A low-cost high throughput microfluidics system for multi-thousand droplets per second sorting based on time resolved fluorescence measurement

Fluorescence lifetimes of rhodamine dyes in vacuo

The non-invasive differentiation of dermal elastic fibres from solar elastosis in vivo is of great interest in dermatologic research, especially for efficacy testing of anti-ageing products. To date, no studies on multiphoton excited fluorescence lifetime characteristics of human elastic fibres and solar elastosis are reported. The goal of the present work was the identification of differential criteria for elastic fibres and solar elastosis by the analysis of fluorescence decay curves acquired by time-correlated single photon counting in vivo multiphoton tomography. For this purpose, fluorescence lifetime measurements (FLIM) were performed with 47 volunteers of different age groups at sun-protected and sun-exposed localizations. Bi-exponential curve fitting was applied to the FLIM data, and characteristic differences between age groups and localizations were found in both relevant fit parameters describing the decay slope. The FLIM analyses have shown that dermal autofluorescence has different lifetimes depending on age and in part on localization.

We report a time-correlated Raman spectroscopy technique based on a silicon photomultiplier (SiPM) and a time-correlated single photon counting (TCSPC) technique to exploit the natural temporal separation between Raman and fluorescence phenomena to alleviate the high fluorescence background with conventional Raman detection. The TCSPC technique employed can greatly reduce the effect of high dark count rate (DCR) and crosstalk of SiPM that seriously hinder its application in low light level detection. The operating principle and performance of the 400 ps time resolution system are discussed along with the improvement of the peak-to-background ratio (PBR) for bulk trinitrotoluene (TNT) Raman spectrum relative to a commercial Raman spectrometer with charge coupled device (CCD). The fluorescence lifetime for solid TNT and Surface Enhanced Raman Scattering (SERS) spectrum for 10(-6) mol/L trace TNT have also been obtained by this system, showing excellent versatility and convenience in spectroscopy measurement.

We have constructed a simple, all solid-state, time-correlated single photon counting device for collecting decay profiles of chromophores attached to DNA fragments moving through a capillary tube filled with a sieving gel under the influence of an applied electric field (capillary electrophoresis). The major components of the instrument consist of an actively pulsed GaAlAs diode laser (λexcitation=780 nm; τp<200 ps; repetition rate=80 MHz; average power=5.0 mW), single photon avalanche diode (dark count rate <50 cps; quantum efficiency=65% at 800 nm) and a PC board containing a constant fraction discriminator, time-to-amplitude converter, and an analog-to-digital converter (maximum processing count rate=3×106 cps). The instrument possessed a response function of approximately 275 ps (full width at half-maximum), adequate for measuring fluorescence lifetimes in the subnanosecond regime. To demonstrate the utility and the sensitivity of the instrument, dynamic measurements of fluorescence lifetimes for near-IR dye-labeled DNA fragments were measured during capillary electrophoresis for the identification of the dye-labeled nucleotide bases via temporal discrimination. The results indicated that in a two-dye experiment, in which only two of the four constituent bases which comprise DNA were labeled with unique fluorescent probes, the characteristic lifetime of the probe could be used to readily identify the terminal nucleotide base. Decay profiles were constructed from roughly 17 500 photoelectrons accumulated over a 2 s counting interval at a loading level of approximately 6.2×10−21 mol (3900 molecules) of DNA per electrophoretic band. The lifetimes of the two labeling dyes were determined to be 669 ps (±42 ps) and 528 ps (±68 ps).

A novel investigation into the fluorescence lifetimes of molecules, both established and newly designed, was performed. These molecules are the basis of a continuous, minimally invasive, glucose sensor based on fluorescence lifetime measurements. This sensor, if coupled with an automated insulin delivery device, would effectively create an artificial pancreas allowing for the constant monitoring and control of glucose levels in a person with diabetes. The proposed sensor includes a fluorescent molecule that changes its' fluorescence properties upon binding selectively and reversibly to glucose. One possible sensor molecule is N-methyl-N-(9-methylene anthryl)-2-methylenephenylboronic acid (AB). The fluorescence intensity of AB was shown to change in response to changing glucose concentrations. (James, 1994) James proposed that when glucose binds to AB the fluorescence intensity increases due to an enhancement of the N{yields}B dative bond which prevents photoinduced electron transfer (PET). PET from the amine (N) to the fluorophore (anthracene) quenches the fluorescence. The dative bond between the boron and the amine can prevent PET by involving the lone pair of electrons on the amine in interactions with the boron rather than allowing them to be transferred to the fluorophore. Results of this research show the average fluorescence lifetime of AB also changes with glucose concentration. It is proposed that fluorescence is due to two components: (1) AB with an enhanced N{yields}B interaction, and no PET, and (2) AB with a weak N{yields}B interaction, resulting in fluorescence quenching by PET. Lifetime measurements of AB as a function of both the pH of the solvent and glucose concentration in the solution were made to characterize this two component system and investigate the nature of the N{yields}B bond. Measurements of molecules similar to AB were also performed in order to isolate behavior of specific AB constituents. These molecules are 9-(Methylaminomethyl)-anthracene (MAMA), and N-benzyl-N-methyl-N-methyl anthracene (AB-B). Fluorescence lifetime measurements confirmed the two species of AB, with and without PET. Fluorescence lifetimes were approximately 11 nsec without PET and 3 nsec with PET. The degree of the interaction between the N and the B atoms was also determined by fluorescence lifetime measurements. Electron transfer rates of AB were measured to be on the order of 10{sup 8} sec{sup -1}. Analysis of AB as a glucose sensor shows it has the potential for measuring glucose concentrations in solution with less than 5% error. Two novel glucose sensing molecules, Chloro-oxazone boronate (COB) and Napthyl-imide boronate (NIB), were synthesized. Both molecules have a N{yields}B dative bond similar to AB, but with longer wavelength fluorophores. COB and NIB were found to be unacceptable for use as glucose sensor molecules due to the small changes in average fluorescence lifetime.

We describe the evolution of HORIBA Jobin Yvon IBH Ltd, and its time-correlated single-photon counting (TCSPC) products, from university research beginnings through to its present place as a market leader in fluorescence lifetime spectroscopy. The company philosophy is to ensure leading-edge research capabilities continue to be incorporated into instruments in order to meet the needs of the diverse range of customer applications, which span a multitude of scientific and engineering disciplines. We illustrate some of the range of activities of a scientific instrument company in meeting this goal and highlight by way of an exemplar the performance of the versatile DeltaFlex instrument in measuring fluorescence lifetimes. This includes resolving fluorescence lifetimes down to 5 ps, as frequently observed in energy transfer, nanoparticle metrology with sub-nanometre resolution and measuring a fluorescence lifetime in as little as 60 μs for the study of transient species and kinetics.

In this study, we describe a time-correlated single photon counting (TCSPC) technique for multi-wavelength lifetime imaging in laser-scanning microscopes. The technique is based on a four-dimensional histogramming process that records the photon density versus the time in the fluorescence decay, the x-y coordinates of the scanning area and the wavelength. It avoids any time gating or wavelength scanning and, therefore, yields a near-ideal counting efficiency. The decay functions are recorded in a large number of time channels, and the components of a multi-exponential decay can be resolved down to less than 30 ps. A single TCSPC imaging channel works with a high detection efficiency up to a photon count rate of about 5 . 10 6 s -1 . A modified version of the TCSPC fluorescence lifetime imaging (FLIM) technique uses several fully parallel detector and TCSPC channels. It operates at a count rate of more than 10 7 photons

Fluorescence Lifetime Microplate Reader for Structural Biology: High Performance and High-Throughput in the Same Tool

We demonstrate a new 512x16 single photon avalanche diode (SPAD) based line sensor with per-pixel TCSPC histogramming for time-resolved, time-zoomable, FRET spectroscopy. The line sensor can operate in single photon counting (SPC) mode as well as time-correlated single photon counting (TCSPC) and per-pixel histogramming modes. TCSPC has been the preferred method for fluorescence lifetime measurements due to its collection of full decays as a histogram of arrival times. However, TCSPC is slow due to only capturing one photon per exposure and large timestamp data transfer requirements for offline histogramming. On-chip histogramming improves the data rate by allowing multiple SPAD pulses (up to one pulse per laser period) to be processed in each exposure cycle, along with secondly reducing the I/O bottleneck as only the final histogram is transferred. This can enable 50x higher acquisition rates (up to 10 billion counts per second), along with time-zoomable histogramming operation from 1.6ns to 205ns with 50ps resolution. A broad spectral range can be interrogated with the sensor (450-900nm). Overall, these sensors provide a unique combination of light sensing capabilities for use in high speed, sensitive, optical instrumentation in the time/wavelength domain. We test the sensor performance by observation of fluorescence resonance energy transfer (FRET) between FAM and TAMRA and between EGFP and RFP FRET standards.

Fluorescence lifetime measurement is a versatile tool for studying the dynamics of biological, chemical, and quantum systems. However, commercially available time-correlated single-photon counting systems are typically very costly and are limited in terms of flexibility. Here, we present the use of a cost-efficient time-to-digital converter chip as a time-correlated single-photon counting module and the integration of the module into a confocal microscopy system to perform fluorescence lifetime imaging microscopy (FLIM). The system can withstand a sustained count rate of 10 000 counts per second with a timing uncertainty less than 170 ps full width at half maximum. FLIM measurement is obtained by scanning the confocal microscope over the sample and acquiring photon arrival times at each position, where the fluorescence lifetime is then computed using the fit-free center of mass method. The combined system is verified by imaging of fluorescence microbeads with known lifetimes, and we demonstrate the use of the system for imaging single-photon emitters with the nitrogen-vacancy defects in diamond. This timing module and calculation method can be used as a standalone upgrade kit for an existing microscope to enable FLIM measurement in biological studies and medical diagnosis applications.

We present an advanced time-correlated single photon counting (TCSPC) technique that delivers traditional fluorescence correlation (FCS) or cross correlation (FCCS) and fluorescence lifetime data simultaneously. Newly developed electronics allow for detection and registration of single photon events over time periods of hours with picoseconds accuracy. Subsequent software-correlation yields correlation curves covering more than 12 orders of magnitude in time. At the same time, the original data, containing all information accessible by single photon counting techniques, can be analyzed conventionally according to common single molecule fluorescence techniques. Potential applications of the new technique using pulsed or cw laser excitation are discussed.

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 describe the integration of techniques and technologies to develop a Point-of-Care for molecular diagnosis PoC-MD, based on a fluorescence lifetime measurement. Our PoC-MD is a low-cost, simple, fast, and easy-to-use general-purpose platform, aimed at carrying out fast diagnostics test through label detection of a variety of biomarkers. It is based on a 1-D array of 10 ultra-sensitive Single-Photon Avalanche Diode (SPAD) detectors made in a 0.18 μm High-Voltage Complementary Metal Oxide Semiconductor (HV-CMOS) technology. A custom microfluidic polydimethylsiloxane cartridge to insert the sample is straightforwardly positioned on top of the SPAD array without any alignment procedure with the SPAD array. Moreover, the proximity between the sample and the gate-operated SPAD sensor makes unnecessary any lens or optical filters to detect the fluorescence for long lifetime fluorescent dyes, such as quantum dots. Additionally, the use of a low-cost laser diode as pulsed excitation source and a Field-Programmable Gate Array (FPGA) to implement the control and processing electronics, makes the device flexible and easy to adapt to the target label molecule by only changing the laser diode. Using this device, reliable and sensitive real-time proof-of-concept fluorescence lifetime measurement of quantum dot QdotTM 605 streptavidin conjugate is demonstrated.

We present an optical depth imaging system suitable for highly scattering underwater environments. The system used the time-correlated single-photon counting (TCSPC) technique and the time-of-flight approach to obtain depth profiles. The single-photon detection was provided by a linear array of single-photon avalanche diode (SPAD) detectors fabricated in a customized silicon fabrication technology for optimized efficiency, dark count rate, and jitter performance. The bi-static transceiver comprised a pulsed laser diode source with central wavelength 670 nm, a linear array of 16 × 1 Si-SPAD detectors, with a dedicated TCSPC acquisition module. Cylindrical lenses were used to collect the light scattered by the target and image it onto the sensor. These laboratory-based experiments demonstrated single-photon depth imaging at a range of 1.65 m in highly scattering conditions, equivalent up to 8.3 attenuation lengths between the system and the target, using average optical powers of up to 15 mW. The depth and spatial resolution of this sensor were investigated in different scattering conditions.

Measurement of fluorescent lifetime is key in many biological analyses. Recently, we proposed a new scheme for fluorescent lifetime measurement utilizing hardware level linearization of the fluorescent decay produced by an excited fluorphore. This method is less sensitive to equivalent voltage and time errors compared to commonly utilized charge modulation schemes and has no upper limit on lifetime measurement. Here we present results for a hardware level implementation of the proposed scheme for lifetime measurement. Preliminary results demonstrate extraction of lifetimes down to 5/µs.