Video-rate two-photon excited fluorescence lifetime imaging system with interleaved digitization.

A fast (up to video rate) two-photon excited fluorescence lifetime imaging system based on interleaved digitization is demonstrated. The system is compatible with existing beam-scanning microscopes with minor electronics and software modification. Proof-of-concept demonstrations were performed using laser dyes and biological tissue.

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

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 multi-channel 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.

Spectral X-Ray Diffraction using a 6 Megapixel Photon Counting Array Detector.

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.

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

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.

Linear fitting of multi-threshold counting data with a pixel-array detector for spectral X-ray imaging.

Experiments and modeling are described to perform spectral fitting of multi-threshold counting measurements on a pixel-array detector. An analytical model was developed for describing the probability density function of detected voltage in X-ray photon-counting arrays, utilizing fractional photon counting to account for edge/corner effects from voltage plumes that spread across multiple pixels. Each pixel was mathematically calibrated by fitting the detected voltage distributions to the model at both 13.5 keV and 15.0 keV X-ray energies. The model and established pixel responses were then exploited to statistically recover images of X-ray intensity as a function of X-ray energy in a simulated multi-wavelength and multi-counting threshold experiment.

Polarization-modulated second harmonic generation ellipsometric microscopy at video rate.

Fast 8 MHz polarization modulation coupled with analytical modeling, fast beam-scanning, and synchronous digitization (SD) have enabled simultaneous nonlinear optical Stokes ellipsometry (NOSE) and polarized laser transmittance imaging with image acquisition rates up to video rate. In contrast to polarimetry, in which the polarization state of the exiting beam is recorded, NOSE enables recovery of the complex-valued Jones tensor of the sample that describes all polarization-dependent observables of the measurement. Every video-rate scan produces a set of 30 images (10 for each detector with three detectors operating in parallel), each of which corresponds to a different polarization-dependent result. Linear fitting of this image set contracts it down to a set of five parameters for each detector in second harmonic generation (SHG) and three parameters for the transmittance of the incident beam. These parameters can in turn be used to recover the Jones tensor elements of the sample. Following validation of the approach using z-cut quartz, NOSE microscopy was performed for microcrystals of both naproxen and glucose isomerase. When weighted by the measurement time, NOSE microscopy was found to provide a substantial (>7 decades) improvement in the signal-to-noise ratio relative to our previous measurements based on the rotation of optical elements and a 3-fold improvement relative to previous single-point NOSE approaches.

Synchronous digitization for high dynamic range lock-in amplification in beam-scanning microscopy.

Digital lock-in amplification (LIA) with synchronous digitization (SD) is shown to provide significant signal to noise (S/N) and linear dynamic range advantages in beam-scanning microscopy measurements using pulsed laser sources. Direct comparisons between SD-LIA and conventional LIA in homodyne second harmonic generation measurements resulted in S/N enhancements consistent with theoretical models. SD-LIA provided notably larger S/N enhancements in the limit of low light intensities, through the smooth transition between photon counting and signal averaging developed in previous work. Rapid beam scanning instrumentation with up to video rate acquisition speeds minimized photo-induced sample damage. The corresponding increased allowance for higher laser power without sample damage is advantageous for increasing the observed signal content.

Digital deconvolution filter derived from linear discriminant analysis and application for multiphoton fluorescence microscopy.

A digital filter derived from linear discriminant analysis (LDA) is developed for recovering impulse responses in photon counting from a high speed photodetector (rise time of ~1 ns) and applied to remove ringing distortions from impedance mismatch in multiphoton fluorescence microscopy. Training of the digital filter was achieved by defining temporally coincident and noncoincident transients and identifying the projection within filter-space that best separated the two classes. Once trained, data analysis by digital filtering can be performed quickly. Assessment of the reliability of the approach was performed through comparisons of simulated voltage transients, in which the ground truth results were known a priori. The LDA filter was also found to recover deconvolved impulses for single photon counting from highly distorted ringing waveforms from an impedance mismatched photomultiplier tube. The LDA filter was successful in removing these ringing distortions from two-photon excited fluorescence micrographs and through data simulations was found to extend the dynamic range of photon counting by approximately 3 orders of magnitude through minimization of detector paralysis.

Real-time dynamic range and signal to noise enhancement in beam-scanning microscopy by integration of sensor characteristics, data acquisition hardware, and statistical methods.

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.

Statistical connection of binomial photon counting and photon averaging in high dynamic range beam-scanning microscopy.

Data from photomultiplier tubes are typically analyzed using either counting or averaging techniques, which are most accurate in the dim and bright signal limits, respectively. A statistical means of adjoining these two techniques is presented by recovering the Poisson parameter from averaged data and relating it to the statistics of binomial counting from Kissick et al. [Anal. Chem. 82, 10129 (2010)]. The point at which binomial photon counting and averaging have equal signal to noise ratios is derived. Adjoining these two techniques generates signal to noise ratios at 87% to approaching 100% of theoretical maximum across the full dynamic range of the photomultiplier tube used. The technique is demonstrated in a second harmonic generation microscope.

Statistical treatment of photon/electron counting: extending the linear dynamic range from the dark count rate to saturation.

An experimentally simple photon counting method is demonstrated providing 7 orders of magnitude in linear dynamic range (LDR) for a single photomultiplier tube (PMT) detector. In conventional photon/electron counting methods, the linear range is dictated by the agreement between the binomially distributed measurement of counted events and the underlying Poisson distribution of photons/electrons. By explicitly considering the log-normal probability distribution in voltage transients as a function of the number of photons present and the Poisson distribution of photons, observed counts for a given threshold can be related to the mean number of photons well beyond the conventional limit. Analytical expressions are derived relating counts and photons that extend the linear range to an average of ∼11 photons arriving simultaneously with a single threshold. These expressions can be evaluated numerically for multiple thresholds extending the linear range to the saturation point of the PMT. The peak voltage distributions are experimentally shown to follow a Poisson weighted sum of log-normal distributions that can all be derived from the single photoelectron voltage peak-height distribution. The LDR that results from this method is compared to conventional single photon counting (SPC) and to signal averaging by analog to digital conversion (ADC).