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Lizards are often found feeding on the tasty insect snacks that gather around lights. It also creates the opportunity for interactions between animals that would never meet in the dark conditions of previous centuries. Some lizards get a double benefit, basking in the warmth given off by artificial lights incandescent lights are really inefficient; most of the energy they use goes to producing heat, not light.

Night lights make birds sing at odd hours, and mess with their mating and migration schedules. During migration, birds are drawn to the light from tall buildings and towers, resulting in deadly collisions. Sea turtles are perhaps the most famous example of animals affected by light. Hatchlings swim towards the light—historically the horizon above the ocean. But now that beaches are backed by well-lit condos and hotels, baby turtles crawl further onshore instead of out to sea.

There is evidence that sea turtles are drawn to light with short wavelengths, and using bulbs with longer wavelengths or using filters that cut out short light wavelengths reduces the number of baby turtles crawling in the wrong direction. Simple fixes, like different bulbs or shades that focus light on the ground, can do a lot to re-darken the night sky, as does turning off unnecessary lighting.

Dimmer, more efficient bulbs that provide enough light for human needs—but not too much—are a step in the right direction, and can save on energy costs. Dark sky conservation and stricter lighting ordinances will help. I was surprised by the negative health effects of exposure to nighttime lighting—something to keep in mind as I work late into the night, basking like a lizard in the glow of my computer screen.

Jennifer Skene develops curriculum on climate change and ocean sciences at the Lawrence Hall of Science and teaches biology and science communication at Mills College and the University of California Berkeley. She has a degree in biology from Brown University and a Ph. Explore: Environment , breast cancer , cancer , ecology , light , light bulb , light pollution , night sky. It is this unpredictable nature that means that adaptive correction, where aberrations are both measured and corrected, is necessary to ensure optimal imaging throughout a specimen.

A current area of interest is how spatial variations in aberration affect AO microscopy. Most demonstrations of AO microscopy have made the implicit assumption that aberrations do not change over the field of view—an assumption that is often not valid. Examples of spatial aberration variations are illustrated in Figure 2. Poland et al. It was shown that higher image quality could be obtained in principle by changing the aberration correction as the microscope scanned across the specimen.

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However, this is unlikely to be a realistic imaging mode as the bandwidth of available correction elements is much slower than the pixel rates of typical scanning microscopes. Zeng et al.

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Multiconjugate adaptive optics, a method already used in astronomy, has been proposed as an alternative method for dealing with spatial aberration variation. Multiconjugate adaptive optics employs multiple correction devices, which are placed in planes conjugate to different depths in the specimen. This configuration means that the correction aberration varies, in a scanning microscope, as the laser is scanned across the specimen. Simulations have shown the potential benefit of this approach, although practical implementations are still to be seen.

Spatial variation of aberrations in specimens. The yellow arrow shows the two depths for which aberration correction was optimized. The signal falls significantly away from the corrected depth. Recent progress in AO microscopy has been enabled by two main factors. The first factor is the commercial availability of compact deformable mirrors and spatial light modulators as adaptive correction elements.

The second factor is advances in aberration measurement techniques and associated control methods. Indeed, aberration measurement and control have long been the limiting factors in the performance of AO microscopes. The past few years have seen significant developments in this area, which can be broadly categorized into direct wavefront measurement or indirect aberration optimisation. In this section we provide a brief review of the advances made in the past few years in these enabling methods. Most AO microscopes have employed a DM as the adaptive correction element.

The main advantages of the DM are its polarisation and wavelength-independent operation and high optical efficiency. This is well suited to use in a microscope, where the design frequently requires the use of multiple illumination and detection wavelengths. This is particularly important in fluorescence mode, where the emission is broadband and unpolarized.

The various available DM models operate on different actuation principles, such as electrostatic, electromagnetic or piezoelectric, and a range of diameters and actuator numbers is available.

Mirror coatings can be chosen to optimize operation at particular wavelengths or across a spectral range. Some models are available with protective windows, which are beneficial in preventing dust contamination; the delicate nature of the DMs means that contact cleaning is effectively impossible without damaging the mirror surface. The choice of drive electronics and computer interface can be important in applications where fast operation is required.

The DM diameter has an influence on the microscope design, as it is important to match the pupils of the DM and the objective lens with a re-imaging system of appropriate magnification. A larger actuator number means more degrees of freedom for aberration correction.

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However, this does not necessarily mean that the aberration correction will be better for a particular application. For example, an actuator on a membrane mirror creates a deformation that spreads across the whole mirror surface. The deformation from each actuator therefore overlaps significantly with the deformation from all of the other actuators. On a microstructured mirror, the influence of the actuator is much more confined, meaning that there is only localized overlap between deformations from adjacent actuators. On the one hand, a membrane DM typically has a large maximum stroke and can produce large amounts of low order aberration modes, which may be advantageous for some applications.

A microstructured DM, on the other hand, can typically produce more limited amplitudes of the low order modes, but with the benefit of a wider range of higher order modes. Due to the coupling between actuators of a DM, it is usually necessary to perform a calibration or training step. This provides the information required to drive the DM with chosen aberration modes, rather than directly with individual actuator signals.

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A range of such methods have been developed specifically for use in microscopes, using interferometry, 23 image based measurements 24 , 25 and phase diversity. They do, however, have many additional uses in microscopy.

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This is applicable, for example, in multiphoton microscopes. SLMs have greater flexibility than DMs in the complexity of phase patterns that they can create due to their large pixel count. Direct wavefront sensing has always been a challenge in microscopy due to the complex three-dimensional nature of specimens. Wavefront sensors, such as the Shack-Hartmann sensor, are designed to operate with well-defined wavefronts from, for example, an isolated point source of light. In microscopes, light typically emanates from multiple points in the specimen simultaneously, whether from scatterers throughout the illumination cone in reflection microscopy, or from multiple fluorophores in fluorescence microscopy.

The superposition of many wavefronts creates ambiguous measurements, as the desired light from the focal point is swamped by out-of-focus background. Several methods have been introduced to overcome this problem when using Shack-Hartmann sensors. Each method relies upon the exclusion of light from outside the focal region. In-focus light can be filtered using a pinhole, much in the same way that the confocal microscope uses a pinhole to enable three-dimensional imaging.

Careful choice of the pinhole diameter is required, as too large a pinhole allows too much background light through to the sensor, whereas a small pinhole would filter out any phase variation due to aberrations. This method has been implemented in scanning microscopes using backscattered light for wavefront sensing, although interpretation of the resulting measurements is not necessarily straightforward. The two-photon excitation fluorescence microscope has inherent three-dimensional resolution due to the confinement of fluorescence emission to the focal region.

As such, the focus provides an ideal point source for wavefront sensing. This can be implemented with fluorescent beads, 6 , 32 , 33 although this is not compatible with many bio-imaging applications.

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A more sophisticated approach relies upon the labelling of sparse structures, such as centrosomes, so that an array of suitable sensing sources is available, throughout a specimen. An alternative way to avoid the effects of out-of-focus light is to use coherence gating. A common approach to aberration sensing in AO microscopy involves indirect measurement, whereby wavefront phase is not measured directly with a dedicated sensor, but rather via a sequence of images. These methods involve simpler hardware implementations than direct sensing systems, as they only require the addition of an adaptive element to the microscope.

However, the software configuration for these systems can be more complicated. Here the aberration is considered as a summation of orthogonal modes. A sequence of images is obtained, each with a pre-determined combination of the modes applied to the adaptive element.

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For each of these images, an image quality metric, such as total intensity or sharpness, is calculated and used in an optimisation process to find the optimum aberration correction. The ability to measure and correct aberrations with the fewest possible image acquisitions is important to avoid over exposure of specimens. This indirect correction method has been implemented in a range of microscope modalities. Methods have been developed to facilitate the in situ calibration of the deformable mirror using image-based feedback.

In principle, one obtains images of the specimen using one segment at a time. Each image is shifted laterally by the local tilt of the wavefront in that particular segment, so the collection of images reveals a spatially sampled approximation to the gradient of the aberration phase. From this information, it is possible to reconstruct the whole phase function, in a manner analogous to that used in the Shack-Hartmann wavefront sensor.

However, this is achieved via a greater number of image measurements. This was somewhat mitigated by the introduction of a parallelized version of the pupil segmentation approach, 45 although it is interesting to note that this modification leads to, in essence, a modal sensing method. The challenges posed by aberrations are ubiquitous, wherever imaging into the bulk of specimens is necessary.


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It should therefore be expected that this technology has found wide applicability in a range of microscope modalities. The first adaptive microscopes were built around laser scanning systems, in particular confocal or two-photon fluorescence microscopes. These microscopes are still the major workhorses of bio-imaging laboratories, enabling three-dimensional imaging of thick tissue specimens.