Flexible polygon-mirror based laser scanning microscope platform for multiphoton in-vivo imaging.

Commercial microscopy systems make use of tandem scanning i.e. either slow or fast scanning. We constructed, for the first time, an advanced control system capable of delivering a dynamic line scanning speed ranging from 2.7 kHz to 27 kHz and achieve variable frame rates from 5 Hz to 50 Hz (512 × 512). The dynamic scanning ability is digitally controlled by a new customized open-source software named PScan1.0. This permits manipulation of scanning rates either to gain higher fluorescence signal at slow frame rate without increasing laser power or increase frame rates to capture high speed events. By adjusting imaging speed from 40 Hz to 160 Hz, we capture a range of calcium waves and transient peaks from soma and dendrite of single fluorescence neuron (CAL-520AM). Motion artifacts arising from respiratory and cardiac motion in small animal imaging reduce quality of real-time images of single cells in-vivo. An image registration algorithm, integrated with PScan1.0, was shown to perform both real time and post-processed motion correction. The improvement is verified by quantification of blood flow rates. This work describes all the steps necessary to develop a high performance and flexible polygon-mirror based multiphoton microscope system for in-vivo biological imaging.

High-accuracy fourier transform interferometry, without oversampling, with a 1-bit analog-to-digital converter.

We demonstrate a new technique for performing accurate Fourier transform interferometry with a 1-bit analog-to-digital (AD) converter that does not require oversampling of the interferogram, unlike in other 1-bit coding schemes that rely on delta-sigma modulation. Sampling aims at locating the intersections {z(i)} of the modulation term s(z) of the interferogram and a reference sinusoid r(z) = A cos(2pif(r)z), where z is the optical path difference. A new autocorrelation-based procedure that includes the accurate recovery of the equally sampled amplitude representation {s(k)} of s(z) from {z(i)} is utilized to calculate the square of the emission spectrum of the light source (sample). The procedure is suitable for interferograms that are corrupted with additive noise. Sinusoid-crossing sampling satisfies the Nyquist sampling criterion, and a z(i) exists within each sampling interval Delta = 1/2f(r), if A >or= |s(z)| for all z, and f(r) >or= f(c), where f(c) is the highest frequency component of s(z). By locating a crossing at an accuracy of 1 part in 2(16), we determine the multimode spectrum of an argon-ion laser with a 1-bit AD converter that performs like a 13-bit amplitude-sampling AD converter.

Excitation with a focused, pulsed optical beam in scattering media: diffraction effects.

To gain a better understanding of the spatiotemporal problems that are encountered in two-photon excitation fluorescence imaging through highly scattering media, we investigate how diffraction affects the three-dimensional intensity distribution of a focused, pulsed optical beam propagating inside a scattering medium. In practice, the full potential of the two-photon excitation fluorescence imaging is unrealized at long scattering depths, owing to the unwanted temporal and spatial broadening of the femtosecond excitation light pulse that reduces the energy density at the geometric focus while it increases the excitation energy density in the out-of-focus regions. To analyze the excitation intensity distribution, we modify the Monte Carlo-based photon-transport model to a semi-quantum-mechanical representation that combines the wave properties of light with the particle behavior of the propagating photons. In our model the propagating photon is represented by a plane wave with its propagation direction in the scattering medium determined by the Monte Carlo technique. The intensity distribution in the focal region is given by the square of the linear superposition of the various plane waves that arrive at different incident angles and optical path lengths. In the absence of scattering, the propagation model yields the intensity distribution that is predicted by the Huygens-Fresnel principle. We quantify the decrease of the energy density delivered at the geometric focus as a function of the optical depth to the mean-free-path ratio that yields the average number of scattering events that a photon encounters as it propagates toward the focus. Both isotropic and anisotropic scattering media are considered. Three values for the numerical aperture (NA) of the focusing lens are considered: NA = 0.25, 0.5, 0.75.

Spectral recovery by analytic continuation in crossing-based spectrum analysis.

By the use of analytic continuation, the correct spectrum of an undersampled analog input signal f(a) (t) of a true bandwidth B is recovered from an aliased Fourier spectrum that is computed directly from a data set consisting of sinusoid-crossing locations {t(i)}, where the signal f(a) (t) intersects with a reference sinusoid r(t) with a frequency of W