Propagation of action potentials arises on millisecond timescales, suggesting the need for advancement of methods capable of commensurate volume rendering for in vivo brain mapping. In practice, beam-scanning multiphoton microscopy is widely used to probe brain function, striking a balance between simplicity and penetration depth. However, conventional beam-scanning platforms generally do not provide access to full volume renderings at the speeds necessary to map propagation of action potentials. By combining a sparse sampling strategy based on Lissajous trajectory microscopy in combination with temporal multiplexing for simultaneous imaging of multiple focal planes, whole volumes of cells are potentially accessible each millisecond.
A beam-scanning microscope is described based on a temporally multiplexed Lissajous trajectory for achieving 1 kHz frame rate 3D imaging. The microscope utilizes two fast-scan resonant mirrors to direct the optical beam on a circuitous, Lissajous trajectory through the field of view. Acquisition of two simultaneous focal planes is achieved by implementation of an optical delay line, producing a second incident beam at a different focal plane relative to the initial incident beam. High frame rates are achieved by separating the full time-domain data into shorter sub-trajectories resulting in undersampling of the field of view. A model-based image reconstruction (MBIR) 3D in-painting algorithm is utilized for interpolating the missing data to recover full images. The MBIR algorithm uses a maximum a posteriori estimation with a generalized Gaussian Markov random field prior model for image interpolation. Because images are acquired using photomultiplier tubes or photodiodes, parallelization for multi-channel imaging is straightforward.
Preliminary results obtained using a Lissajous trajectory beam-scanning approach coupled with temporal multiplexing by the implementation of an optical delay line demonstrate the ability to acquire 2 distinct focal planes simultaneously at frame rates >450 Hz for full 512 × 512 images. The use of multi-channel data acquisition cards allows for simultaneous multimodal image acquisition with perfect image registry between all imaging modalities. Also discussed here is the implementation of Lissajous trajectory beam-scanning on commercially available microscope hardware.
A beam-scanning microscope based on Lissajous trajectory imaging is described for achieving streaming 2D imaging with continuous frame rates up to 1.4 kHz. The microscope utilizes two fast-scan resonant mirrors to direct the optical beam on a circuitous trajectory through the field of view. By separating the full Lissajous trajectory time-domain data into sub-trajectories (partial, undersampled trajectories) effective frame-rates much higher than the repeat time of the Lissajous trajectory are achieved with many unsampled pixels present. A model-based image reconstruction (MBIR) 3D in-painting algorithm is then used to interpolate the missing data for the unsampled pixels to recover full images. The MBIR algorithm uses a maximum a posteriori estimation with a generalized Gaussian Markov random field prior model for image interpolation. Because images are acquired using photomultiplier tubes or photodiodes, parallelization for multi-channel imaging is straightforward. Preliminary results show that when combined with the MBIR in-painting algorithm, this technique has the ability to generate kHz frame rate images across 6 total dimensions of space, time, and polarization for SHG, TPEF, and confocal reflective birefringence data on a multimodal imaging platform for biomedical imaging. The use of a multichannel data acquisition card allows for multimodal imaging with perfect image overlay. Image blur due to sample motion was also reduced by using higher frame rates.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.