A simple optical change to existing beam-scanning microscopy is described enabling up to kHz frame-rate imaging across multiple channels of acquisition simultaneously. Two fast-scan resonant mirrors are used to direct the optical beam in a Lissajous trajectory. By dicing the raw time-domain data into sub-trajectories, the Lissajous pattern can be re-binned to achieve faster imaging periods, effectively. A consequence of re-binning the data into sub-trajectories is regions of the field of view not sampled for every sub-frame generated. To overcome this, Model-based Image Reconstruction (MBIR) techniques were explored to recover the missing data using the entire 3D data set (two spatial dimensions plus time). Since re-binning the data is done post analysis, both lower frame-rate high resolution imaging and higher frame-rate lower resolution imaging can be produce from the data, running the entire gamut in-between the two extremes. Preliminary work was based on laser transmittance imaging and polarization-dependent second harmonic generation (SHG) microscopy, supporting the viability of the technique for detection of subtle changes in large signal as well as for trace-light detection or transient fluctuations. The linchpin mechanism of this technique is the commercial availability of modern PCI express based digital oscilloscope cards. In brief, the laser behaves as the master clock (80 MHz), and the voltage level from the signal transient from the detectors are flash-digitized at 80 million times per second across multiple channels. Quantitative analysis of the reconstructed images was performed. The in-painting results are demonstrated here from a video of a dog (Marco, panel A) in which data was artificially removed to represent a fill pattern similar to the prototype microscope at 250 Hz imaging (B). The missing data was reconstructed using the MBIR technique (C), and the difference in the original and in-painted results are demonstrated in D. Future work will be extending the optics to 3 spatial dimensions for video-rate voxel rendering.

Multichannel high frame-rate beam-scanning microscopy based on Lissajous scanning trajectories and model based image reconstruction

249th National Meeting (2015)

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