Comparison of Time Resolved Optical Turbidity Measurements for Water Monitoring to Standard Real-Time Techniques.

Environmental water monitoring requires the estimation of the suspended solids load. In this paper, we compare the concentration range accessible through three different techniques: optical turbidity, acoustic backscattering and the newly in-lab developed time resolved optical turbidity. We focus on their comparison on measurements made in the laboratory on water suspensions of known particles and concentrations. We used laboratory grade kieselguhr, wheat starch and kaolin as suspended solid surrogates. The explored concentration domains are the ones, for the total suspended solid load, commonly encountered in wastewater and rivers in standard (less than 1 g/L to a few g/L) or extreme conditions such as floods or storm events (up to several dozen g/L). Regarding the operable concentration domain, the time resolved optical turbidity shows a clear advantage upon the other methods, whatever the kind of particle is.

Single Photon Avalanche Diode Arrays for Time-Resolved Raman Spectroscopy.

The detection of peaks shifts in Raman spectroscopy enables a fingerprint reconstruction to discriminate among molecules with neither labelling nor sample preparation. Time-resolved Raman spectroscopy is an effective technique to reject the strong fluorescence background that profits from the time scale difference in the two responses: Raman photons are scattered almost instantaneously while fluorescence shows a nanoseconds time constant decay. The combination of short laser pulses with time-gated detectors enables the collection of only those photons synchronous with the pulse, thus rejecting fluorescent ones. This review addresses time-gating issues from the sensor standpoint and identifies single photon avalanche diode (SPAD) arrays as the most suitable single-photon detectors to be rapidly and precisely time-gated without bulky, complex, or expensive setups. At first, we discuss the requirements for ideal Raman SPAD arrays, particularly focusing on the design guidelines for optimized on-chip processing electronics. Then we present some existing SPAD-based architectures, featuring specific operation modes which can be usefully exploited for Raman spectroscopy. Finally, we highlight key aspects for future ultrafast Raman platforms and highly integrated sensors capable of undistorted identification of Raman peaks across many pixels.

Time-Resolved Fluorescence Anisotropy of a Molecular Rotor Resolves Microscopic Viscosity Parameters in Complex Environments.

Understanding viscosity in complex environments remains a largely unanswered question despite its importance in determining reaction rates in vivo. Here, time-resolved fluorescence anisotropy imaging (TR-FAIM) is combined with fluorescent molecular rotors (FMRs) to simultaneously determine two non-equivalent viscosity-related parameters in complex heterogeneous environments. The parameters, FMR rotational correlation time and lifetime, are extracted from fluorescence anisotropy decays, which in heterogeneous environments show dip-and-rise behavior due to multiple dye populations. Decays of this kind are found both in artificially constructed adiposomes and in live cell lipid droplet organelles. Molecular dynamics simulations are used to assign each population to nano-environments within the lipid systems. The less viscous population corresponds to the state showing an average 25° tilt to the lipid membrane normal, and the more viscous population to the state showing an average 55° tilt. This combined experimental and simulation approach enables a comprehensive description of the FMR probe behavior within viscous nano-environments in complex, biological systems.

Fluorescence Lifetime and Intensity of Thioflavin T as Reporters of Different Fibrillation Stages: Insights Obtained from Fluorescence Up-Conversion and Particle Size Distribution Measurements.

Thioflavin T (ThT) assay is extensively used for studying fibrillation kinetics in vitro. However, the differences in the time course of ThT fluorescence intensity and lifetime and other physical parameters of the system, such as particle size distribution, raise questions about the correct interpretation of the aggregation kinetics. In this work, we focused on the investigation of the mechanisms, which underlay the difference in sensitivity of ThT fluorescence intensity and lifetime to the formation of protein aggregates during fibrillation by the example of insulin and during binding to globular proteins. The assessment of aggregate sizes and heterogeneity was performed using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). Using the sub-nanosecond resolution measurements, it was shown that the ThT lifetime is sensitive to the appearance of as much as a few percent of ThT bound to the high-affinity sites that occur simultaneously with an abrupt increase of the average particle size, particles concentration, and size heterogeneity. The discrepancy between ThT fluorescence intensity and a lifetime can be explained as the consequence of a ThT molecule fraction with ultrafast decay and weak fluorescence. These ThT molecules can only be detected using time-resolved fluorescence measurements in the sub-picosecond time domain. The presence of a bound ThT subpopulation with similar photophysical properties was also demonstrated for globular proteins that were attributed to non-specifically bound ThT molecules with a non-rigid microenvironment.

0.16 µm⁻BCD Silicon Photomultipliers with Sharp Timing Response and Reduced Correlated Noise.

Silicon photomultipliers (SiPMs) have improved significantly over the last years and now are widely employed in many different applications. However, the custom fabrication technologies exploited for commercial SiPMs do not allow the integration of any additional electronics, e.g., on-chip readout and analog (or digital) processing circuitry. In this paper, we present the design and characterization of two microelectronics-compatible SiPMs fabricated in a 0.16 µm⁻BCD (Bipolar-CMOS-DMOS) technology, with 0.67 mm × 0.67 mm total area, 10 × 10 square pixels and 53% fill-factor (FF). The photon detection efficiency (PDE) surpasses 33% (FF included), with a dark-count rate (DCR) of 330 kcps. Although DCR density is worse than that of state-of-the-art SiPMs, the proposed fabrication technology enables the development of cost-effective systems-on-chip (SoC) based on SiPM detectors. Furthermore, correlated noise components, i.e., afterpulsing and optical crosstalk, and photon timing response are comparable to those of best-in-class commercial SiPMs.

Distance-Resolving Raman Radar Based on a Time-Correlated CMOS Single-Photon Avalanche Diode Line Sensor.

Remote Raman spectroscopy is widely used to detect minerals, explosives and air pollution, for example. One of its main problems, however, is background radiation that is caused by ambient light and sample fluorescence. We present here, to the best of our knowledge, the first time a distance-resolving Raman radar device that is based on an adjustable, time-correlated complementary metal-oxide-semiconductor (CMOS) single-photon avalanche diode line sensor which can measure the location of the target sample simultaneously with the normal stand-off spectrometer operation and suppress the background radiation dramatically by means of sub-nanosecond time gating. A distance resolution of 3.75 cm could be verified simultaneously during normal spectrometer operation and Raman spectra of titanium dioxide were distinguished by this system at distances of 250 cm and 100 cm with illumination intensities of the background of 250 lux and 7600 lux, respectively. In addition, the major Raman peaks of olive oil, which has a fluorescence-to-Raman signal ratio of 33 and a fluorescence lifetime of 2.5 ns, were distinguished at a distance of 30 cm with a 250 lux background illumination intensity. We believe that this kind of time-correlated CMOS single-photon avalanche diode sensor could pave the way for new compact distance-resolving Raman radars for application where distance information within a range of several metres is needed at the same time as a Raman spectrum.

Chlorophyll fluorescence lifetime imaging provides new insight into the chlorosis induced by plant virus infection.

KEY MESSAGE: Leaf chlorosis induced by plant virus infection has a short fluorescence lifetime, which reflects damaged photosynthetic complexes and degraded chloroplasts. Plant viruses often induce chlorosis and necrosis, which are intimately related to photosynthetic functions. Chlorophyll fluorescence lifetime measurement is a valuable noninvasive tool for analyzing photosynthetic processes and is a sensitive indicator of the environment surrounding the fluorescent molecules. In this study, our central goal was to explore the effect of viral infection on photosynthesis by employing chlorophyll fluorescence lifetime imaging (FLIM), steady-state fluorescence, non-photochemical quenching (NPQ), transmission electron microscopy (TEM), and pigment analysis. The data indicated that the chlorophyll fluorescence lifetime of chlorotic leaves was significantly shorter than that of healthy control leaves, and the fitted short lifetime component of chlorophyll fluorescence of chlorotic leaves was dominant. This dominant short lifetime component may result from damage to the structure of thylakoid, which was confirmed by TEM. The NPQ value of chlorotic leaves was slightly higher than that of healthy green leaves, which can be explained by increased neoxanthin, lutein and violaxanthin content relative to chlorophyll a. The difference in NPQ is slight, but FLIM can provide simple and direct characterization of PSII structure and photosynthetic function. Therefore, this technique shows great potential as a simple and rapid method for studying mechanisms of plant virus infection.

Quantitative Live Cell FLIM Imaging in Three Dimensions.

In this chapter, the concept of fluorescence lifetime and its utility in quantitative live cell imaging will be introduced, along with methods to record and analyze FLIM data. Relevant applications in 3D tissue and live cell imaging, including multiplexed FLIM detection, will also be detailed.

Quantitative Imaging of Ca2+ by 3D-FLIM in Live Tissues.

The calcium concentration within living cells is highly dynamic and, for many cell types, a reliable indicator of the functional state of the cells-both of isolated cells, but even, more important, of cells in tissue. In order to dynamically quantify intracellular calcium levels, various genetically encoded calcium sensors have been developed-the best of which are those based on Förster resonant energy transfer (FRET). Here we present a fluorescence lifetime imaging (FLIM) method to measure FRET in such a calcium sensor (TN L15) in neurons of hippocampal slices and of the brain stem of anesthetized mice. The method gives the unique opportunity to determine absolute neuronal calcium concentrations in the living organism.

Interpenetrated Donor-Acceptor Heterojunctions in 2D Conjugated Dibenzo[g,p]chrysene-Based Kagome Covalent Organic Frameworks.

Dibenzo[g,p]chrysene can be viewed as a constrained propeller-shaped tetraphenylethylene with reduced curvature and has been utilized to construct dual-pore kagome covalent organic frameworks (COFs) with tightly packed two-dimensional (2D) layers owing to its rigid and more planar structural characteristics. Here, we introduce 2D COFs based on the node 4,4',4″,4‴-(dibenzo[g,p]chrysene-2,7,10,15-tetraphenyl)tetraamine (DBCTPTA) featuring extended conjugation compared to the dibenzo[g,p]chrysene-3,6,11,14-tetraamine (DBCTA) node. We establish two exceptionally crystalline imine-linked 2D COFs with a hexagonal dual-pore kagome structure based on the DBCTPTA core. The newly synthesized thienothiophene (TT) and benzodithiophene (BDT)-based DBCTPTA COFs show a tight stacking behavior between adjacent layers. Furthermore, we obtained an unprecedented, interpenetrated electron-donor/acceptor host-guest system with an electron-donating BDT DBCTPTA COF synthesized in situ with the soluble fullerene derivative [6,6]-phenyl-C61-butyric acid methyl ester (PCBM) serving as molecular acceptor. The BDT DBCTPTA COF@PCBM film shows a much shorter amplitude-averaged PL lifetime of 7 ± 2 ps compared to 30 ± 4 ps of the BDT DBCTPTA COF film, indicating the light-induced charge transfer process. The successful in situ formation of interpenetrated donor-acceptor heterojunctions within 2D COFs offers a promising strategy for establishing D-A heterojunctions in diverse framework materials with open channel systems.

Visualising varnish removal for conservation of paintings by fluorescence lifetime imaging (FLIM).

The removal of varnish from the surface is a key step in painting conservation. Varnish removal is traditionally monitored by examining the painting surface under ultraviolet illumination. We show here that by imaging the fluorescence lifetime instead, much better contrast, sensitivity, and specificity can be achieved. For this purpose, we developed a lightweight (4.8 kg) portable instrument for macroscopic fluorescence lifetime imaging (FLIM). It is based on a time-correlated single-photon avalanche diode (SPAD) camera to acquire the FLIM images and a pulsed 440 nm diode laser to excite the varnish fluorescence. A historical model painting was examined to demonstrate the capabilities of the system. We found that the FLIM images provided information on the distribution of the varnish on the painting surface with greater sensitivity, specificity, and contrast compared to the traditional ultraviolet illumination photography. The distribution of the varnish and other painting materials was assessed using FLIM during and after varnish removal with different solvent application methods. Monitoring of the varnish removal process between successive solvent applications by a swab revealed an evolving image contrast as a function of the cleaning progress. FLIM of dammar and mastic resin varnishes identified characteristic changes to their fluorescence lifetimes depending on their ageing conditions. Thus, FLIM has a potential to become a powerful and versatile tool to visualise varnish removal from paintings.

Improved transient electroluminescence technique based on time-correlated single-photon counting technology to evaluate organic mobility.

The transient electroluminescence (EL) technique is widely used to evaluate the carrier mobility in the field of organic light emitting diodes. The traditional analog detection strategy using oscilloscopes is generally limited since the background noise causes an underestimation of the mobility value. In this paper, we utilize time-correlated single-photon counting (TCSPC) to probe the transient EL for mobility calculation. The measurements on tris(8-hydroxyquinoline) aluminum (Alq3) show that the electron mobilities obtained using the TCSPC technique are slightly higher than those obtained from the analog method at all the investigated voltages. Moreover, the TCSPC mobilities demonstrate weaker dependence on the root of electrical field compared to the oscilloscope mobilities. These improvements are attributed to the unique principle of TCSPC, which quantifies the EL intensity by counting the number of single-photon pulses, improving its single-photon sensitivity and eliminating the negative impacts of electrical noise. These advantages make TCSPC a powerful technique in the characterization of time-resolved electroluminescence.

Lightsheet fluorescence lifetime imaging microscopy with wide-field time-correlated single photon counting.

We report on wide-field time-correlated single photon counting (TCSPC)-based fluorescence lifetime imaging microscopy (FLIM) with lightsheet illumination. A pulsed diode laser is used for excitation, and a crossed delay line anode image intensifier, effectively a single-photon sensitive camera, is used to record the position and arrival time of the photons with picosecond time resolution, combining low illumination intensity of microwatts with wide-field data collection. We pair this detector with the lightsheet illumination technique, and apply it to 3D FLIM imaging of dye gradients in human cancer cell spheroids, and C. elegans.

Silicon technologies for arrays of Single Photon Avalanche Diodes.

In order to fulfill the requirements of many applications, we recently developed a new technology aimed at combining the advantages of traditional thin and thick silicon Single Photon Avalanche Diodes (SPAD). In particular we demonstrated single-pixel detectors with a remarkable improvement in the Photon Detection Efficiency in the red/near-infrared spectrum (e.g. 40% at 800nm) while maintaining a timing jitter better than 100ps. In this paper we discuss the limitations of such Red-Enhanced (RE) technology from the point of view of the fabrication of small arrays of SPAD and we propose modifications to the structure aimed at overcoming these issues. We also report the first preliminary experimental results attained on devices fabricated adopting the improved structure.

Initial photophysical characterization of the proteorhodopsin optical proton sensor (PROPS).

Fluorescence is not frequently used as a tool for investigating the photocycles of rhodopsins, largely because of the low quantum yield of the retinal chromophore. However, a new class of genetically encoded voltage sensors is based upon rhodopsins and their fluorescence. The first such sensor reported in the literature was the proteorhodopsin optical proton sensor (PROPS), which is capable of indicating membrane voltage changes in bacteria by means of changes in fluorescence. However, the properties of this fluorescence, such as its lifetime decay components and its origin in the protein photocycle, remain unknown. This paper reports steady-state and nanosecond time-resolved emission of this protein expressed in two strains of Escherichia coli, before and after membrane depolarization. The voltage-dependence of a particularly long lifetime component is established. Additional work to improve quantum yields and improve the general utility of PROPS is suggested.

The influence of dead time related distortions on live cell fluorescence lifetime imaging (FLIM) experiments.

Recent developments in the field of fluorescence lifetime imaging microscopy (FLIM) techniques allow the use of high repetition rate light sources in live cell experiments. For light sources with a repetition rate of 20-100 MHz, the time-correlated single photon counting (TCSPC) FLIM systems suffer serious dead time related distortions, known as "inter-pulse pile-up". The objective of this paper is to present a new method to quantify the level of signal distortion in TCSPC FLIM experiments, in order to determine the most efficient laser repetition rate for different FLT ranges. Optimization of the F -value, which is the relation between the relative standard deviation (RSD) in the measured FLT to the RSD in the measured fluorescence intensity (FI), allows quantification of the level of FI signal distortion, as well as determination of the correct FLT of the measurement. It is shown that by using a very high repetition rate (80 MHz) for samples characterized by high real FLT's (4-5 ns), virtual short FLT components are added to the FLT histogram while a F -value that is higher than 1 is obtained. For samples characterized with short real FLT's, virtual long FLT components are added to the FLT histogram with the lower repetition rate (20-50 MHz), while by using a higher repetition rate (80 MHz) the "inter-pulse pile-up" is eliminated as the F -value is close to 1.

New silicon technologies enable high-performance arrays of Single Photon Avalanche Diodes.

In order to fulfill the requirements of many applications, we recently developed a new technology aimed at combining the advantages of traditional thin and thick silicon Single Photon Avalanche Diodes (SPAD). In particular we demonstrated single-pixel detectors with a remarkable improvement in the Photon Detection Efficiency at the longer wavelengths (e.g. 40% at 800nm) while maintaining a timing jitter better than 100ps. In this paper we will analyze the factors the currently prevent the fabrication of arrays of SPADs by adopting such a Red-Enhanced (RE) technology and we will propose further modifications to the device structure that will enable the fabrication of high performance RE-SPAD arrays for photon timing applications.