We demonstrate wavefront sensorless aberration correction in a two-photon excited fluorescence microscope. Using analysis of the image-formation process, we have developed an optimized correction scheme permitting image-quality improvement with minimal additional exposure of the sample. We show that, as a result, our correction process induces little photobleaching and significantly improves the quality of images of biological samples. In particular, increased visibility of small structures is demonstrated. Finally, we illustrate the use of this technique on various fresh and fixed biological tissues.

We describe a method of optical refocusing for high numerical aperture (NA) systems that is particularly relevant for confocal and multiphoton microscopy. This method avoids the spherical aberration that is common to other optical refocusing systems. We show that aberration-free images can be obtained over an axial scan range of 70 mum for a 1.4 NA objective lens. As refocusing is implemented remotely from the specimen, this method will enable high axial scan speeds without mechanical interference between the objective lens and the specimen.

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Multiphoton microscopy is a powerful tool in neuroscience, promising to deliver important data on the spatiotemporal activity within individual neurons as well as in networks of neurons. A major limitation of current technologies is the relatively slow scan rates along the z direction compared to the kHz rates obtainable in the x and y directions. Here, we describe a custom-built microscope system based on an architecture that allows kHz scan rates over hundreds of microns in all three dimensions without introducing aberration. We further demonstrate how this high-speed 3D multiphoton imaging system can be used to study neuronal activity at millisecond resolution at the subcellular as well as the population level.fluorescence microscopy | multiphoton imaging | multiphoton microscopy | three-dimensional microscopy N eurons process information by integrating many thousands of synaptic inputs arriving at thin processes called dendrites and translating them into action potential output. In order to understand the neural code it is important to study both the activity in dendrites and the action potential outputs of large populations of neurons. However, direct electrical recordings from thin dendrites and simultaneous recordings from multiple identified neurons are difficult to achieve and often not feasible, especially in the in vivo setting. Instead, optical sectioning techniques using two-photon excitation of fluorescent indicators of neuronal activity have established themselves as the method of choice to monitor the activity in thin dendrites and large neuronal populations (1-6). Current technology, however, does not allow the spot of light to be scanned sufficiently fast in all three dimensions to fully address the questions of dendritic integration and neuronal population activity. While it is relatively straightforward to scan the focal spot in the x-y plane at kHz rates, scan rates along the z-axis are limited to approximately 20 Hz with conventional imaging approaches (7). This is due to the mechanical inertia of the objective lens and specimen during refocusing. Higher-speed refocusing was recently achieved using a carefully designed electrically tunable lens (8), but at the expense of the numerical aperture.Recently we overcame these fundamental problems and showed that neither speed nor numerical aperture needs to be compromised if we use a different microscope architecture (9). Using this method, the spot can now be scanned along the z axis at high speed while still maintaining diffraction-limited performance. In our design, scanning is carried out by moving a lightweight mirror instead of the objective and high-speed axial scanning is achieved without introducing significant spherical aberration. To demonstrate the practical feasibility of this principle, we have implemented our unique scan approach in a custombuilt two-photon microscope. With it, we monitored calcium transients both along the dendritic arbor of individual pyramidal neurons and in a population of bulk-loaded neurons from a large volum...

We implement wave front sensor-less adaptive optics in a structured illumination microscope. We investigate how the image formation process in this type of microscope is affected by aberrations. It is found that aberrations can be classified into two groups, those that affect imaging of the illumination pattern and those that have no influence on this pattern. We derive a set of aberration modes ideally suited to this application and use these modes as the basis for an efficient aberration correction scheme. Each mode is corrected independently through the sequential optimisation of an image quality metric. Aberration corrected imaging is demonstrated using fixed fluorescent specimens. Images are further improved using differential aberration imaging for reduction of background fluorescence.