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

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.

Single-Chain Lanthanide Luminescence Biosensors for Cell-Based Imaging and Screening of Protein-Protein Interactions.

Automated Systems.

Introduction: Fluorescence lifetime assays complement the portfolio of established assay formats available in drug discovery, particularly with the recent advances in microplate readers and the commercial availability of novel fluorescent labels. Fluorescence lifetime assists in lowering complexity of compound screening assays, affording a modular, toolbox-like approach to assay development and yielding robust homogeneous assays.Areas covered: To date, materials and procedures have been reported for biochemical assays on proteases, as well as on protein kinases and phosphatases. This article gives an overview of two assay families, distinguished by the origin of the fluorescence signal modulation.Expert opinion: The pharmaceutical industry demands techniques with a robust, integrated compound profiling process and short turnaround times. Fluorescence lifetime assays have already helped the drug discovery field, in this sense, by enhancing productivity during the hit-to-lead and lead optimization phases. Future work will focus on covering other biochemical molecular modifications by investigating the detailed photo-physical mechanisms underlying the fluorescence signal.

We present fluorescence lifetime imaging (FLIM) and fluorescence anisotropy imaging along with translational diffusion measurements of living cells labelled with green fluorescent protein (GFP) recorded in a single experiment. The experimental set-up allows for time and polarization-resolved fluorescence images to be measured in every frame of a fluorescence recovery after photobleaching (FRAP) series. We have validated the method using rhodamine 123 in homogeneous solution prior to measurements of living A431 cells labelled with cdc42-GFP, for which the FRAP recovery exhibits an immobile fraction and the rotational mobility of the protein is hindered while the fluorescence lifetime fairly homogeneous across the cell. By eliminating the need for sequential measurements to extract fluorescence lifetimes and molecular diffusion coefficients we remove artefacts arising from changes in sample morphology and excessive photobleaching during sequential experiments.

Excited-state lifetimes and rotational reorientation times were obtained for trans-4,4‘-diphenylstilbene (DPS) in a series of n-alkanes and nonalkane solvents as a function of temperature using picosecond time-resolved fluorescence lifetime and anisotropy measurements. The fluorescence lifetimes do not have a significant solvent or temperature dependence, indicating that isomerization via activated barrier crossing is not a dominant decay pathway for S1 DPS. However, the temperature dependence of the lifetime is shown to be larger in polar solvents than nonpolar solvents. Arrhenius plots for polar solvents give two different decay channels in the low-temperature and high-temperature regions. The rotational reorientation time of DPS is sensitive to solvent environments. In a given solvent the rotational reorientation times depend linearly on η/T in agreement with the Debye−Stokes−Einstein theory. The data in n-alkane solvents show that the solute−solvent coupling, as evidenced by the friction coefficient, ...

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.

A high-throughput direct fluorescence resonance energy transfer-based assay for analyzing apoptotic proteases using flow cytometry and fluorescence lifetime measurements

The fluorescence from disordered phenanthrene adlayers on Al2O3(0001) was examined in ultrahigh vacuum at 20 K using laser-induced fluorescence techniques. The fluorescence spectra was consistent with emission from a phenanthrene excimer. The fluorescence lifetime of the phenanthrene excimer was studied versus phenanthrene coverage and versus distance from the Al2O3(0001) surface using xenon spacers. As a function of coverage on Al2O3(0001), the fluorescence lifetime decreased from τ=34±0.5 ns at phenanthrene coverages of Θ≥20 ML to τ=7±0.5 ns at Θ=1 ML. As a function of xenon spacer distance from the Al2O3(0001) surface, the fluorescence lifetime also decreased from τ=34±0.5 ns at distances of d>100 Å to τ=7±0.5 ns at d=7 Å. Fluorescence measurements versus phenanthrene coverage on xenon, butane, acetonitrile and methanol multilayers revealed that the phenanthrene excimer fluorescence lifetime was constant at τ≊35 ns on the molecular multilayer surfaces. These results indicated that the reduction of the fluorescence lifetime was particular to the Al2O3(0001) surface. The fluorescence lifetimes versus phenanthrene coverage and xenon spacer distance on Al2O3(0001) were equivalent when the phenanthrene coverage was converted to total adlayer thickness. This correspondence suggested that the excited electronic energy in disordered phenanthrene adlayers transfers rapidly to phenanthrene excimers at the phenanthrene–vacuum interface. Subsequently, competition occurs between fluorescence quenching by the Al2O3(0001) surface and phenanthrene excimer fluorescence. In addition, fluorescence lifetime measurements vs phenanthrene coverage on CaF2 thin films displayed similar fluorescence quenching. Fluorescence lifetimes versus phenanthrene coverage on O2 molecular multilayers also revealed fluorescence quenching that was attributed to a charge-transfer mechanism. The observed fluorescence quenching on Al2O3(0001) indicates that the surface states of this ionic crystal may be accessible for electronic energy transfer even though Al2O3 is a known insulator. A Förster electronic energy transfer mechanism was used to analyze the observed fluorescence lifetimes vs phenanthrene coverage or xenon spacer distance on Al2O3(0001).

The fluorescence lifetime and spectrum of pyrene in fluid solution depend on experimental conditions, including sample concentration, solvent viscosity, and the presence of dissolved oxygen. Students are introduced to nanosecond-domain transient spectroscopy in fluid solution by using a pulsed-laser apparatus to make direct measurements of the effects of these variables. They become familiar with the operation of a nitrogen laser and an associated apparatus for fluorescence spectra and lifetime measurements, including the optical and electronic hardware and the computer software used to acquire and analyze the data. The experiment specifically illustrates the role of sample concentration and solvent viscosity on fluorescence lifetimes and spectra, and the effects of argon purging to remove dissolved oxygen.

Considerable efforts have been devoted to the development of spectroscopic probes that are sensitive to water and can be used to monitor, for example, biological and chemical processes involving dehydration or hydration. Continuing this line of research, herein we show that 7-cyanoindole can serve as a sensitive fluorescence probe of hydration as its fluorescence properties, including intensity, peak wavelength and lifetime, depend on the amount of water in nine water-organic solvent mixtures. Our results indicate that 7-cyanoindole is not only able to reveal the underlying microheterogeneity of these binary solvent systems, but also offers distinct advantages. These include: (1) its fluorescence intensity increases more than ten times upon going from a hydrated to a dehydrated environment; (2) its peak wavelength shifts as much as 35 nm upon dehydration; (3) its single-exponential fluorescence decay lifetime increases from 2.0 ns in water to 8-16 ns in water-organic binary mixtures, making it viable to distinguish between differently hydrated environments via fluorescence lifetime measurements; and (4) its absorption spectrum is significantly red-shifted from that of indole, making selective excitation of its fluorescence possible in the presence of naturally occurring amino-acid fluorophores. Moreover, we find that for seven binary mixtures the fluorescence lifetimes of 7-cyanoindole measured at solvent compositions where maximum microheterogeneity occurs correlate linearly with the peak wavenumbers of its fluorescence spectra obtained in the respective pure organic solvents. This suggests that the microheterogeneities of these binary mixtures bear certain similarity, a phenomenon that warrants further investigation.

Picosecond fluorescence anisotropy and lifetime measurements are used to investigate the orientational dynamics and steady state order of the fluorescent probe oxazine 4 in the nematic and isotropic phases of 5, 6 and 7 cyanobiphenyl. Variation of the excitation polarization angle β with respect to the nematic director allows the preparation of both cylindrically symmetric and asymmetrically aligned probe distributions whose relaxation dynamics are sensitive to both θ and φ motions yielding two characteristic relaxation times: τ₂₀ (pure θ-diffusion) and τ₂₂ (predominantly φ-diffusion). Analysis of the fluorescence intensity decays for excitation polarisation angles of β=0° and β=54.7° allows a determination of the effect of local field and differential reflection losses without the measurement of sample refractive indices. A striking feature of oxazine 4 dynamics in the approach to the nematic-isotropic phase transition temperature (T_NI) is that whilst θ diffusion shows a characteristic Arrhenius temperature dependence, the rate of diffusion in the φ coordinate is reduced as the system becomes less ordered. In the isotropic phase over a 50°C temperature range above T_NI the fluorescence anisotropy is characterised by two correlation times consistent with restricted rotational diffusion (intra-domain relaxation τ_f) within a slowly relaxing (pseudo-domain) structure (τ_s). The temperature dependence of τ_f and τ_s was in good agreement with recent theoretical models for intra- and inter-domain relaxation.

Optical spectroscopy, Spectrophotometry and fluorimetry can be used to monitor processes occurring in living cells provided that suitable chromophores are present which ‘report’ on the events in which they participate. The advantages of optical techniques are manifold. Firstly they can be fast—with appropriate apparatus events in the pico- and nano-second domains can be studied by fluorescence spectroscopy. Secondly they are continuous—instant feedback from the experimental system can guide the most complex of experimental protocols, and allow the experimenter to adjust system parameters as necessary. Thirdly they are convenient, and most laboratories have access to equipment that can provide quantitative analysis of optical signals; examples include conventional spectrophotometers/fluorimeters, dedicated instruments (e.g. for fluorescence lifetime and polarization measurements), cameras, microscopes and plate readers. Significantly detectors from one apparatus can often be used on others to open up new experimental protocols. Fortunately the principles underlying the use of such a diverse array of optical devices are straightforward and universal—they apply just as much to laboratory ‘work-horse’ instruments as they do to the most specialized, laser-illuminated fluorescence microscope. The availability of fast laboratory computers with large storage capacities means that most modern spectrometers are microprocessor controlled and digitization of signals opens up the full range of possibilities of data accumulation, storage, analysis and interpretation. The main problem with optical measurements is not the acquisition but rather the interpretation of the data obtained. Straightforward analysis of the results depends on the clarity of the experimental design and the appropriate choice of chromophore. This chapter describes some of the problems that can be addressed by spectroscopic techniques and attempts to give guidance on good experimental design. Optical spectroscopy requires either spectrophotometers, to measure absorbance, fluorimeters, to measure fluorescence, or microscopes, which can measure fluorescence or absorbance of single cells or small groups of cells. Fluorimeters and spectrophotometers usually require solutions or suspensions of material in conventional cuvettes; microscopes provide two-dimensional images from smears, slices or surfaces. Other devices that record signals resolved in two-dimensions include gel scanners and microplate readers.

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.

This doctoral thesis addresses two novel approaches in fluorescence microscopy. The first part introduces a new method that can be used to enhance the effect of photoselection. This method, termed ExPAN (excitation polarization angle narrowing), exploits stimulated emission to narrow the angular distribution of excited fluorophores. ExPAN could be of significant interest for applications like fluorescence anisotropy measurements, which are commonly applied in life science, e.g. to study receptor-ligand interactions or investigate protein dynamics. In the context of this work, ExPAN was applied in conjunction with a new fluorescence microscopy approach that utilizes molecular orientation as a contrast mechanism. Using excitation light with continuously rotating polarization, it is possible to obtain information about the orientation of dye molecules. When excited in this manner, molecules with a preferred orientation exhibit a periodically modulated fluorescence signal as a result of photoselection. This work showed that the additional modulation information can be utilized to distinguish molecules with different orientations even when their signals overlap spatially. The approach was explored by experiments with single molecules on a surface using a modified widefield microscope and additionally by simulation studies. The results indicated that it is possible to distinguish signals from dye molecules with a distance down to 80 nm. It was demonstrated that ExPAN enhances the effect of photoselection and can be used to distinguish molecules even when their orientations are very similar. Furthermore, a modulated fluorescence signal was also observed in fixed biological samples and with surface labeled microparticles in aqueous solution. In the second part of this thesis a method is presented for improving the resolution of confocal laser scanning microscopes. This method, termed multi-detector scanning (MDS), is based on the concept of image scanning microscopy (ISM). Using ISM it is theoretically possible to double the resolution of a fluorescence microscope. Since ISM requires an array detector, most of the previously developed implementations use CCD or CMOS cameras for detection. Instead of a camera, several single-photon detectors that are combined to an array detector by a fiber optic bundle, are used in this work.This configuration allowed it for the first time to use the method in conjunction with Fluorescence-lifetime imaging microscopy (FLIM). FLIM has proven to be an important microscopy technique in life science and has been applied in a wide variety of studies including protein-protein interaction and cell metabolism research. Therefore, the improvement in spatial resolution of FLIM through ISM is of potential interest for numerous biological applications. Within the scope of this work, a multi-detector scanning microscope was constructed and characterized. Resolution improvements of 168 nm with MDS and 146 nm with MDS and deconvolution were achieved in fixed biological samples. Furthermore, the measurement of fluorescence lifetimes was demonstrated with the experimental setup. Thus, the application of MDS has the potential to increase the spatial resolution of FLIM and could serve as a tool for measuring biological systems in more detail.