This review presents a selection of advanced microscope designs employing acousto-optical deflectors (AODs). In the designs covered, AODs are used as tunable diffraction gratings to control complex illumination patterns at the Fourier plane of an objective lens. This approach allows us to generate desired illumination patterns at the focal plane of a light microscope. In simple terms, I will describe two established designs, the 3D Random-Access Multi-Photon Microscope and the Standing-Wave Super-Resolution Microscope, as well as two new schemes including the Random-Access STED Microscope and the Frequency-Encoded Multi-Beam Microscope. All instruments mentioned here were designed to overcome the throughput limitations of previously used light microscopes in experimental Neuroscience.
KEYWORDS: Amplitude modulation, Femtosecond phenomena, Microscopy, Neurons, Two photon imaging, Multiplexing, Laser scanners, Interferometers, Luminescence, Signal to noise ratio, Data acquisition, Photons, Signal detection, Interferometry
We present a frequency-multiplexed multi-site two-photon imaging method utilizing amplitude modulation of femtosecond laser pulses in the MHz range to tag each excitation beam and the corresponding fluorescence signals with specific frequencies. The frequency tags are generated with an interferometric setup employing acousto-optic deflectors (AODs) to achieve precise spatial overlap of femtosecond pulses with acoustically-shifted frequencies. Creating matching excitation beam patterns in each interferometer arm using multiple AOD driving frequencies and subsequently overlapping these matching patterns results in multiple excitation beams with unique beat frequencies available for scanning. As a proof-of-concept, we demonstrate multiplexed two-photon image acquisition using test samples, and compare the performance of this approach to conventional two-photon laser scanning microscopy.
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