaptive aberration correction for specimen induced spherical aberration