Polarization-Enabled Multidimensional Optical Microscopy

Polarization-dependence provides a unique handle for extending the dimensionality of optical microscopy, with particular benefits in nonlinear optical imaging. Polarization- dependent second order nonlinear optical processes such as second harmonic generation (SHG) provide rich qualitative and quantitative information on local molecular orientation distribution. By bridging Mueller and Jones tensor, a theoretical framework was introduced to experimentally extend the application of polarization-dependent SHG microscopy measurements toward in vivo imaging, in which partial polarization or depolarization of the beam can complicate polarization analysis. In addition, polarization wavefront shaping was demonstrated to enable a new quantitative phase contrast imaging strategy for thin transparent samples. The axially-offset differential interference contrast microscopy (ADIC) was achieved as a combination of classic Zernike phase contrast and Nomarski differential interference contrast (DIC) methods. The fundamentally unique manner of this strategy also inspired rapid volumetric analysis in time dimension that is accessible for most existing microscopy systems. Finally, the dimensionality of high speed two-photon fluorescence imaging was extended to the spectral domain by spatial/spectral multiplexing, enabling beam scanning two photon fluorescence microscopy with 17 frames per second rate and over 2000 effective spectral data points

Ratiometric Fluorescence Probe for Monitoring Hydroxyl Radical in Live Cells Based on Gold Nanoclusters

Determination of hydroxyl radical (^•OH) with high sensitivity and accuracy in live cells is a challenge for evaluating the role that ^•OH plays in the physiological and pathological processes. In this work, a ratiometric fluorescence biosensor for ^•OH was developed, in which gold nanocluster (AuNC) protected by bovine serum albumin was employed as a reference fluorophore and the organic molecule 2-[6-(4′-hydroxy)­phenoxy-3<i>H</i>-xanthen-3-on-9-yl]­benzoic acid (HPF) acted as both the response signal and specific recognition element for ^•OH. In the absence of ^•OH, only one emission peak at 637 nm ascribed to AuNCs was observed, because HPF was almost nonfluorescent. However, fluorescence emission at 515 nm attributed to the HPF product after reaction with ^•OHdianionic fluoresceingradually increased with the continuous addition of ^•OH, while the emission at 637 nm stays constant, resulting in a ratiometric determination of ^•OH. The developed fluorescent sensor exhibited high selectivity for ^•OH over other reactive oxygen species (ROS), reactive nitrogen species (RNS), metal ions, and other biological species, as well as high accuracy and sensitivity with low detection limit to ∼0.68 μM, which fulfills the requirements for detection of ^•OH in a biological system. In addition, the AuNC-based inorganic–organic probe showed long-term stability against light illumination and pH, good cell permeability, and low cytotoxicity. As a result, the present ratiometric sensor was successfully used for bioimaging and monitoring of ^•OH changes in live cells upon oxidative stress

Single Probe for Imaging and Biosensing of pH, Cu²⁺ Ions, and pH/Cu²⁺ in Live Cells with Ratiometric Fluorescence Signals

It is very essential to disentangle the complicated inter-relationship between pH and Cu in the signal transduction and homeostasis. To this end, reporters that can display distinct signals to pH and Cu are highly valuable. Unfortunately, there is still no report on the development of biosensors that can simultaneously respond to pH and Cu²⁺, to the best of our knowledge. In this work, we developed a single fluorescent probe, AuNC@FITC@DEAC (AuNC, gold cluster; FITC, fluorescein isothiocyanate; DEAC, 7-diethylaminocoumarin-3-carboxylic acid), for biosensing of pH, Cu²⁺, and pH/Cu²⁺ with different ratiometric fluorescent signals. First, 2,2′,2″-(2,2′,2″-nitrilotris­(ethane-2,1-diyl)­tris­((pyridin-2-yl-methyl)­azanediyl))­triethanethiol (TPAASH) was designed for specific recognition of Cu²⁺, as well as for organic ligand to synthesize fluorescent AuNCs. Then, pH-sensitive molecule, FITC emitting at 518 nm, and inner reference molecule, DEAC with emission peak at 472 nm, were simultaneously conjugated on the surface of AuNCs emitting at 722 nm, thus, constructing a single fluorescent probe, AuNC@FITC@DEAC, to sensing pH, Cu²⁺, and pH/Cu²⁺ excited by 405 nm light. The developed probe exhibited high selectivity and accuracy for independent determination of pH and Cu²⁺ against reactive oxygen species (ROS), other metal ions, amino acids, and even copper-containing proteins. The AuNC-based inorganic–organic probe with good cell-permeability and high biocompatibility was eventually applied in monitoring both pH and Cu²⁺ and in understanding the interplaying roles of Cu²⁺ and pH in live cells by ratiometric multicolor fluorescent imaging

Carbon-Dot-Based Ratiometric Fluorescent Probe for Imaging and Biosensing of Superoxide Anion in Live Cells

In this article, a ratiometric fluorescent biosensor for O₂^•– was developed, by employing carbon dots (C-Dots) as the reference fluorophore and hydroethidine (HE), a specific organic molecule toward O₂^•–, playing the role as both specific recognition element and response signal. The hybrid fluorescent probe CD-HE only emitted at 525 nm is ascribed to C-Dots, while HE was almost nonfluorescent, upon excitation at 488 nm. However, after reaction with O₂^•–, a new emission peak ascribed to the reaction products of HE and O₂^•– was clearly observed at 610 nm. Meanwhile, this peak gradually increased with the increasing concentration of O₂^•– but the emission peak at 525 nm stayed constant, leading to a ratiometric detection of O₂^•–. The inorganic–organic fluorescent sensor exhibited high sensitivity, a broad dynamic linear range of ∼5 × 10^–7–1.4 × 10^–4 M, and low detection limit down to 100 nM. The present probe also showed high accuracy and excellent selectivity for O₂^•– over other reactive oxygen species (ROS), metal ions, and so on. Moreover, the C-Dot-based inorganic–organic probe demonstrated long-term stability against pH changes and continuous light illumination, good cell-permeability, and low cytotoxicity. Accordingly, the developed fluorescent biosensor was eventually applied for intracellular bioimaging and biosensing of O₂^•– changes upon oxidative stress

Two-Photon Ratiometric Fluorescent Sensor Based on Specific Biomolecular Recognition for Selective and Sensitive Detection of Copper Ions in Live Cells

In this work, we develop a ratiometric two-photon fluorescent probe, ATD@QD-E₂Zn₂SOD (ATD = amino triphenylamine dendron, QD = CdSe/ZnSe quantum dot, E₂Zn₂SOD = Cu-free derivative of bovine liver copper–zinc superoxide dismutase), for imaging and sensing the changes of intracellular Cu²⁺ level with clear red-to-yellow color change based on specific biomolecular recognition of E₂Zn₂SOD for Cu²⁺ ion. The inorganic–organic nanohybrided fluorescent probe features two independent emission peaks located at 515 nm for ATD and 650 nm for QDs, respectively, under two-photon excitation at 800 nm. Upon addition of Cu²⁺ ions, the red fluorescence of QDs drastically quenches, while the green emission from ATD stays constant and serves as a reference signal, thus resulting in the ratiometric detection of Cu²⁺ with high accuracy by two-photon microscopy (TPM). The present probe shows high sensivity, broad linear range (10^–7–10^–3 M), low detection limit down to ∼10 nM, and excellent selectivity over other metal ions, amino acids, and other biological species. Meanwhile, a QD-based inorganic-organic probe demonstrates long-term photostability, good cell-permeability, and low cytotoxicity. As a result, the present probe can visualize Cu²⁺ changes in live cells by TPM. To the best of our knowledge, this is the first report for the development of a QD-based two-photon ratiometric fluorescence probe suitable for detection of Cu²⁺ in live cells