Jinling Lu

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.

Dual-wavelength laser speckle imaging to simultaneously access blood flow, blood volume, and oxygenation using a color CCD camera

We developed a dual-wavelength laser speckle imaging system using a single industrial-grade color CCD camera with Bayer filters to simultaneously image changes in blood flow, blood volume, and oxygenation. One frame of a color image recorded with dual-wavelength laser illumination provides not only the intensity fluctuation of the speckle pattern, but also the dual-wavelength optical reflectance signal. The method was validated using a tissue phantom and cuff ischemia experiments in the human arm. This system achieves complete time synchronization, unlike conventional time-sharing systems. Compared with a multicamera system, it also avoids the problem of image registration and can be less expensive.

Fluorescence imaging to assess the matrix metalloproteinase activity and its inhibitor in vivo.

Matrix metalloproteinases (MMPs) are a kind of secretory proteinases. Degradation of the extracellular matrix (ECM) by MMPs enhances tumor invasion and metastasis. To monitor MMPs activity and assess the MMP inhibitor effects in vivo, we constructed a plasmid that encoded a secretory fluorescent sensor named DMC (DsRed2-MSS-CFP expressed from pDisplay vector) that DsRed2 and cyan fluorescent protein (CFP) linked by MMP substrate site (MSS). MDA-MB 435s cells highly expressing endogenetic secretory MMP were transfected with the DMC plasmid so that the DMC could be cleaved by endogenetic MMP and the fluorescence ratio of DsRed2 to CFP was decreased. Treating the cells with GM6001, an MMP inhibitor, blocked the cleavage of DMC and caused an increase of the DsRed2/CFP ratio. The same result was achieved by using an in vivo tumor model that stable DMC-expressing MDA-MB 435s cells inoculated onto the chorioallantoic membrane of developing chick embryos to form primary tumors on the membrane. Thus, the fluorescent sensor DMC is able to sensitively monitor MMP activity and assess MMP inhibitors for anticancer research in vivo. This proves a novel method to efficiently screen and assess the anticancer drug MMP inhibitor in living cells and in vivo tumor models.

Visualization of β-secretase cleavage in living cells using a genetically encoded surface-displayed FRET probe

The human beta-secretase, BACE, plays a key role in the generation of pathogenic amyloid beta-peptide (Abeta) in Alzheimers disease and has been identified as an ideal target for therapy. Previous studies reported the monitoring of BACE activity in vitro utilizing chemical synthesized sensors. Here we describe the first genetically encoded FRET probe that can detect BACE activity in vivo. The FRET probe was constructed with the BACE substrate site (BSS) and two mutated green fluorescent proteins. In living cell, the FRET probe was directed to the secretory pathway and anchored on the cell surface to measure BACE enzymatic activity. The results show that the FRET probe can be cleaved by BACE effectively in vivo, suggesting that the probe can be used for real-time monitoring of BACE activity. This assay provides a novel platform for BACE inhibitor screening in vivo.

mBeRFP, an Improved Large Stokes Shift Red Fluorescent Protein

Herein, we describe the generation of a monomeric large Stokes shift (LSS) red fluorescent protein, mBeRFP, with excitation and emission peaks at 446 and 615 nm, respectively. Compared with two previously reported LSS-RFPs (mKeima and LSS-mKate2), mBeRFP is approximately three times brighter. In addition, mBeRFP is characterized by improved photostability, rapid maturation, an extended lifetime, and a monomeric nature. Additionally, mBeRFP can be paired with the Alexa 647 dye as a FRET donor to detect caspase 3 activity. This FRET pair has an extremely dynamic range and a large Förster radius (approximately 6.5 nm). To demonstrate the applicability of mBeRFP for imaging in living cells, we performed dual-color imaging of mBeRFP and CFP simultaneously excited by a single excitation source, and we demonstrated that these fluorescent proteins allow the clear visualization of the dynamics of Bax during cancer cell apoptosis. Thus, mBeRFP appears to be particularly useful for cellular imaging applications.

Enhanced dynamic range in a genetically encoded Ca2+ sensor

Genetically encoded fluorescence resonance energy transfer (FRET) indicators are powerful tools for real-time detection of second messenger molecules and activation of signal proteins. However, these fluorescent protein-based sensors typically display marginal FRET efficiency. To improve their FRET efficiency for optical imaging and screening, we developed a number of fluorescent protein mutants based on cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). To improve FRET ratios, which were initially within a narrow dynamic range, we used DNA shuffling to develop a new FRET pair called 3xCFP/Venus. The optimized 3xCFP/Venus pair exhibited higher FRET ratios than CyPet/YPet, which has one of the greatest dynamic ranges of protein-based FRET pairs. We converted this FRET pair to a Ca(2+) FRET indicators using circular permutation Venus (cpVenus) linked with 3xCFP to form 3xCFP/cpVenus, which displayed an ∼11-fold change in dynamic range in response to Ca(2+) binding. The enhanced dynamic range for Ca(2+) concentration detection using 3xCFP/cpVenus was confirmed in PC12 cells using previously established indicators (TN-XXL, ECFP/cpCitrine). To our knowledge, this FRET pair displays the largest dynamic range so far among genetically-encoded sensors, and can be used for sensitive FRET detection.

Coherent slow cortical potentials reveal a superior localization of resting-state functional connectivity using voltage-sensitive dye imaging

The resting-state functional connectivity (RSFC) of spontaneous hemodynamic fluctuations is widely used to investigate large-scale functional brain networks based on neurovascular mechanisms. However, high-resolution RSFC networks based on neural activity have not been disclosed to explore the neural basis of these spontaneous hemodynamic signals. The present study examines the neural RSFC networks in mice at high spatial resolution using optical imaging with voltage-sensitive dyes (VSDs). Our results show that neural RSFC networks for the slow cortical potentials (0.1-4Hz) showed similar correlation patterns to the RSFC networks for the spontaneous hemodynamic signals, indicating a tight coupling between the slow cortical potential and the spontaneous hemodynamic signals during rest, but the bilateral symmetry of the RSFC networks for the slow cortical potentials was significantly lower than that for the spontaneous hemodynamic signals. Moreover, similar asymmetric neural activation patterns could also be found between the bilateral cortexes after stimulating the paws of mice. By increasing anesthetic levels to induce the reduction of consciousness, the RSFC networks for the slow cortical potentials persisted, but those for the spontaneous hemodynamic signals became discrete. These results suggest that the coherent slow cortical potentials underlie the spontaneous hemodynamic fluctuations and reveal a superior localization of RSFC networks. VSD imaging may potentially be used to examine the RSFC of neural activity, particularly under conditions of impaired neurovascular coupling.

Comparison of caspase-3 activation in tumor cells upon treatment of chemotherapeutic drugs using capillary electrophoresis

Caspases play important roles in cell apoptosis. Measurement of the dynamics of caspase activation in tumor cells not only facilitates understanding of the molecular mechanisms of apoptosis but also contributes to the development, screening, and evaluation of anticancer drugs that target apoptotic pathways. The fluorescence resonance energy transfer (FRET) technique provides a valuable approach for defining the dynamics of apoptosis with high spatio-temporal resolution. However, FRET generally functions in the single-cell level and becomes ineffective when applied in the high throughput detection of caspase activation. In the current study, a FRET sensor was combined with capillary electrophoresis (CE) to achieve a high throughput method for cellular caspase detection. The FRET-based CE system is composed of a homemade CE system and a laser source for detecting the dynamics of caspase-3 in various cells expressing sensors of caspase-3 that have been treated with anticancer drugs, such as cell cycle-independent drug cisplatin and specific cell cycle drugs camptothecin and etoposide, as well as their combination with tumor necrosis factor (TNF). A positive correlation between the caspase-3 activation velocity and drug concentration was observed when the cells were treated with cisplatin, but cells induced by camptothecin and etoposide did not show any apparent correlation with their concentrations. Moreover, different types of cells presented distinct sensitivities under the same drug treatment, and the combination treatment of TNF and anticancer drugs significantly accelerated the caspase-3 activation process. Its high throughput capability and detection sensitivity make the FRET-based CE system a useful tool for investigating the mechanisms of anticancer drugs and anticancer drug screening.

Improving the estimation of flow speed for laser speckle imaging with single exposure time

Laser speckle contrast imaging is a full-field imaging technique for measuring blood flow by mapping the speckle contrast with high spatial and temporal resolution. However, the statically scattered light from stationary tissues seriously degrades the accuracy of flow speed estimation. In this Letter, we present a simple calibration approach to calculate the proportions of dynamically scattered light and correct the effect of static scattering with single exposure time. Both the phantom and animal experimental results suggest that this calibration approach has the ability to improve the estimation of the relative blood flow in the presence of static scattering.

Improving the sensitivity of velocity measurements in laser speckle contrast imaging using a noise correction method

We demonstrate that noise is an important factor contributing to the decline of sensitivity and linear response range of velocity measurements for laser speckle contrast imaging. We propose to use a noise correction method to improve the sensitivity of velocity measurements. For a kind of camera in which the mean values of the dark noise have been subtracted and negative counts have been set to zero, we propose a method to estimate the true dark noise based on the maximum likelihood estimation, which expands the application scope of the noise correction method.