High frame-rate multichannel beam-scanning microscopy based on Lissajous trajectories

Abstract

A simple beam-scanning optical design based on Lissajous trajectory imaging is described for achieving up to kHz frame-rate optical imaging on multiple simultaneous data acquisition channels. In brief, two fast-scan resonant mirrors direct the optical beam on a circuitous trajectory through the field of view, with the trajectory repeat-time given by the least common multiplier of the mirror periods. Dicing the raw time-domain data into sub-trajectories combined with model-based image reconstruction (MBIR) 3D in-painting algorithms allows for effective frame-rates much higher than the repeat time of the Lissajous trajectory. Since sub-trajectory and full-trajectory imaging are simply different methods of analyzing the same data, both high-frame rate images with relatively low resolution and low frame rate images with high resolution are simultaneously acquired. The optical hardware required to perform Lissajous imaging represents only a minor modification to established beam-scanning hardware, combined with additional control and data acquisition electronics. Preliminary studies based on laser transmittance imaging and polarization-dependent second harmonic generation microscopy support the viability of the approach both for detection of subtle changes in large signals and for trace-light detection of transient fluctuations.

Multi-channel beam-scanning imaging at kHz frame rates by Lissajous trajectory microscopy

“…The Lissajous beam-scanning microscope has been detailed previously 7 and will only be described briefly here. The Lissajous microscope utilized a MaiTai HP Ti:Sapphire laser (Spectra Physics) to produce ~100 femtosecond pulses centered around 800 nm, with an 80 MHz repetition rate, and a maximum power of ~3.0 W. The period of the pulsed laser source served as the master clock dictating all the subsequent timing of the resonant mirrors, feedback controls, and data acquisition electronics.…”

“…The interpolation algorithm used in this paper has been described in detail elsewhere 7 and will only be discussed briefly here. The in-painting algorithm used is an iterative model-based image reconstruction algorithm combining a maximum a posteriori (MAP) estimation with a generalized Gaussian Markov random field (GGMRF) prior model, adopted within a 3 dimensional space-time neighborhood.…”

“…The last row contains the in-painted results for the subtrajectories. Figure adapted from Sullivan et al 7 …”

“…The greatest advantage that Lissajous trajectory imaging provides is the ability to achieve frame rates much higher than video rate, as previously discussed by Sullivan et al . 7 While increasing the frame rate allows for the visualization of highly dynamic, rapidly changing samples (i.e. visualizing the action potential as a neuron fires), arguably the greatest benefit to in vivo imaging is the reduction in image blurring as a result of sample motion due to organism movement.…”

Volumetric Lissajous Confocal Microscopy

“…First, a large increase in laser scanning speed is achieved, up to 45-fold faster for our experimental conditions, compared with conventional scanning methods with a single resonant mirror (see Supplementary information and Figure S1). Even if Lissajous trajectories have been reported in different microscopy techniques, including atomic force microscopy or two-photon microscopy [12][13][14], they have been limited to 2D scans along the xy plane. Instead, our system extends their use into 3D imaging utilizing the superior scanning speed in the z-axis.…”