Proc. SPIE. 10070, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXIV
KEYWORDS: Signal to noise ratio, Microscopes, Mirrors, Microscopy, Luminescence, Light scattering, Multiplexing, Spatial light modulators, Objectives, Electron multiplying charge coupled devices, High speed imaging, Spatial resolution, 3D scanning, Two photon excitation microscopy, Signal detection, 3D image processing
Optical sectioning techniques using two-photon excitation of fluorescent indicators are central to diverse imaging applications. The limitations of the technique are low speed and undesirable specimen agitation. In our design, high-speed axial scanning is carried out by moving a reference objective to axially displace the focal spot without introducing significant spherical aberration and any agitation of the specimen. Further, the system is configured to allow switching between single spot and multiple focal spot remote focusing to allow either high dynamic range or high speed imaging.
Networks of neurons are inherently three-dimensional in nature, whereas conventional imaging methods, such as laser scanning two-photon microscopy, usually provide only fast two-dimensional imaging. Rapid volumetric imaging would however be preferable for imaging neurons. To get a more complete picture of the dynamics of the neuron-to-neuron interactions, we have developed a pseudo-parallelised multi-plane two-photon excitation imaging system through the incorporation of an acousto-optic switching and a remote focusing technique into a resonant scanning microscope. This permits the recording of millisecond scale fluorescence transients of calcium indicators from large populations of neurons upon neural firing events at multiple chosen axial planes in very short time frame. While the remote focusing system offers aberration-free axial scanning over a few hundreds of micrometres of depth, the acousto-optic deflector provides high speed optical switching between different laser beam paths in sub-microsecond timescale which in turn, controls the axial focal plane to be targeted. Here, we report on the development of the high temporal resolution multi-plane targeted microscope and its potential application.