Shaoqun Zeng

Modified laser speckle imaging method with improved spatial resolution.

A two-dimensional map of blood flow is crucial for physiological studies. We present a modified laser speckle imaging method (LSI) that is based on the temporal statistics of a time-integrated speckle. A model experiment was performed for the validation of this technique. The spatial and temporal resolutions of this method were studied in theory and compared with current laser speckle contrast analysis (LASCA); the comparison indicates that the spatial resolution of the modified LSI is five times higher than that of current LASCA. Cerebral blood flow under different temperatures was investigated by our modified LSI. Compared with the results obtained by LASCA, the blood flow map obtained by the modified LSI possessed higher spatial resolution and provided additional information about changes in blood perfusion in small blood vessels. These results suggest that this is a suitable method for imaging the full field of blood flow without scanning and provides much higher spatial resolution than that of current LASCA and other laser Doppler perfusion imaging methods.

Imaging cerebral blood flow through the intact rat skull with temporal laser speckle imaging

We discovered that laser speckle temporal contrast analysis (LSTCA) is able to access the two-dimensional (2D) cerebral blood flow velocity and vessel structure through the intact rat skull. It is demonstrated that LSTCA can significantly suppress the influence of the laser speckle from the stationary structure, such as the skull, and thus reveal the blood flow and morphology of blood vessels through the laser speckle images recorded from the intact rat skull.

Continuously tracing brain-wide long-distance axonal projections in mice at a one-micron voxel resolution.

Revealing neural circuit mechanisms is critical for understanding brain functions. Significant progress in dissecting neural connections has been made using optical imaging with fluorescence labels, especially in dissecting local connections. However, acquiring and tracing brain-wide, long-distance neural circuits at the neurite level remains a substantial challenge. Here, we describe a whole-brain approach to systematically obtaining continuous neuronal pathways in a fluorescent protein transgenic mouse at a one-micron voxel resolution. This goal is achieved by combining a novel resin-embedding method for maintaining fluorescence, an automated fluorescence micro-optical sectioning tomography system for long-term stable imaging, and a digital reconstruction-registration-annotation pipeline for tracing the axonal pathways in the mouse brain. With the unprecedented ability to image a whole mouse brain at a one-micron voxel resolution, the long-distance pathways were traced minutely and without interruption for the first time. With advancing labeling techniques, our method is believed to open an avenue to exploring both local and long-distance neural circuits that are related to brain functions and brain diseases down to the neurite level.

Laser speckle imaging of blood flow in microcirculation

Monitoring the spatio-temporal characteristics of microcirculation is crucial for studying the functional activities of biotissue and the mechanism of disease. However, conventional methods used to measure blood flow suffer from limited spatial resolution or the injection of exogenous substances or the need of scanning to obtain the dynamic of regional blood flow. Laser speckle imaging (LSI) technique makes up these disadvantages by obtaining the regional blood flow distribution with high spatio-temporal resolution without the need to scan. In this paper, LSI was introduced to investigate the dynamic responses of the rat mesenteric microcirculation to an incremental dose of phentolamine. The results showed that when the dose of phentolamine was less than 4 microg ml(-1), local application of phentolamine on the mesentery would increase the blood perfusion as the concentration increased. When the dose increased further, the improvement decreased. At a dose of 200 microg ml(-1), a microcirculation impediment was caused. At the same time, different responses between veinules and arterioles were manifested. These suggested that LSI is promising to be a useful contribution to drug development and testing.

Simultaneous compensation for spatial and temporal dispersion of acousto-optical deflectors for two-dimensional scanning with a single prism

The dispersive nature of the acousto-optical deflector (AOD) presents a challenge to applications of two sequential orthogonal AODs (a two-dimensional AOD) as XY scanners in multiphoton microscopy. Introducing a prism before the two-dimensional (2D) AOD allows both temporal and spatial dispersion to be compensated for simultaneously. A 90 fs laser pulse was broadened to 572 fs without compensation, and 143 fs with compensation. The ratio of long axis to short axis of the exiting laser beam spot was 3.50 without compensation and 1.14 with compensation. The insertion loss was 37%. Two-photon fluorescence microscopy used the compensated 2D AOD scanner to image a fluorescent microsphere, which improves signal intensity -15-fold compared with the uncompensated scanner.

A novel far-red bimolecular fluorescence complementation system that allows for efficient visualization of protein interactions under physiological conditions

Fluorescent protein (FP) has enabled the analysis of biomolecular interactions in living cells, and bimolecular fluorescence complementation (BiFC) represents one of the newly developed imaging technologies to directly visualize protein-protein interactions in living cells. Although 10 different FPs that cover a broad range of spectra have been demonstrated to support BiFC, only Cerulean (cyan FP variant), Citrine and Venus (yellow FP variants)-based BiFC systems can be used under 37 degrees C physiological temperature. The sensitivity of two mRFP-based red BiFC systems to higher temperatures (i.e., 37 degrees C) limits their applications in most mammalian cell-based studies. Here we report that mLumin, a newly isolated far-red fluorescent protein variant of mKate with an emission maximum of 621 nm, enables BiFC analysis of protein-protein interactions at 37 degrees C in living mammalian cells. Furthermore, the combination of mLumin with Cerulean- and Venus-based BiFC systems allows for simultaneous visualization of three pairs of protein-protein interactions in the same cell. The mLumin-based BiFC system will facilitate simultaneous visualization of multiple protein-protein interactions in living cells and offer the potential to visualize protein-protein interactions in living animals.

High-density localization of active molecules using Structured Sparse Model and Bayesian Information Criterion

Localization-based super-resolution microscopy (or called localization microscopy) rely on repeated imaging and localization of active molecules, and the spatial resolution enhancement of localization microscopy is built upon the sacrifice of its temporal resolution. Developing algorithms for high-density localization of active molecules is a promising approach to increase the speed of localization microscopy. Here we present a new algorithm called SSM_BIC for such purpose. The SSM_BIC combines the advantages of the Structured Sparse Model (SSM) and the Bayesian Information Criterion (BIC). Through simulation and experimental studies, we evaluate systematically the performance between the SSM_BIC and the conventional Sparse algorithm in high-density localization of active molecules. We show that the SSM_BIC is superior in processing single molecule images with weak signal embedded in strong background.

Ultra-fast, high-precision image analysis for localization-based super resolution microscopy

Localization-based super resolution microscopy holds superior performances in live cell imaging, but its widespread use is thus far mainly hindered by the slow image analysis speed. Here we show a powerful image analysis method based on the combination of the maximum likelihood algorithm and a Graphics Processing Unit (GPU). Results indicate that our method is fast enough for real-time processing of experimental images even from fast EMCCD cameras working at full frame rate without compromising localization precision or field of view. This newly developed method is also capable of revealing movements from the images immediately after data acquisition, which is of great benefit to live cell imaging.

The development and application of the visible Chinese human model for Monte Carlo dose calculations.

A new whole-body computational phantom, the Visible Chinese Human (VCH), was developed using high-resolution transversal photographs of a Chinese adult male cadaver. Following the segmentation and tridimensional reconstruction, a voxel-based model that faithfully represented the average anatomical characteristics of the Chinese population was established for radiation dosimetry. The vascular system of VCH was fully preserved, and the cadaver specimen was processed in the standing posture. A total of 8,920 slices were obtained by continuous sectioning at 0.2 mm intervals, and 48 organs and tissues were segmented from the tomographic color images at 5440 × 4080 pixel resolution, corresponding to a voxel size of 0.1 × 0.1 × 0.2 mm3. The resulting VCH computational phantom, consisting of 230 × 120 × 892 voxels with a unit volume of 2 × 2 × 2 mm3, was ported into Monte Carlo code MCNPX2.5 to calculate the conversion coefficients from kerma free-in-air to absorbed dose and to effective dose for external monoenergetic photon beams from 15 keV to 10 MeV under six idealized external irradiation geometries (anterior-posterior, posterior-anterior, left lateral, right lateral, rotational, and isotropic). Organ masses of the VCH model are fairly different from other human phantoms. Differences of up to 300% are observed between doses from ICRP 74 data and those of VIP-Man. Detailed information from the VCH model is able to improve the radiological datasets, particular for the Chinese population, and provide insights into the research of various computational phantoms.

Localization-based super-resolution microscopy with an sCMOS camera

In the community of localization-based super-resolution microscopy (or called localization microscopy), it is generally believed that the emission of single molecules is so weak that an EMCCD (electron multiplying charge coupled device) camera is necessary to be used as the detector by eliminating read noise. Here we evaluate the possibility of a new kind of low light detector, scientific complementary metal-oxide-semiconductor (sCMOS) camera in localization microscopy. We demonstrate experimentally that sCMOS is capable of imaging actin bundles with FWHM diameter of 37 nm, evidencing the capability of sCMOS in localization microscopy. We further characterize the noise performance of sCMOS and find out that, with the use of a bright fluorescence probe such as d2EosFP, localization microscopy imaging is now working in the shot noise limited region.