Zhenqiao Zhou

Chemical reactivation of quenched fluorescent protein molecules enables resin-embedded fluorescence microimaging

Resin embedding is a well-established technique to prepare biological specimens for microscopic imaging. However, it is not compatible with modern green-fluorescent protein (GFP) fluorescent-labelling technique because it significantly quenches the fluorescence of GFP and its variants. Previous empirical optimization efforts are good for thin tissue but not successful on macroscopic tissue blocks as the quenching mechanism remains uncertain. Here we show most of the quenched GFP molecules are structurally preserved and not denatured after routine embedding in resin, and can be chemically reactivated to a fluorescent state by alkaline buffer during imaging. We observe up to 98% preservation in yellow-fluorescent protein case, and improve the fluorescence intensity 11.8-fold compared with unprocessed samples. We demonstrate fluorescence microimaging of resin-embedded EGFP/EYFP-labelled tissue block without noticeable loss of labelled structures. This work provides a turning point for the imaging of fluorescent protein-labelled specimens after resin embedding.

Visualization of brain circuits using two-photon fluorescence micro-optical sectioning tomography

Neural circuits are fundamental for brain functions. However, obtaining long range continuous projections of neurons in the entire brain is still challenging. Here a two-photon fluorescence micro-optical sectioning tomography (2p-fMOST) method is developed for high-throughput, high-resolution visualization of the brain circuits. Two-photon imaging technology is used to obtain high resolution, and acoustical optical deflector (AOD), an inertia-free beam scanner is used to realize fast and prolonged stable imaging. The combination of these techniques with imaging and then sectioning method of a plastic-embedded mouse brain facilitated the acquisition of a three-dimensional data set of a fluorescent mouse brain with a resolution adequate to resolve the spines. In addition, the brain circuit tracing ability is showed by several neurons projecting across different brain regions. Besides brain imaging, 2p-fMOST could be used in many studies that requires sub-micro resolution or micro resolution imaging of a large sample.

Fluorescence holography with improved signal-to-noise ratio by near image plane recording

Holographic fluorescence imaging is very promising, as it can obtain three-dimensional fluorescence imaging without scanning. However, the current method usually records holograms far from the image plane, with the fluorescence decaying when spreading broadly. Here we show that the signal-to-noise ratio (SNR) of fluorescence holography can be improved by recording the high-contrast interferogram near the image plane. We found that this can be achieved by setting the focal length of the lens for the reference wave (f(2)) close to that for the object wave (f(1)). With experiments, we demonstrate an example of an increase of about 21 times in SNR by changing f(2) from infinity to 226 mm, which is close to f(1) (323 mm).

Spatial and temporal thermal analysis of acousto-optic deflectors using finite element analysis model

Thermal effects greatly influence the optical properties of the acousto-optic deflectors (AODs). Thermal analysis plays an important role in modern AOD design. However, the lack of an effective method of analysis limits the prediction in the thermal performance. In this paper, we propose a finite element analysis model to analyze the thermal effects of a TeO(2)-based AOD. Both transducer heating and acoustic absorption are considered as thermal sources. The anisotropy of sound propagation is taken into account for determining the acoustic absorption. Based on this model, a transient thermal analysis is employed using ANSYS software. The spatial temperature distributions in the crystal and the temperature changes over time are acquired. The simulation results are validated by experimental results. The effect of heat source and heat convection on temperature distribution is discussed. This numerical model and analytical method of thermal analysis would be helpful in the thermal design and practical applications of AODs.

Reducing low-frequency residual motion in vibration isolation to the nanometre level

If the low frequency residual motion of a vibration isolated test mass can be reduced to the nanometre level, then the forces required to control its position may be applied directly to the test mass without injecting noise and the control and acquisition of lock in an interferometer is rendered trivial. We show that two stages of already demonstrated pre-isolation elements, combined with tilt control and a novel self-damped isolator structure, are capable of achieving this performance. The designs and performance requirements of the individual components making up this system are discussed and various experimental measurements presented.

Beam deformation within an acousto-optic lens.

The acousto-optic lens (AOL) is becoming a popular tool in the neuroscience field. Here we analyzed the deformation of the diffraction beam after passage through an AOL consisting of a pair of acousto-optic deflectors using both theoretical and experimental data. The results showed that, because of the high sensitivity of optical spatial frequencies of acousto-optic deflectors, the boundary strength of the diffraction beam of the AOL decreases significantly. When the focal length of AOL diminishes, the deformation of the diffraction beam becomes more serious with a smaller beam size. This deformation of the diffraction beam finally leads to a decreased illuminative numerical aperture, which worsens the images spatial resolution.

Wide-band acousto-optic deflectors for large field of view two-photon microscope

Acousto-optic deflector (AOD) is an attractive scanner for two-photon microscopy because it can provide fast and versatile laser scanning and does not involve any mechanical movements. However, due to the small scan range of available AOD, the field of view (FOV) of the AOD-based microscope is typically smaller than that of the conventional galvanometer-based microscope. Here, we developed a novel wide-band AOD to enlarge the scan angle. Considering the maximum acceptable acoustic attenuation in the acousto-optic crystal, relatively lower operating frequencies and moderate aperture were adopted. The custom AOD was able to provide 60 MHz 3-dB bandwidth and 80% peak diffraction efficiency at 840 nm wavelength. Based on a pair of such AOD, a large FOV two-photon microscope was built with a FOV up to 418.5 μm (40× objective). The spatiotemporal dispersion was compensated simultaneously with a single custom-made prism. By means of dynamic power modulation, the variation of laser intensity within the FOV was reduced below 5%. The lateral and axial resolution of the system were 0.58-2.12 μm and 2.17-3.07 μm, respectively. Pollen grain images acquired by this system were presented to demonstrate the imaging capability at different positions across the entire FOV.

IMPROVED LOW FREQUENCY SEISMIC NOISE ISOLATION SYSTEM FOR GRAVITATIONAL WAVE DETECTORS

An improved three-stage low frequency seismic noise isolation system for gravitational wave detectors, based on the cantilever spring, is presented. The measurement results show that the vertical and horizontal displacements of the test mass, at 10 Hz, reach 7×10−13 m/Hz and 1×10−12 mHz, respectively, and the isolation ratios of the system come to about 60 dB vertically and over 50 dB horizontally.

A gyro-stabilized platform leveling loop for marine gravimeter

An ultra-low-frequency platform leveling loop based on a mixed sensitivity H∞ approach, which considers both the system bandwidth and response speed, was designed and applied to a prototype, two-axis gyro-stabilized platform marine gravimeter CHZ-II. The instrument was developed for regional surveys in deep ocean areas where high-resolution gravity measurements with accuracy 1 mGal are required. Horizontal accelerations in the surge and sway directions are suppressed about 60 dB in the frequency range 0.05 to 0.5 Hz. This typically improves the quality of the gravity data before any processing corrections. The time required for stabilizing the platform at the beginning of a survey line or course change is about 3 min, which improves the data collection efficiency. In May 2015, the first test was conducted in open sea conditions aboard the Chinese State Oceanic Administrations R/V Xiangyanghong 10. Sixteen traverses were run in the South China Sea to evaluate the loop performance. Platform motion tracks and gravity data from the survey were of satisfactory quality. According to analyses of 16 sets of calculated errors, the root mean square repeatability of the pitch and roll off-level angles were less than 10 and 20 arc sec, respectively, with a horizontal acceleration of about 50 Gal. Errors derived from the inability of the platform to maintain perfect sensor leveling during the survey cruise were less than 0.3 mGal.

Compensation of spatial dispersion of an acousto-optic deflector with a special Keplerian telescope

Compensation of spatial dispersion caused by the acousto-optic deflector (AOD) when using a femtosecond laser is difficult across the whole scanning range of the system, and this is a significant impediment to its use. In conventional methods, the dispersion of the AOD was compensated only when it was at a particular position, while at other positions, the quality of the light beam was reduced. We developed a novel method for compensating the spatial dispersion within the entire scanning range using a special Keplerian telescope. Our experimental results show that the residual dispersion of the AOD is compensated sufficiently, and the focal spots of the laser reach the diffraction limit within a 40-MHz ultrasound bandwidth.