Time-gated Mode Research Articles

Sensitization of Europium Oxide Nanoparticles Enhances Signal-to-Noise over Autofluorescence with Time-Gated Luminescence Detection.

Clinical translation of nanoparticle-based therapeutics has been limited, and a lack of preclinical delivery characterization is partly to blame, limiting our understanding of the mechanisms of failure. The improvement of the preclinical delivery assessment requires nanoparticles with higher detectability. This work focused on the exploration of several aromatic carboxylic ligands (terephthalic acid, quinaldic acid, and kynurenic acid) for the sensitization of europium oxide nanoparticles with a long emission lifetime to overcome cellular autofluorescence, a key confounder of detection in luminescence-based bioimaging. A facile one-pot synthesis and ligand exchange process generated and sensitized ultrasmall Eu2O3 cores. As reflected in the emission spectra and lifetimes, ligand binding yielded unique coordination environments around Eu3+. Then, the efficacy of sensitization was tested against the autofluorescence provided by tissue lysate. Normal (simultaneous excite-read) measurements showed integrated signal improvements over autofluorescence of 2.2-, 3.9-, and 14.0-fold for EuTA, EuQA, and EuKA, respectively. In time-gated mode, the improvements over autofluorescence were more dramatic with fold differences of 75-, 89-, and 108-fold for EuTA, EuQA, and EuKA, respectively. The investigation of novel sensitizers expands the breadth of the field of sensitized lanthanide oxide nanoparticles, and the signal enhancement observed with sensitization and time-gating supports the utility of the generated samples for future bioimaging applications.

Inorganic Chemistry in Dalian University of Technology: Fundamental Science and Solution for Renewable Energy, Environment, Health, and Materials

Inorganic Chemistry in Dalian University of Technology: Fundamental Science and Solution for Renewable Energy, Environment, Health, and Materials

Gate-Width Optimisation Based on Time-Gated Single Photon Avalanche Diode Receiver for Optical Wireless Communications

Using a single photon avalanche diode (SPAD) as a receiver in an optical wireless communications (OWC) system can effectively expand the transmission distance. However, the performance of the SPAD receiver is usually affected with the bit error rate (BER) lower bound determined by background light and the inter-symbol interference (ISI) distortion caused by dead time. In this paper, external time-gated technology is employed, and the SPAD is only activated within the set gate-ON time to alleviate the influence of background light and ISI distortion. The SPAD photon counting model and the communication BER model are established according to the external time-gated characteristics. Considering the functional relationship among the gate-ON time, signal light flux, background light flux and blocking time, we take the minimum BER as the optimization target, the optimal gate-ON time is derived. The numerical results show that whenever the signal light flux or the background light flux is higher, the BER performance of the time-gated mode is apparently better than the free-running mode. For example, when signal photons and background photons are 30 and 10 per symbol time, respectively, the BER of free-running SPAD converges to 0.1, while the BER of the time-gated scheme is about 10−6.

Imaging luminescent tattoo inks for direct visualization of linac and cobalt irradiation.

Tattoo fiducials are commonly used in radiotherapy patient alignment, and recent studies have examined the use of UV-excited luminescent tattoo ink as a cosmetic substitute to make these visible under UV illumination. The goal of this study was to show how luminescent tattoo inks could be excited with MV radiation and imaged during beam delivery for direct visualization of field position. A survey of nine UV-sensitive tattoo inks with various emission spectra were investigated using both UV and MV excitation. Images of liquid solutions were collected under MV excitation using an intensified-CMOS imager. Solid skin-simulating phantoms were imaged with both surface-painted ink and in situ tattooing during dose delivery by both a clinical linear accelerator and cobalt-60 source. The UV inks have peak fluorescence emission ranging from approximately 440 to 600nm with lifetimes near 11-16μs. The luminescence intensity is approximately 6x higher during the x-ray pulse than after the pulse, however, the signal-to-noise is only approximately twice as large. Spatial resolution for imaging was achieved at 1.6mm accuracy in a skin test phantom. Optical filtering allows for continuous imaging using a cobalt source and provides a mechanism to discriminate ink colors using a monochromatic image sensor. This study demonstrates how low-cost inks can be used as fiducial markers and imaged both using time-gated and continuous modes during MV dose delivery. Phantom studies demonstrate the potential application of real-time field verification. Further studies are required to understand if this technique could be used as a tool for radiation dosimetry.

A folic acid-functionalized dual-emissive nanoprobe for "double-check" luminescence imaging of cancer cells.

Development of luminescent probes for rapid and effective discrimination and detection of cancer cells has the potential to address the current challenges in early diagnosis and treatment monitoring of cancer diseases. In this work, we report the preparation of a unique folic acid (FA)-functionalized dual-emissive nanoprobe, CTMR@BHHBCB-Eu-FA, for steady-state and time-gated luminescence "double-check" imaging of cancer cells. The nanoprobe was engineered by covalently doping two luminescent dyes, 5-carboxytetramethylrhodamine (CTMR) and BHHBCB-Eu3+, in core and shell of silica nanoparticles, followed by surface modification of the nanoparticles with FA, a cancer cell-targeting molecule. As-prepared nanoprobe is monodisperse and highly stable in buffer displaying two strong emissions, short-lived emission from CTMR at 584 nm and long-lived emission from BHHBCB-Eu3+ at 612 nm. The nanoprobe is biocompatible, and can specifically recognize folate receptor (FR)-overexpressed cancer cells through the FA-FR binding interaction. Using the nanoprobe, the "double-check" imaging of HeLa cells was successfully achieved at steady-state and time-gated luminescence modes, indicating the capability of the nanoprobe for cancer cell imaging.

Quantitative Monitoring and Visualization of Hydrogen Sulfide In Vivo Using a Luminescent Probe Based on a Ruthenium(II) Complex.

Development of novel bioanalytical methods for monitoring of H2 S is key toward understanding the physiological and pathological functions of this gasotransmitter in live organisms. A ruthenium(II)-complex-based luminescence probe, Ru-MDB (MDB: 4'-methyl-[2,2'-bipyridine]-4-yl)methyl 2-((2,4-dinitrophenyl)thio)benzoate), was developed by introducing a new H2 S responsive masking moiety to a red-emitting RuII luminophore. Cleavage of this masking group by a H2 S-triggered reaction leads to a luminescence "off-on" response. The long-lived emissions of Ru-MDB and its reaction product with H2 S allowed quantitative detection of H2 S in autofluorescence-rich human sera and adult zebrafish organs using the time-gated luminescence mode. Ru-MDB exhibits red emission, a large Stokes shift, high specificity and sensitivity for H2 S detection, and low cytotoxicity, which enables imaging and flow cytometry analysis of lysosomal H2 S generation in live inflamed cells under drug stimulation. Monitoring of H2 S in live Daphnia magna, zebrafish embryos, adult zebrafish, and mice, was conducted by in vivo imaging using Ru-MDB as a probe.

Development of a novel europium complex-based luminescent probe for time-gated luminescence imaging of hypochlorous acid in living samples

Luminescent lanthanide complexes are key reagents used in the time-gated luminescence bioassay technique, but functional lanthanide complexes that can act as luminescent probes for specifically responding to analytes are very limited. In this work, we designed and synthesized a novel Eu3+ complex-based luminescence probe for hypochlorous acid (HOCl), NPPTTA-Eu3+, by using terpyridine polyacid-Eu3+, dinitrophenyl, and hydrazine as luminophore, quencher and HOCl-recognizer moieties, respectively. In the absence of HOCl, the probe is non-luminescent due to the strong luminescence quenching of the dinitrophenyl group in the complex. However, upon reaction with HOCl, the dinitrophenyl moiety is rapidly cleaved from the probe, which affords a strongly luminescent Eu3+ complex CPTTA-Eu3+, accompanied by a ∼900-fold luminescence enhancement with a long luminescence lifetime of 1.41 ms. This unique luminescence response of NPPTTA-Eu3+ to HOCl allowed NPPTTA-Eu3+ to be conveniently used as a probe for highly selective and sensitive detection of HOCl under the time-gated luminescence mode. In addition, by loading NPPTTA-Eu3+ into RAW 264.7 macrophage cells and Daphnia magna, the generation of endogenous HOCl in RAW 264.7 cells and the uptake of exogenous HOCl by Daphnia magna were successfully imaged on a true-color time-gated luminescence microscope. The results demonstrated the practical applicability of NPPTTA-Eu3+ as an efficient probe for time-gated luminescence imaging of HOCl in living cells and organisms.

A β-diketonate–europium(iii) complex-based fluorescent probe for highly sensitive time-gated luminescence detection of copper and sulfide ions in living cells

A strongly fluorescent β-diketonate–europium(iii) complex was developed for highly sensitive imaging of intracellular copper and sulfide ions with time-gated luminescence mode.

A visible-light-excited Eu3+ complex-based luminescent probe for highly sensitive time-gated luminescence imaging detection of intracellular peroxynitrite.

Although considerable efforts have been made for the development of lanthanide complex-based luminescent probes, a major drawback of the current probes is the requirement for UV excitation. In this work, a visible-light-excited Eu3+ complex-based luminescent probe, [Eu(HDPH)3(DPBT)] (HDPH: 1,1,1,2,2,3,3-heptafluoro-6-(2,4-dimethoxyphenyl)-4,6-hexanedione; DPBT: 2-(N,N-diethylanilin-4-yl)-4,6-bis(3,5-dimethylpyrazol-1-yl)-1,3,5-triazine), has been designed and synthesized for the highly sensitive and selective time-gated luminescence imaging detection of peroxynitrite (ONOO-) in living cells. The probe exhibits a strong visible excitation peak at 400 nm, with the excitation window extending to beyond 460 nm. Upon reaction with ONOO-, the luminescence of the probe at 610 nm is greatly quenched because the excited state of the Eu3+ complex is deactivated by the interaction between the 2,4-dimethoxyphenyl moiety and ONOO-. The dose-dependent luminescence decrease of the probe shows good linearity with a detection limit of 1.8 μM for ONOO-, and the probe can specifically distinguish ONOO- from other reactive oxygen/nitrogen species (ROS/RNS). The [Eu(HDPH)3(DPBT)]-loaded HepG2 cells were prepared, and then the exogenous ONOO- in HepG2 cells was successfully imaged in time-gated luminescence mode. The photostability results of the [Eu(HDPH)3(DPBT)]-loaded HepG2 cells demonstrate the practical applicability of the visible-light-excited probes for long-term cell imaging.

Comparison of Cherenkov excited fluorescence and phosphorescence molecular sensing from tissue with external beam irradiation

Ionizing radiation delivered by a medical linear accelerator (LINAC) generates Cherenkov emission within the treated tissue. A fraction of this light, in the 600–900 nm wavelength region, propagates through centimeters of tissue and can be used to excite optical probes in vivo, enabling molecular sensing of tissue analytes. The success of isolating the emission signal from this Cherenkov excitation background is dependent on key factors such as: (i) the Stokes shift of the probe spectra; (ii) the excited state lifetime; (iii) the probe concentration; (iv) the depth below the tissue surface; and (v) the radiation dose used. Previous studies have exclusively focused on imaging phosphorescent dyes, rather than fluorescent dyes. However there are only a few biologically important phosphorescent dyes and yet in comparison there are thousands of biologically relevant fluorescent dyes. So in this study the focus was a study of efficacy of Cherenkov-excited luminescence using fluorescent commercial near-infrared probes, IRDye 680RD, IRDye 700DX, and IRDye 800CW, and comparing them to the well characterized phosphorescent probe Oxyphor PtG4, an oxygen sensitive dye. Each probe was excited by Cherenkov light from a 6 MV external radiation beam, and measured in continuous wave or time-gated modes. The detection was performed by spectrally resolving the luminescence signals, and measuring them with spectrometer-based separation on an ICCD detector. The results demonstrate that IRDye 700DX and PtG4 allowed for the maximal signal to noise ratio. In the case of the phosphorescent probe, PtG4, with emission decays on the microsecond (μs) time scale, time-gated acquisition was possible, and it allowed for higher efficacy in terms of the probe concentration and detection depth. Phantoms containing the probe at 5 mm depth could be detected at concentrations down to the nanoMolar range, and at depths into the tissue simulating phantom near 3 cm. In vivo studies showed that 5 nmol of dye was readily detected with radiation doses less than 5 cGy. Since concentration, radiation dose and depth each contribute to the level of the detected signal, it may be possible to improve any of these parameters at expense of the others. This paradigm of nanoMolar sensitivity for optical reporters in vivo introduces the concept of molecular sensing of tumors during therapy or diagnostically with biologically relevant concentrations of fluorescent reporters.

Afterpulsing Characteristics of Free-Running and Time-Gated Single-Photon Avalanche Diodes in 130-nm CMOS

The temperature-dependent afterpulsing characteristics of single-photon avalanche diodes (SPADs) fabricated in a standard digital 130-nm CMOS technology are described. Avalanche interarrival time statistics are analyzed to obtain the afterpulsing probabilities (APs) for 81- and 110- $\mu \text{m}^{2}$ active area SPAD pixels using free running (FR) and time-gated (TG) front-end circuits. A strong reduction in AP was evident in the TG mode compared with the FR SPAD pixels. Optimal operating conditions in terms of hold-off time and temperature were found for <1% AP.

100 000 Frames/s 64 × 32 Single-Photon Detector Array for 2-D Imaging and 3-D Ranging

We report on the design and characterization of a multipurpose 64 × 32 CMOS single-photon avalanche diode (SPAD) array. The chip is fabricated in a high-voltage 0.35-μm CMOS technology and consists of 2048 pixels, each combining a very low noise (100 cps at 5-V excess bias) 30-μm SPAD, a prompt avalanche sensing circuit, and digital processing electronics. The array not only delivers two-dimensional intensity information through photon counting in either free-running (down to 10-μs integration time) or time-gated mode, but can also perform smart light demodulation with in-pixel background suppression. The latter feature enables phase-resolved imaging for extracting either three-dimensional depth-resolved images or decay lifetime maps, by measuring the phase shift between a modulated excitation light and the reflected photons. Pixel-level memories enable fully parallel processing and global-shutter readout, preventing motion artifacts (e.g., skew, wobble, motion blur) and partial exposure effects. The array is able to acquire very fast optical events at high frame-rate (up to 100 000 fps) and at single-photon level. Low-noise SPADs ensure high dynamic range (up to 110 dB at 100 fps) with peak photon detection efficiency of almost 50% at 410 nm. The SPAD imager provides different operating modes, thus, enabling both time-domain applications, like fluorescence lifetime imaging (FLIM) and fluorescence correlation spectroscopy, as well as frequency-domain FLIM and lock-in 3-D ranging for automotive vision and lidar.

A highly sensitive protocol for the determination of Hg2+ in environmental water using time-gated mode

In this paper, a sensitive time-gated fluorescent sensing strategy for mercury ions (Hg2+) monitoring is developed based on Hg2+-mediated thymine (T)–Hg2+–T structure and the mechanism of fluorescence resonance energy transfer from Mn-doped CdS/ZnS quantum dots to graphene oxide. The authors employ two T-rich single-stranded DNA (ssDNA) as the capture probes for Hg2+, and one of them is modified with Mn-doped CdS/ZnS quantum dots. The addition of Hg2+ makes the two T-rich ssDNA hybrids with each other to form stable T-Hg2+-T coordination chemistry, which makes Mn-doped CdS/ZnS quantum dots far away from the surface of graphene oxide. As a result, the fluorescence signal is increased obviously compared with that without Hg2+. The time-gated fluorescence intensities are linear with the concentrations of Hg2+ in the range from 0.20 to 10nM with a limit of detection of 0.11nM. The detection limit is much lower than the U.S. Environmental Protection Agency limit of the concentration of Hg2+ for drinking water. The time-gated fluorescent sensing strategy is specific for Hg2+ even with interference by other metal ions based on the results of selectivity experiments. Importantly, the proposed sensing strategy is applied successfully to the determination of Hg2+ in environmental water samples.

Time-gated fluorescence sensor for silver ions using Mn:CdS/ZnS quantum dots/DNA/gold nanoparticle complexes

A fluorescent sensor for the determination of Ag+ by time-gated mode is proposed. The method is based on Ag+-induced formation of quantum dot/DNA/gold nanoparticle complexes. Two ssDNA strands (strand A and strand B) are designed which contain C–C mismatched base pairs that can specifically react with Ag+. The water-soluble long-lifetime fluorescence quantum dots (Mn:CdS/ZnS) functionalized with strand A are selected as the fluorophore. The gold nanoparticles (GNPs) which are labeled with strand B, acted as the quencher. When Ag+ is absent in the sample solution, DNA-modified GNPs and Mn:CdS/ZnS were in the freedom state, the fluorescence signal of Mn:CdS/ZnS is obviously strong. When Ag+ are present in the sample solution, a DNA duplex is formed because of the strong binding ability between Ag+ and cytosine bases and formed stable C–Ag+–C structures. As a result, the Mn:CdS/ZnS and the GNPs are brought into close proximity, which caused the fluorescence quenching of the Mn:CdS/ZnS due to the nanometal surface energy transfer (NSET) between the Mn:CdS/ZnS and GNPs. This fluorescent sensor could present a satisfactory specificity and selectivity for Ag+. Meanwhile, the detection limit of Ag+ was estimated to be 7.9 nM.

SPAD Image Sensor With Analog Counting Pixel for Time-Resolved Fluorescence Detection

This paper presents a 32 × 32 pixel image sensor for time-gated fluorescence lifetime detection based on single-photon avalanche diodes. The sensor, fabricated in a high-voltage 0.35- μm CMOS technology, uses an analog counting approach to minimize the area occupation of pixel electronics while maintaining a nanosecond timing resolution and shot-noise-limited operation. The all nMOS pixel is formed by 12 transistors and features 25- μm pitch and 20.8% fill factor. The chip includes a phase-locked loop circuit for gating window generation, working at a maximum repetition frequency of 40 MHz, while the sensor can be gated at frequency up to 80 MHz using an external delay generator. Optical characterization with a picosecond-pulsed laser showed a minimum gating window width of 1.1 ns. Example images acquired in both continuous and time-gated mode are presented, together with a lifetime image obtained with the sensor mounted on a fluorescence microscope.

A low-noise time-gated single-photon detector in a HV-CMOS technology for triggered imaging

An optical imager for triggered applications is presented. The detector consists of a 10×43 array of single-photon avalanche pixels and exhibits an unusual fill-factor of 67%. It has been fabricated in a conventional 0.35μm HV-CMOS process. The array presents an average dark count rate of 67kHz at a reverse bias overvoltage of 1V. Due to the large sensor area of 20μm×100μm, the array is operated in a time-gated mode to reduce the probability of detecting the sensor noise down to 10−4 noise counts per frame with an active period of 4ns. The crosstalk can be minimized to negligible levels also by means of the time-gated operation. A number of experiments have been conducted on the detector to show that the proposed technique is advantageous in improving the imager signal-to-noise ratio, dynamic range, contrast and spatial resolution.

Simple no-chromatography procedure for amine-reactive Eu3+ luminescent chelates optimal for bioconjugation

Lanthanide ions luminescence has long life time enabling highly sensitive detection in time-gated mode. The synthesis of reactive lanthanide probes for covalent labeling of the objects of interest is cumbersome task due to the large size of the probes, complex multi-step procedures and the presence of sensitive groups, which often prevents introduction of reactive cross-linking functions optimal for conjugation. We suggest simple synthetic protocol for luminescent europium chelates based on serendipitous reaction yielding acylating compounds, whose reactivity is comparable to that of commonly used N-hydroxysuccinimide (NHS) esters. The probes react with proteins at pH 7.0 within several minutes at ambient temperature displaying high coupling efficiency. The resulting conjugates survive electrophoretic separation under denaturing conditions, which makes the labels useful in proteomic studies that rely on high detection sensitivity.

“Turn-On” Fluorescent Sensor for Hg2+ Based on Single-Stranded DNA Functionalized Mn:CdS/ZnS Quantum Dots and Gold Nanoparticles by Time-Gated Mode

An ultrasensitive "turn-on" fluorescent sensor was presented for determination of Hg(2+). This method is mainly based on Hg(2+)-induced conformational change of a thymine-rich single-stranded DNA. The water-soluble long-lifetime fluorescence quantum dot (Mn:CdS/ZnS) acted as the fluorophore, which was labeled on a 33-mer thymine-rich single-stranded DNA (strand A). The gold nanoparticles (GNPs) functionalized 10-mer single-stranded DNA (strand B) is selected as the quencher to quench the fluorescence of Mn:CdS/ZnS. Without Hg(2+) in the sample solution, strands A and B could form hybrid structures, resulting in the fluorescence of Mn:CdS/ZnS being decreased sharply. When Hg(2+) is present in the sample solution, Hg(2+)-mediated base pairs induced the folding of strand A into a hairpin structure, leading to the release of GNPs-tagged strand B from the hybrid structures. The fluorescence signal is then increased obviously compared with that without Hg(2+). The sensor exhibits two linear response ranges between fluorescence intensity and Hg(2+) concentration. Meanwhile, a detection limit of 0.18 nM is estimated based on 3α/slope. Selectivity experiments reveal that the fluorescent sensor is specific for Hg(2+) even with interference by high concentrations of other metal ions. This sensor is successfully applied to determination of Hg(2+) in tap water and lake water samples. This sensor offers additional advantage to efficiently reduce background noise using long-lifetime fluorescence quantum dots by a time-gated mode. With excellent sensitivity and selectivity, this sensor is potentially suitable for monitoring of Hg(2+) in environmental applications.

Lanthanide Complex-Based Luminescent Probes for Highly Sensitive Time-Gated Luminescence Detection of Hypochlorous Acid

Two novel lanthanide complex-based luminescent probes, ANMTTA-Eu(3+) and ANMTTA-Tb(3+) {ANMTTA, [4'-(4-amino-3-nitrophenoxy)methylene-2,2':6',2"-terpyridine-6,6"-diyl] bis(methylenenitrilo) tetrakis(acetic acid)}, have been designed and synthesized for the highly sensitive and selective time-gated luminescence detection of hypochlorous acid (HOCl) in aqueous media. The probes are almost nonluminescent due to the photoinduced electron transfer (PET) process from the 4-amino-3-nitrophenyl moiety to the terpyridine-Ln(3+) moiety, which quenches the lanthanide luminescence effectively. Upon reaction with HOCl, the 4-amino-3-nitrophenyl moiety is rapidly cleaved from the probe complexes, which affords strongly luminescent lanthanide complexes HTTA-Eu(3+) and HTTA-Tb(3+) {HTTA, (4'-hydroxymethyl-2,2':6',2"-terpyridine-6,6"-diyl) bis(methylenenitrilo) tetrakis(acetic acid)}, accompanied by the remarkable luminescence enhancements. The dose-dependent luminescence enhancements show good linearity with detection limits of 1.3 nM and 0.64 nM for HOCl with ANMTTA-Eu(3+) and ANMTTA-Tb(3+), respectively. In addition, the luminescence responses of ANMTTA-Eu(3+) and ANMTTA-Tb(3+) to HOCl are pH-independent with excellent selectivity to distinguish HOCl from other reactive oxygen/nitrogen species (ROS/RNS). The ANMTTA-Ln(3+)-loaded HeLa and RAW 264.7 macrophage cells were prepared, and then the exogenous HOCl in HeLa cells and endogenous HOCl in macrophage cells were successfully imaged with time-gated luminescence mode. The results demonstrated the practical applicability of the probes for the cell imaging application.

Cell‐Penetrating Peptides as Delivery Vehicles for a Protein‐Targeted Terbium Complex

Release after transmission: Arginine-rich, cell-penetrating peptides (CPPs) mediate cytoplasmic delivery of trimethoprim (TMP)-terbium complex conjugates and selective, intracellular labeling of E. coli dihydrofolate reductase (eDHFR) fusion proteins. A disulfide bond linking CPP and cargo is reduced following uptake. CPP conjugation can be used to deliver otherwise cell-impermeable, ligand-fluorophore conjugates.