Fluorescence lifetime imaging with cost-efficient time-correlated photon counter

The nitrogen-vacancy (NV) defect in diamond is an efficient quantum sensor of randomly fluctuating signals via relaxometry measurements. In particular, the longitudinal spin relaxation of the NV defect accelerates in the presence of magnetic noise with a spectral component at its electron spin resonance frequency. We look into this effect quantitatively by applying a calibrated and tunable magnetic noise on a single NV defect. We show that an increase of the longitudinal spin relaxation rate translates into a reduction of the photoluminescence (PL) signal emitted under continuous optical illumination, which can be explained using a simplified three-level model of the NV defect. This PL quenching mechanism offers a simple, all-optical method to detect magnetic noise sources at the nanoscale.

Protein kinases of the microtubule affinity regulating kinase (MARK)/Par-1 family play important roles in the establishment of cellular polarity, cell cycle control, and intracellular signal transduction. Disturbance of their function is linked to cancer and brain diseases, e.g. lissencephaly and Alzheimer disease. To understand the biological role of MARK family kinases, we searched for specific inhibitors and a biosensor for MARK activity. A screen of the ChemBioNet library containing ~18,000 substances yielded several compounds with inhibitory activity in the low micromolar range and capable of inhibiting MARK activity in cultured cells and primary neurons, as judged by MARK-dependent phosphorylation of microtubule-associated proteins and its consequences for microtubule integrity. Four of the compounds share a 9-oxo-9H-acridin-10-yl structure as a basis that will serve as a lead for optimization of inhibition efficiency. To test these inhibitors, we developed a cellular biosensor for MARK activity based on a MARK target sequence attached to the 14-3-3 scaffold protein and linked to enhanced cyan or teal and yellow fluorescent protein as FRET donor and acceptor pairs. Transfection of the teal/yellow fluorescent protein sensor into neurons and imaging by fluorescence lifetime imaging revealed that MARK was particularly active in the axons and growth cones of differentiating neurons.

We developed a time-correlated single-photon counting (TCSPC) system based on the low-jitter superconducting nanowire single-photon detection (SNSPD) technology. The causes of jitters in the TCSPC system were analyzed. Owing to the low jitter of the SNSPD technology, a system jitter of 26.8ps full width at half-maximum was achieved after optimizing the system. We demonstrated time-of-flight laser ranging at 1550nm wavelength at a standoff distance of 115m based on this TCSPC system. A depth resolution of 4mm was achieved directly by locating the centroids of each of the two return signals. Laser imaging was also performed using the TCSPC system. This low-jitter TCSPC system using the SNSPD technology presents great potential in long-range measurements and imaging applications for low-energy-level and eye-safe laser systems.

Diffuse optical tomography resorting to time-domain measurements (TD-DOT) provides information-rich data that have not yet been fully exploited for image reconstruction, notably to increase imaging spatial resolution. Current TD-DOT scanners suffer from a very low sensitivity owing to their small number of detection channels. This leads to excessively long acquisition times for in vivo imaging. To obtain a higher number of detection channels, thus increasing detection density, a low-cost time-correlated single photon counting (TCSPC) system dedicated to TD-DOT was designed, resorting solely to off-the-shelf electronic components to reduce costs, in distinction to custom application-specific integrated circuit solutions. The system features four input channels, each of them including a leading-edge discriminator for direct interfacing with off-the-shelf photodetector modules and a programmable delay line to compensate for undesired propagation delays, thus also avoiding manual adjustment of cable lengths. The system supports a large number of TCSPC channels using a daisy-chain configuration through an onboard Ethernet switch. It also features a 13.02 ps bin width with a 12.5 ns dynamic range. Results show excellent linearity, with a $\sigma _{\mathrm{ DNL}}$ of 1.7% least significant bit (LSB), peak differential nonlinearity of 5% LSB, and peak integral nonlinearity of 10% LSB and an average accuracy of 19 ps full-width at half-maximum (FWHM) throughout two separate four-channel systems. The system was tested in a TD-DOT acquisition setup, and 37 ps FWHM instrument response functions were obtained using a single-photon avalanche diode (SPAD) detector. Diffuse intrinsic measurements were also acquired and compared with a reference TCSPC system showing similar results.

Fluorescence lifetime imaging microscopy (FLIM) is now routinely used for dynamic measurements of signaling events inside living cells, including detection of protein-protein interactions. An understanding of the basic physics of fluorescence lifetime measurements is required to use this technique. In this protocol, we describe both the time-correlated single photon counting and the frequency-domain methods for FLIM data acquisition and analysis. We describe calibration of both FLIM systems, and demonstrate how they are used to measure the quenched donor fluorescence lifetime that results from Förster resonance energy transfer (FRET). We then show how the FLIM-FRET methods are used to detect the dimerization of the transcription factor CCAAT/enhancer binding protein-α in live mouse pituitary cell nuclei. Notably, the factors required for accurate determination and reproducibility of lifetime measurements are described. With either method, the entire protocol including specimen preparation, imaging and data analysis takes ∼2 d.

The method of fluorescence lifetime imaging microscopy (FLIM) is a quantitative approach that can be used to detect Forster resonance energy transfer (FRET). The use of FLIM to measure the FRET that results from the interactions between proteins labeled with fluorescent proteins (FPs) inside living cells provides a non-invasive method for mapping interactomes. Here, the use of the phasor plot method to analyze frequency domain (FD) FLIM measurements is described, and measurements obtained from cells producing the ‘FRET standard’ fusion proteins are used to validate the FLIM system for FRET measurements. The FLIM FRET approach is then used to measure both homologous and heterologous protein–protein interactions (PPI) involving the CCAAT/enhancer-binding protein alpha (C/EBPα). C/EBPα is a transcription factor that controls cell differentiation, and localizes to heterochromatin where it interacts with the heterochromatin protein 1 alpha (HP1α). The FLIM-FRET method is used to quantify the homologous interactions between the FP-labeled basic leucine zipper (BZip) domain of C/EBPα. Then the heterologous interactions between the C/EBPa BZip domain and HP1a are quantified using the FRET-FLIM method. The results demonstrate that the basic region and leucine zipper (BZip) domain of C/EBPα is sufficient for the interaction with HP1α in regions of heterochromatin.

The development of stable vesicular assemblies and the understanding of their interaction and dynamics in aqueous solution are long-standing topics in the research of chemistry and biology. Fatty acids are known to form vesicle structure in aqueous solution depending on the pH of the medium. Protic ionic liquid of fatty acid with ethyl amine (oleate ethyl amine, OEA) as a component spontaneously forms a vesicle in aqueous solution. The general comparison of dynamics and interaction of these two vesicles have been drawn using fluorescence correlation spectroscopy (FCS) and fluorescence lifetime imaging microscopy (FLIM) measurements. Further, FLIM images of a single vesicle are taken at multiple wavelengths, and the solvation of the probe molecules has been observed from the multiwavelength FLIM images. The lifetime of the probe molecule in OEA vesicle is higher than that in simple fatty acid vesicles. Therefore, it suggests that the membrane of the OEA vesicle is more dehydrated compared to that of fatty acid vesicles, and it facilitates OEA vesicles to fuse themselves in the presence of electrolyte, sodium chloride (NaCl). However, under the same conditions, only fatty acid vesicles do not fuse. The fusion of OEA vesicles is successfully demonstrated by the time scan FLIM measurements. The different events in the fusion process are analyzed in the light of the reported model of vesicle fusion. Finally, the local viscosity of the water pool of the vesicle is determined using kiton red, as a molecular rotor. With addition of NaCl, the fluidity in the interior of the vesicle is increased which leads to disassembly of vesicle. The rich dynamic properties of this vesicular assembly and the FLIM based approach of vesicle fusion will provide better insight into the growth of a protocell membrane.

In the present work, we report the imaging of Au nanostars nanoparticles (AuNSt) and their multifunctional applications in biomedical research and theranostics applications. Their optical and spectroscopic properties are considered for the multimodal imaging purpose. The AuNSt are prepared by the seed-meditated method and characterized for use as an agent for bio-imaging. To demonstrate imaging with AuNSt, penetration and localization in different biological models such as cancer cell culture (A549 lung carcinoma cell), 3D tissue model (multicellular tumor spheroid on the base of human oral squamous carcinoma cell, SAS) and murine skin tissue are studied. AuNSt were visualized using fluorescence lifetime imaging (FLIM) at two-photon excitation with a pulse duration 140 fs, repetition rate 80 MHz and 780 nm wavelength femtosecond laser. Strong emission of AuNSt at two-photon excitation in the near infrared range and fluorescence lifetime less than 0.5 ns were observed. It allows using AuNSt as a fluorescent marker at two-photon fluorescence microscopy and lifetime imaging (FLIM). It was shown that AuNSt can be observed inside a thick sample (tissue and its model). This is the first demonstration using AuNSt as an imaging agent for FLIM at two-photon excitation in biosystems. Increased scattering of near-infrared light upon excitation of AuNSt surface plasmon oscillation was also observed and rendered using a possible contrast agent for optical coherence tomography (OCT). AuNSt detection in a biological system using FLIM is compared with OCT on the model of AuNSt penetrating into animal skin. The AuNSt application for multimodal imaging is discussed.

Time-resolved fluorescence measurement screening and sorting has been proven to be interesting for biological applications. Fluorescence lifetime (FLT) measurement provides additional information to the measurement of the intensity or polarization of the fluorescence emission. Furthermore, droplet microfluidics enables higher throughput than well plate readers. Few researches have been recently done about the microfluidic droplet sorting based on the fluorescence lifetime measurement. Some of the proposed solutions support high throughput but they do not extract the FLT directly from the fluorescence decay. In this paper, we present an alternative low-cost system for the microfluidic droplets sorting. We implemented a FLT measurement system based on the time-correlated single photon counting (TCSPC) technique on a cyclone V SoC-FPGA. For the excitation light source, we use a simple pulsed laser diode and a single photon avalanche diode (SPAD) as a photodetector. The optical system is an ad hoc microscope. The droplet generation is done with flow focusing technique in a PDMS-based microfluidics chip. This system was successfully tested in real-time at a droplet rate of more than 3000 droplets per second.

Here, we report the characterization of cholesterol levels on membrane fluidity with a twisted intramolecular charge transfer (TICT) membrane dye, namely DI-8-ANEPPS, using fluorescence lifetime techniques such as time-correlated single photon counting (TCSPC) and fluorescence lifetime imaging microscopy (FLIM). The characterized liposomes comprised a 3 : 1 ratio of POPC and POPG, respectively, 1% DI-8-ANEPPS, and increasing cholesterol levels from 0% to 50%. Fluorescence lifetime characterization revealed that increasing the cholesterol levels from 0% to 50% increases the fluorescence lifetime of DI-8-ANEPPS from 2.36 ns to 3.65 ns, a 55% increment. Such lengthening in the fluorescence lifetime is concomitant with reduced Stokes shifts and higher quantum yield, revealing that localized excitation (LE) dominates over TICT states with increased cholesterol levels. Fluorescence anisotropy measurements revealed a less isotropic environment in the membrane upon increasing cholesterol levels, suggesting a shift from liquid-disorder (Lα) to liquid-order (LO) upon adding cholesterol. Local electrostatic and dipole characterization experiments revealed that changes in the zeta-potential (ζ-potential) and transmembrane dipole potential (Ψd) induced by changes in cholesterol levels or the POPC : POPG ratio play a minimal role in the fluorescence lifetime outcome of DI-8-ANEPPS. Instead, these results indicate that the cholesterol's effect in restricting the degree of movement of DI-8-ANEPPS dominates its photophysics over the cholesterol effect on the local dipole strength. We envision that time-resolved spectroscopy and microscopy, coupled with TICT dyes, could be a convenient tool in exploring the complex interplay between membrane lipids, sterols, and proteins and provide novel insights into membrane fluidity, organization, and function.

Fluorescence lifetime imaging microscopy (FLIM) has been widely used in the field of biological research because of its high specificity, sensitivity, and quantitative ability in the sensing cellular microenvironment. The most commonly used FLIM technology is based on time-correlated single photon counting (TCSPC). Although the TCSPC method has the highest temporal resolution, the data acquisition time is usually long, and the imaging speed is slow. In this work, we proposed a fast FLIM technology for fluorescence lifetime tracking and imaging of single moving particles, named single particle tracking FLIM (SPT-FLIM). We used feedback-controlled addressing scanning and Mosaic FLIM mode imaging to reduce the number of scanned pixels and the data readout time, respectively. Moreover, we developed a compressed sensing analysis algorithm based on alternating descent conditional gradient (ADCG) for low-photon-count data. We applied the ADCG-FLIM algorithm on both simulated and experimental datasets to evaluate its performance. The results showed that ADCG-FLIM could achieve reliable lifetime estimation with high accuracy and precision in the case of a photon count less than 100. By reducing the photon count requirement for each pixel from, typically, 1000 to 100, the acquisition time for a single frame lifetime image could be significantly shortened, and the imaging speed could be improved to a great extent. On this basis, we obtained lifetime trajectories of moving fluorescent beads using the SPT-FLIM technique. Overall, our work offers a powerful tool for fluorescence lifetime tracking and imaging of single moving particles, which will promote the application of TCSPC-FLIM in biological research.

We intend to develop an efficient method of measuring respiratory function of the cornea. With this purpose, we resorted to fluorescence lifetime imaging microscopy (FLIM) to monitor the metabolic co-factor flavin adenine dinucleotide (FAD). FAD and nicotinamide adenine dinucleotide (NADH) are co-factors of the electron transport chain. Therefore alterations in amount of these molecules reflect alterations in the metabolism. For assessing the potential of FLIM for metabolic imaging of the cornea, we performed a series of experiments using a time-correlated single photon counting (TCSPC) fluorescence lifetime microscope (Picoquant to MicroTime 100 coupled to an Olympus BX51 Microscope). In this technique, the acquired signal is the convolution between the instrument response function (IRF) and the fluorescence signal from the sample. IRF was acquired using an Erythrosin B solution. In this work we show that it is possible to acquire fluorescence lifetime images of rat and bovine corneas using FAD autofluorescence.

This project will test the coupling of light emitted from silicon vacancy and nitrogen vacancy defects in diamond into additively manufactured photonic wire bonds toward integration into an "on-chip quantum photonics platform". These defects offer a room-temperature solid state solution for quantum information technologies but suffer from issues such as low activation rate and variable local environments. Photonic wire bonding will allow entanglement of pre-selected solid-state defects alleviating some of these issues and enable simplified integration with other photonic devices. These developments could prove to be key technologies to realize quantum secured networks for national security applications.

Fluorescence lifetime imaging microscopy (FLIM) techniques are widely used in auto-fluorescence imaging to investigate dynamic metabolic states of the cells. Traditional FLIM imaging requires the cells to adhere to the coverslip to collect enough photons for FLIM data processing. Such conditions pose challenges to non-adherent cells, such as acute myeloid leukemia (AML) cells, because of cell motility. We developed a proto-type micro cell trapping array (MCTA) to immobilize cells in picoliter-size wells with location references. The array keeps non-adherent cells in referenced well locations, allows treatment on stage and re-imaging after time for ultimate cell segmentation analysis. Individual wells are analyzed by a pixel-based region-of-interest (ROI) to analyze cellular redox states. This single well trapping and analysis method allows to isolate treatment responses of a small number of cells, compare their range and predict early effect, which may have clinical applications in the context of cancer aggressiveness and treatment outcomes. We expanded the common intensity-based assay for cellular redox state by a Fluorescence Lifetime measurement, NAD(P)H-a2%, serving as an alternative metric. The new assay is flexible and can be applied to other non-adherent cell lines, expanding FLIM applications in both research and the clinic.

Point-like defects in wide-bandgap materials are at the heart of a broad range of emerging applications including quantum information processing and metrology [1]. A well-known example is the nitrogen-vacancy (NV) defect in diamond, which can be used as a solid-state qubit to perform elaborate quantum information protocols [2] and highly sensitive magnetic field sensing [3]. These results motivate the search of new defects in other wide-bandgap materials, which would offer an expanded range of functionalities compared to NV defects in diamond. In that context, hexagonal boron nitride (hBN) appears as an appealing material. First, it has a 6-eV bandgap, which is ideally suited to host optically active defects with energy levels deeply buried between the valence band and the conduction band. Second, hBN is an electrical insulator with a two-dimensional (2D) honeycomb structure, which is a key element of Van der Waals heterostructures. Such "artificial" materials are currently attracting a great interest owing to their unique mechanical, electrical and optical properties [4]. Combining these properties with individual quantum systems would likely open new perspectives in quantum technologies. In this talk, I will report on the optical detection of individual defects hosted in a high-purity hBN crystal. Stable single photon emission is demonstrated under ambient conditions by means of photon correlation measurements [5]. A detailed analysis of the photophysical properties of the defect reveals a highly efficient radiative transition, leading to one of the brightest single photon source reported to date from a bulk, unpatterned, material. These results make a bridge between the physics of 2D materials and quantum technologies, and pave the way towards applications of van der Waals heterostructures in photonic-based quantum information science, metrology and optoelectronics.