Fluorophores In Solution Research Articles

Dimensional metrology of lab-on-a-chip internal structures: a comparison of optical coherence tomography with confocal fluorescence microscopy.

The characterization of internal structures in a polymeric microfluidic device, especially of a final product, will require a different set of optical metrology tools than those traditionally used for microelectronic devices. We demonstrate that optical coherence tomography (OCT) imaging is a promising technique to characterize the internal structures of poly(methyl methacrylate) devices where the subsurface structures often cannot be imaged by conventional wide field optical microscopy. The structural details of channels in the devices were imaged with OCT and analyzed with an in-house written ImageJ macro in an effort to identify the structural details of the channel. The dimensional values obtained with OCT were compared with laser-scanning confocal microscopy images of channels filled with a fluorophore solution. Attempts were also made using confocal reflectance and interferometry microscopy to measure the channel dimensions, but artefacts present in the images precluded quantitative analysis. OCT provided the most accurate estimates for the channel height based on an analysis of optical micrographs obtained after destructively slicing the channel with a microtome. OCT may be a promising technique for the future of three-dimensional metrology of critical internal structures in lab-on-a-chip devices because scans can be performed rapidly and noninvasively prior to their use.

Application of Fluorescence Correlation Spectroscopy in the Characterization of Particle Size Distributions of Colloidal Silica Abrasives Used in Chemical-Mechanical Planarization

Recent studies have demonstrated that fluorescence correlation spectroscopy (FCS) can be used to analyze the adsorption of dissolved chemical compounds onto the dispersed abrasive particles in a CMP slurry (1, 2). The technique exploits competitive adsorption at the particle’s surface between a fluorophore and CMP-relevant, dissolved chemical compounds. As chemical compounds in a slurry adsorb to a fluorescent abrasive particle and de-sorb the fluorophore from the particle, FCS-measured diffusion coefficients of the fluorophores will indicate increasing concentration of the free fluorophore in solution as the concentration of adsorbed chemical compounds increases.In the present study, the FCS method is extended for the analysis of particle size distributions of smaller-sized colloidal silica CMP abrasives. This new application of the FCS technique is motivated by the commercial need to further reduce wafer defect formation in conjunction with increased removal of slurry and wafer particulates produced during CMP processes. A key analytical capability gating this processing requirement is the necessity to analyze, quantitatively, the smallest abrasive particles in a CMP slurry which are present at extreme dilution. Established methods for particle size analysis, such as disc centrifugation and dynamic light scattering, typically lack the sensitivity required to analyze a minute fraction of abrasive particles, such as colloidal silica, with hydrodynamic diameters less than 20 nm in a CMP slurry. Because FCS has sizing capability for small molecules equivalent in hydrodynamic diameter (d h) to fluorescent dyes (d h = 1.0 to 1.5 nm) coupled with its analytical sensitivity in the nanomolar range, particle size distribution analysis of silica abrasive particles with mean diameters less than 10 nm, is potentially accessible via FCS.Representative particle size distributions determined by FCS are shown in Figure 1 for a pair of related fluorescently-labeled silica dispersions. The data overlay in the figure compares the particle size distributions of a parent silica dispersion (solid line, mean hydrodynamic diameter = 45 nm) and that of the isolated supernatant of the dispersion obtained by ultracentrifugation of the parent dispersion in order to isolate the smallest particles (dashed line, mean hydrodynamic diameter = 11 nm) in the dispersion. These data demonstrate the ability of the FCS technique to detect and quantity the fraction of smallest particles in abrasive colloidal silica dispersions. Additional examples illustrating the range and applicability of the technique in the quantitative analysis of particle size distributions for representative silica particles used commonly in CMP abrasives are discussed in this presentation.1. Moinpour, M.; Wayman, A.; Rawat, R.; Carver, C. T.; Remsen, E. E. Surface Adsorption of CMP Slurry Additives on Abrasive Particles. Electrochem. Soc. Trans. 2013, 52,489-494.2. England, A.N.; Rawat, A.; Moinpour, M.; Remsen, E.E. Additive/Abrasive Interactions in Solution: Investigation of the Surface Chemistry and Adsorption Behaviour of CMP Abrasives. Proc. Int. Conf. Planar. Tech. 2012, 1-6.

Secondary emission influenced fluorescence decay of a homogeneous fluorophore solution

An analytical expression is found allowing the calculation of the secondary emission influenced fluorescence decay of a homogeneous fluorophore solution. Before starting the calculation one has to know the shape of the primary fluorescence decay of the fluorophore and the value of the parameter κ denoting the ratio of the secondary to primary steady-state fluorescence intensities. The method elaborated by Budó and Ketskeméty is recommended for evaluation of the parameter κ. The main importance of the obtained expression is that it can be used to recover parameters characterizing the fluorescence decay in the absence of secondary emission. The cases of monoexponential, biexponential and multiexponential primary fluorescence decays are discussed in detail.

Absorption and Emission Spectra of a Flexible Dye in Solution: a Computational Time-Dependent Approach.

The spectroscopic properties of the organic chromophore 4-naphthoyloxy-1-methoxy-2,2,6,6-tetramethylpiperidine (NfO-TEMPO-Me) in toluene solution are explored through an integrated computational strategy combining a classical dynamic sampling with a quantum mechanical description within the framework of the time-dependent density functional theory (TDDFT) approach. The atomistic simulations are based on an accurately parametrized force field, specifically designed to represent the conformational behavior of the molecule in its ground and bright excited states, whereas TDDFT calculations are performed through a selected combination of hybrid functionals and basis sets to obtain optical spectra closely matching the experimental findings. Solvent effects, crucial to obtain good accuracy, are taken into account through explicit molecules and polarizable continuum descriptions. Although, in the case of toluene, specific solvation is not fundamental, the detailed conformational sampling in solution has confirmed the importance of a dynamic description of the molecular geometry for a reliable description of the photophysical properties of the dye. The agreement between theoretical and experimental data is established and a robust protocol for the prediction of the optical behaviour of flexible fluorophores in solution is set.

Bioprobes Based on AIE Fluorogens

Fluorescent bioprobes are powerful tools for analytical sensing and optical imaging, which allow direct visualization of biological analytes at the molecular level and offer useful insights into complex biological structures and processes. The sensing and imaging sensitivity of a bioprobe is determined by the brightness and contrast of its fluorescence before and after analyte binding. Emission from a fluorophore is often quenched at high concentration or in aggregate state, which is notoriously known as concentration quenching or aggregation-caused quenching (ACQ). The ACQ effect limits the label-to-analyte ratio and forces researchers to use very dilute solutions of fluorophores. It compels many probes to operate in a fluorescence "turn-off" mode with a narrow scope of practical applications. The unique aggregation-induced emission (AIE) process offers a straightforward solution to the ACQ problem. Typical AIE fluorogens are characterized by their propeller-shaped rotorlike structures, which undergo low-frequency torsional motions as isolated molecules and emit very weakly in solutions. Their aggregates show strong fluorescence mainly due to the restriction of their intramolecular rotations in the aggregate state. This fascinating attribute of AIE fluorogens provides a new platform for the development of fluorescence light-up molecules and photostable nanoaggregates for specific analyte detection and imaging. In this Account, we review our recent AIE work to highlight the utility of AIE effect in the development of new fluorescent bioprobes, which allows the use of highly concentrated fluorogens for biosensing and imaging. The simple design and fluorescence turn-on feature of the molecular AIE bioprobes offer direct visualization of specific analytes and biological processes in aqueous media with higher sensitivity and better accuracy than traditional fluorescence turn-off probes. The AIE dot-based bioprobes with different formulations and surface functionalities show advanced features over quantum dots and small molecule dyes, such as large absorptivity, high luminosity, excellent biocompatibility, free of random blinking, and strong photobleaching resistance. These features enable cancer cell detection, long term cell tracing, and tumor imaging in a noninvasive and high contrast manner. Recent research has significantly expanded the scope of biological applications of AIE fluorogens and offers new strategies to fluorescent bioprobe design. We anticipate that future development on AIE bioprobes will combine one- or multiphoton fluorescence with other modalities (e.g., magnetic resonance imaging) or functionalities (e.g. therapy) to fully demonstrate their potential as a new generation of theranostic reagent. In parallel, the advances in molecular biology will provide more specific bioreceptors, which will enable the development of next generation AIE bioprobes with high selectivity and sensitivity for molecular sensing and imaging.

Wireless capsule for autofluorescence detection in biological systems

Variations in tissue autofluorescence (AF) can be exploited to detect early signs of intestinal cancer, however current endoscopic AF systems are only able to inspect the oesophegus and large intestine. We present the design, fabrication and testing of a pill capable of inducing and detecting AF from mammalian intestinal tissue. The prototype comprises an application specific integrated circuit (ASIC), illumination LED, optical filters to minimise sensor response to crosstalk from the illumination wavelength, a pulse counter/control unit and a radio transmitter. The ASIC implements a single photon avalanche diode detector (SPAD), and integrated high voltage (up to 37.9V) charge pump power supply. The SPAD is biased above its breakdown voltage to operate in Geiger mode, and exhibits a detection efficiency peak at 465nm, sufficiently close to human tissue autofluorescence's peak of 520±10nm. The ASIC was fabricated using a commercial high-voltage CMOS process. The complete device uses an average power of only 21.4mW. The implemented system has been characterised against controlled solutions of fluorophores and tested in vitro with biological samples. It has been proven to be capable of inducing and detecting fluorescence in fluorophore solution concentration as low as 1nM, and with mammalian intestinal tissue.

Nanocavity‐Based Determination of Absolute Values of Photoluminescence Quantum Yields

We present a new method for determining absolute values of quantum yield of luminescent emitters, which is based on the modification of the radiative transition of emitters within a tunable metallic nanocavity. The method presented is easy to set up and works without any calibration. It will thus be useful for all applications where absolute and calibration-free measurements of luminescence quantum yields are needed. Moreover, it requires only a minute amount of low-concentration fluorophore solution. We give a detailed description of the theory and data evaluation of the nanocavity measurements, and report experimental results for several common dyes in aqueous solution.

Studying Interfacial Transport With Evanescent Wave-Based Particle Velocimetry and Thermometry

Interfacial phenomena due to surface forces are important in microfluidic devices with their relatively large surface areas and small volumes. This article reviews our recent studies measuring fluid velocities in Poiseuille and electrokinetically driven flows over the first ∼0.4 μm next to the wall from the motion of an ensemble of O(105) fluorescent particles illuminated by evanescent waves. Because the evanescent-wave intensity decays exponentially with wall-normal distance, the particle–wall separation can be determined from the brightness of each particle image, and used to estimate the steady-state distribution of the tracers near the wall. More recently, evanescent-wave illumination has been combined with fluorescence thermometry, to estimate water temperature fields from changes in the fluorescence intensity of aqueous fluorophore solutions. Poiseuille flow of a fluorescein solution at Reynolds numbers of 3.3 and 8.3 through a heated minichannel was illuminated with evanescent waves to measure solution temperatures at an average distance of 74 nm from the wall. The temperature results obtained over three different regions were in good agreement with numerical predictions of the wall surface temperature, even in the presence of temperature variations exceeding 9°C over the 1-mm width of the channel.

Lifetime and Spectrally Resolved Characterization of the Photodynamics of Single Fluorophores in Solution Using the Anti-Brownian Electrokinetic Trap

We report simultaneous determination of fluorescence intensity, lifetime, and emission spectrum over time scales on the order of seconds for single molecules in solution, using the anti-Brownian electrokinetic trap. We demonstrate the technique with trapped single fluorophores of Atto647N and Alexa647. Three emission states with distinct intensities, lifetimes, and emission peaks are found in the case of Atto647N. Transitions between states happen occasionally. We characterize the three states and quantify the transition probabilities between states using concurrent intensity, lifetime, and spectrum data. Alexa647, on the other hand, showed little dynamics. These results represent a significant advance in the ability to identify and characterize different dynamical states of single molecules in aqueous solution with high precision and millisecond time resolution.

Gelatin–nanogold bioconjugates as effective plasmonic platforms for SERS detection and tagging

It is well known that standard citrate-reduced gold nanoparticles (AuNPs) are unstable at high ionic strength solution, which limits their applications in the biomedical field. In this work we present an environmentally friendly approach for the stabilization of citrate-reduced AuNPs in aqueous solution. Specifically, the stability of the AuNPs against salt-induced aggregation was greatly improved in the presence of gelatin biopolymer and stabilization of individual or small assemblies of nanoparticles can be controlled by the amount of gelatin. Furthermore, the gelatin–nanogold bioconjugates were demonstrated to be operational as highly sensitive surface-enhanced Raman scattering (SERS) active substrate for the detection of Rose Bengal fluorophore in solution at very low concentration. The results suggest that such bioconjugates can be successfully employed not only for detection of analytes, but more interestingly for building SERS-active tags in view of imaging purpose. The stabilization of bioconjugates was analyzed by localized surface plasmon resonance spectroscopy (LSPR), transmission electron microscopy (TEM), dynamic light scattering (DLS) and zeta-potential, and the chemical interaction of gelatin with AuNPs was inferred from Fourier transform infrared spectroscopy (FT-IR).

Tuning photophysical properties of triphenylamine and aromatic cyano conjugate-based wavelength-shifting compounds by manipulating intramolecular charge transfer strength

Abstract A series of triphenylamine-based aromatic cyano compounds have been synthesized as red-emitting fluorophores with large Stokes shifts in both solution (>100 nm in CHCl3) and solid state (>150 nm in film). Intramolecular charge transfer (ICT) properties of the synthesized compounds are examined using UV–Vis absorptions, photoluminescence measurements and solvatochromic studies. Our studies suggest that Stokes shifts of these compounds can be fine-tuned by manipulating the ICT strength between donor and acceptor with various electronic donating groups, and the largest Stokes shifts are typically associated with compounds that have the strong ICT characters. The observed spectroscopic properties of the compounds are consistent with theoretical calculations using density function theory (DFT) or time-dependent density function theory (TD-DFT). The calculations suggest that the ICT occurs from localized HOMO to localized LUMO with magnitudes of 60–80%. The relative quantum yields of these fluorophores in solution are various and highly solvent dependent. In solid state, the quantum yields of the compounds are significantly increased and some can reach to 0.40.

NIST/ISAC standardization study: Variability in assignment of intensity values to fluorescence standard beads and in cross calibration of standard beads to hard dyed beads

Results from a standardization study cosponsored by the International Society for Advancement of Cytometry (ISAC) and the US National Institute of Standards and Technology (NIST) are reported. The study evaluated the variability of assigning intensity values to fluorophore standard beads by bead manufacturers and the variability of cross calibrating the standard beads to stained polymer beads (hard-dyed beads) using different flow cytometers. Hard dyed beads are generally not spectrally matched to the fluorophores used to stain cells, and spectral response varies among flow cytometers. Thus if hard dyed beads are used as fluorescence calibrators, one expects calibration for specific fluorophores (e.g., FITC or PE) to vary among different instruments. Using standard beads surface-stained with specific fluorophores (FITC, PE, APC, and Pacific Blue™), the study compared the measured intensity of fluorophore standard beads to that of hard dyed beads through cross calibration on 133 different flow cytometers. Using robust CV as a measure of variability, the variation of cross calibrated values was typically 20% or more for a particular hard dyed bead in a specific detection channel. The variation across different instrument models was often greater than the variation within a particular instrument model. As a separate part of the study, NIST and four bead manufacturers used a NIST supplied protocol and calibrated fluorophore solution standards to assign intensity values to the fluorophore beads. Values assigned to the reference beads by different groups varied by orders of magnitude in most cases, reflecting differences in instrumentation used to perform the calibration. The study concluded that the use of any spectrally unmatched hard dyed bead as a general fluorescence calibrator must be verified and characterized for every particular instrument model. Close interaction between bead manufacturers and NIST is recommended to have reliable and uniformly assigned fluorescence standard beads.

Application of ‘multivariate curve resolution alternating least square (MCR–ALS)’ analysis to extract pure component synchronous fluorescence spectra at various wavelength offsets from total synchronous fluorescence spectroscopy (TSFS) data set of dilute aqueous solutions of fluorophores

In the present study, the possibility of using the multivariate curve resolution alternating least square (MCR–ALS) analysis for the simultaneous extraction of the pure synchronous fluorescence spectra at various wavelength offsets (Δλ) for each fluorophore from the total synchronous fluorescence spectroscopy (TSFS) data set of the dilute aqueous mixtures of the three fluorophores, was explored. The present work was based on the assumption that unfolded TSFS data has a bilinear structure and therefore it can be subjected to MCR–ALS analysis. Three fluorophores, benzo[a]pyrene(BaP), perylene(PE), and pyrene(PY), were chosen. These three fluorophores show fluorescence at all the seven wavelength offsets (Δλ) used to create the TSFS data set. In addition, Raman scattering due to solvent molecules (i.e. water) also appear in the wavelength ranges where these fluorophores show fluorescence. These two factors make the simultaneous extraction of synchronous spectral profile at various Δλs from the TSFS data set relatively difficult. The appearance of the diagonal signals in the three‐dimensional landscapes of TSFS shows the presence of the Raman scattering. The Raman signal due to solvent molecules was found to influence the synchronous profile of a fluorophore to different extents at different Δλs. TSFS data set of dimension, sample×wavelength×Δλ, was unfolded along the first mode to obtain the unfolded TSFS data set. Pure synchronous spectral profiles at various Δλs were obtained for each fluorophore by performing the MCR–ALS analysis on the unfolded TSFS data. However Raman scattering signals could not be eliminated from the synchronous spectral profiles of the PE and PY. For the mitigation of Raman scattering from the calculated spectral profiles, TSFS data of solvent blank were subtracted from all the samples before performing the MCR–ALS analysis. The obtained spectral profiles of BaP, PE, and PY were found to match with their actual spectral profiles which verifies that unfolded TSFS data set has a bilinear structure. To test the strength of the present work, MCR–ALS analysis was also performed on the unfolded TSFS data set of 12 groundwater samples which were contaminated with the BaP‐ and PY‐spiked gasoline. The obtained results show that it is possible to monitor the presence of BaP and PY in groundwater samples.

Fluorescence correlation spectroscopy at high concentrations using gold bowtie nanoantennas

Fluorescence correlation spectroscopy (FCS) measures the fluorescence fluctuations of fluorophores in solution, but is restricted to extremely low concentrations. Plasmonic gold bowtie nanoantennas enhance a single molecule’s fluorescence relative to a large background of unenhanced molecules, and here we show that bowties can extend FCS measurements to much higher concentrations. In this demonstration, the bowtie-FCS signal is dominated by molecules that transiently stick to the substrate near the bowtie gap, and photobleaching/photoblinking dynamics for two fluorophores are measured on the 10–100ms time scale.

Control of circularly polarized photoluminescent property via dihedral angle of binaphthyl derivatives

The chiroptical signs in circularly polarized luminescence (CPL) and circular dichroism (CD) spectra of chiral binaphthyl fluorophores in solution were found to be controllable by changing the dihedral angle of the binaphthyl unit regardless of the same axial chirality.

Testing the use of molecular dynamics to simulate fluorophore motions and FRET

Fluorescence resonance energy transfer (FRET) is commonly used to determine the proximity of fluorophores, but usually many assumptions are required to gain a quantitative relationship between the likelihood of energy transfer and fluorophore separation. Molecular Dynamics (MD) simulations provide one way of checking these assumptions, but before using simulations to study complex systems it is important to make sure that they can correctly model the motions of fluorophores and the likely FRET efficiency in a simple system. Here we simulate a well characterised situation of independent fluorophores in solution so that we can compare the predictions with expected values. Our simulations reproduce the experimental fluorescence anisotropy of Alexafluor488 and predict that of AlexaFluor568. At the ensemble level we are able to reproduce the expected isotropic and dynamic motion of the fluorophores as well as the FRET efficiency of the system. At the level of single donor-acceptor pairs, however, very long simulations are required to adequately sample the translational motion of the fluorophores and more surprisingly also the rotational motion. Our studies demonstrate how MD simulations can be used in more complex systems to check if the dynamic orientation averaging regime applies, if the fluorophores have isotropic orientational motion, to calculate the likely values of the orientation factor κ(2) and to determine the FRET efficiency of the system in both dynamic and static orientational averaging regimes. We also show that it is possible in some situations to create system specific relationships between FRET efficiency and fluorophore separation that can be used to interpret experimental data and find any correlations between κ(2) and separation that may influence distance measurements.

Raster image correlation spectroscopy in live cells.

Raster image correlation spectroscopy (RICS) is a noninvasive technique to detect and quantify events in a live cell, including concentration of molecules and diffusion coefficients of molecules; in addition, by measuring changes in diffusion coefficients, RICS can indirectly detect binding. Any specimen containing fluorophores that can be imaged with a laser scanning microscope can be analyzed using RICS. There are other techniques to measure diffusion coefficients and binding; however, RICS fills a unique niche. It provides spatial information and can be performed in live cells using a conventional confocal microscope. It can measure a range of diffusion coefficients that is not accessible with any other single optical correlation-based technique. In this article we describe a protocol to obtain raster scanned images with an Olympus FluoView FV1000 confocal laser scanning microscope using Olympus FluoView software to acquire data and SimFCS software to perform RICS analysis. Each RICS measurement takes several minutes. The entire procedure can be completed in ∼2 h. This procedure includes focal volume calibration using a solution of fluorophores with a known diffusion coefficient and measurement of the diffusion coefficients of cytosolic enhanced green fluorescent protein (EGFP) and EGFP-paxillin.

Measuring and imaging diffusion with multiple scan speed image correlation spectroscopy

The intracellular mobility of biomolecules is determined by transport and diffusion as well as molecular interactions and is crucial for many processes in living cells. Methods of fluorescence microscopy like confocal laser scanning microscopy (CLSM) can be used to characterize the intracellular distribution of fluorescently labeled biomolecules. Fluorescence correlation spectroscopy (FCS) is used to describe diffusion, transport and photo-physical processes quantitatively. As an alternative to FCS, spatially resolved measurements of mobilities can be implemented using a CLSM by utilizing the spatio-temporal information inscribed into the image by the scan process, referred to as raster image correlation spectroscopy (RICS). Here we present and discuss an extended approach, multiple scan speed image correlation spectroscopy (msICS), which benefits from the advantages of RICS, i.e. the use of widely available instrumentation and the extraction of spatially resolved mobility information, without the need of a priori knowledge of diffusion properties. In addition, msICS covers a broad dynamic range, generates correlation data comparable to FCS measurements, and allows to derive two-dimensional maps of diffusion coefficients. We show the applicability of msICS to fluorophores in solution and to free EGFP in living cells.

A dual-modality optical coherence tomography and fluorescence lifetime imaging microscopy system for simultaneous morphological and biochemical tissue characterization.

Most pathological conditions elicit changes in the tissue optical response that may be interrogated by one or more optical imaging modalities. Any single modality typically only furnishes an incomplete picture of the tissue optical response, hence an approach that integrates complementary optical imaging modalities is needed for a more comprehensive non-destructive and minimally-invasive tissue characterization. We have developed a dual-modality system, incorporating optical coherence tomography (OCT) and fluorescence lifetime imaging microscopy (FLIM), that is capable of simultaneously characterizing the 3-D tissue morphology and its biochemical composition. The Fourier domain OCT subsystem, at an 830 nm center wavelength, provided high-resolution morphological volumetric tissue images with an axial and lateral resolution of 7.3 and 13.4 µm, respectively. The multispectral FLIM subsystem, based on a direct pulse-recording approach (upon 355 nm laser excitation), provided two-dimensional superficial maps of the tissue autofluorescence intensity and lifetime at three customizable emission bands with 100 µm lateral resolution. Both subsystems share the same excitation/illumination optical path and are simultaneously raster scanned on the sample to generate coregistered OCT volumes and FLIM images. The developed OCT/FLIM system was capable of a maximum A-line rate of 59 KHz for OCT and a pixel rate of up to 30 KHz for FLIM. The dual-modality system was validated with standard fluorophore solutions and subsequently applied to the characterization of two biological tissue types: postmortem human coronary atherosclerotic plaques, and in vivo normal and cancerous hamster cheek pouch epithelial tissue.

Real-time fluorescence lifetime imaging system with a 32 × 32 013μm CMOS low dark-count single-photon avalanche diode array

A compact real-time fluorescence lifetime imaging microscopy (FLIM) system based on an array of low dark count 0.13microm CMOS single-photon avalanche diodes (SPADs) is demonstrated. Fast background-insensitive fluorescence lifetime determination is achieved by use of a recently proposed algorithm called 'Integration for Extraction Method' (IEM) [J. Opt. Soc. Am. A 25, 1190 (2008)]. Here, IEM is modified for a wider resolvability range and implemented on the FPGA of the new SPAD array imager. We experimentally demonstrate that the dynamic range and accuracy of calculated lifetimes of this new camera is suitable for widefield FLIM applications by imaging a variety of test samples, including various standard fluorophores covering a lifetime range from 1.6ns to 16ns, microfluidic mixing of fluorophore solutions, and living fungal spores of Neurospora Crassa. The calculated lifetimes are in a good agreement with literature values. Real-time fluorescence lifetime imaging is also achieved, by performing parallel 32 x 16 lifetime calculations, realizing a compact and low-cost FLIM camera and promising for bigger detector arrays.