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.
Nonlinear optical Stokes ellipsometric (NOSE) microscopy was demonstrated for the analysis of collagen structure in a mouse tail section. NOSE is based on polarization-dependent second harmonic generation (SHG) imaging. The fast polarization-modulation was achieved using an electro-optic modulator (EOM), allowing for the potential of video-rate NOSE analysis. The signal to noise advantages associated with suppression of 1/f noise by rapid polarization modulation allowed reliable recovery of the local-frame tensor on a per-pixel basis. An iterative approach involving laboratory to local frame coordinate transformation was developed to recover the spatial distribution of local-frame nonlinear susceptibility tensor elements of collagen as well as the polar and azimuthal orientation angles of the collagen structure.
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.
Pixel-array array detectors allow single-photon counting to be performed on a massively parallel scale, with several million counting circuits and detectors in the array. Because the number of photoelectrons produced at the detector surface depends on the photon energy, these detectors offer the possibility of spectral imaging. In this work, a statistical model of the instrument response is used to calibrate the detector on a per-pixel basis. In turn, the calibrated sensor was used to perform separation of dual-energy diffraction measurements into two monochromatic images. Targeting applications include multi-wavelength diffraction to aid in protein structure determination and X-ray diffraction imaging.
Fast beam-scanning non-linear optical microscopy, coupled with fast (8 MHz) polarization modulation and analytical modeling have enabled simultaneous nonlinear optical Stokes ellipsometry (NOSE) and linear Stokes ellipsometry imaging at video rate (15 Hz). NOSE enables recovery of the complex-valued Jones tensor that describes the polarization-dependent observables, in contrast to polarimetry, in which the polarization stated of the exciting beam is recorded. Each data acquisition consists of 30 images (10 for each detector, with three detectors operating in parallel), each of which corresponds to polarization-dependent results. Processing of this image set by linear fitting contracts down each set of 10 images to a set of 5 parameters for each detector in second harmonic generation (SHG) and three parameters for the transmittance of the fundamental laser beam. Using these parameters, it is possible to recover the Jones tensor elements of the sample at video rate. Video rate imaging is enabled by performing synchronous digitization (SD), in which a PCIe digital oscilloscope card is synchronized to the laser (the laser is the master clock.) Fast polarization modulation was achieved by modulating an electro-optic modulator synchronously with the laser and digitizer, with a simple sine-wave at 1/10th the period of the laser, producing a repeating pattern of 10 polarization states. This approach was validated using Z-cut quartz, and NOSE microscopy was performed for micro-crystals of naproxen.
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.
Photon counting represents the Poisson limit in signal to noise, but can often be complicated in imaging
applications by detector paralysis, arising from the finite rise / fall time of the detector upon photon absorption.
We present here an approach for reducing dead-time by generating a deconvolution digital filter based on
optimizing the Fisher linear discriminant. In brief, two classes are defined, one in which a photon event is
initiated at the origin of the digital filter, and one in the photon event is non-coincident with the filter origin.
Linear discriminant analysis (LDA) is then performed to optimize the digital filter that best resolves the
coincident and non-coincident training set data.1 Once trained, implementation of the filter can be performed
quickly, significantly reducing dead-time issues and measurement bias in photon counting applications.
Experimental demonstration of the LDA-filter approach was performed in fluorescence microscopy
measurements using a highly convolved impulse response with considerable ringing. Analysis of the counts
supports the capabilities of the filter in recovering deconvolved impulse responses under the conditions
considered in the study. Potential additional applications and possible limitations are also considered.
Despite the ubiquitous use of multi-photon and confocal microscopy measurements in biology, the core techniques
typically suffer from fundamental compromises between signal to noise (S/N) and linear dynamic range (LDR). In this
study, direct synchronous digitization of voltage transients coupled with statistical analysis is shown to allow S/N
approaching the theoretical maximum throughout an LDR spanning more than 8 decades, limited only by the dark counts
of the detector on the low end and by the intrinsic nonlinearities of the photomultiplier tube (PMT) detector on the high
end. Synchronous digitization of each voltage transient represents a fundamental departure from established methods in
confocal/multi-photon imaging, which are currently based on either photon counting or signal averaging. High
information-density data acquisition (up to 3.2 GB/s of raw data) enables the smooth transition between the two
modalities on a pixel-by-pixel basis and the ultimate writing of much smaller files (few kB/s). Modeling of the PMT
response allows extraction of key sensor parameters from the histogram of voltage peak-heights. Applications in second
harmonic generation (SHG) microscopy are described demonstrating S/N approaching the shot-noise limit of the detector
over large dynamic ranges.
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