Alternative, non-electrochemistry-based technologies for continuous glucose monitoring are needed for eventual use in diabetes mellitus. As part of a programme investigating fluorescent glucose sensors, we have developed fibre-optic biosensors using glucose/galactose binding protein (GBP) labelled with the environmentally sensitive fluorophore, Badan. GBP–Badan was attached via an oligohistidine-tag to the surface of Ni–nitrilotriacetic acid (NTA)-functionalized agarose or polystyrene beads. Fluorescencelifetime increased in response to glucose, observed by fluorescencelifetime imaging microscopy of the GBP–Badan-beads. Either GBP–Badan agarose or polystyrene beads were loaded into a porous chamber at the end of a multimode optical fibre. Fluorescencelifetime responses were recorded using pulsed laser excitation, high speed photodiode detection and time-correlated single- photon counting. The maximal response was at 100 mM glucose with an apparent K d of 13 mM
Photodynamic Therapy (PDT) provides an opportunity for treatment of various invasive tumors by the use of a cancer targeting photosensitizing agent and light of specific wave- lengths. However, real-time monitoring of drug localization is desirable because the induction of the phototoxic effect relies on interplay between the dosage of localized drug and light. Fluorescence emission in PDT may be used to monitor the uptake process but fluorescence intensity is subject to variability due to scattering and absorption; the addition of fluorescencelifetime may be beneficial to probe site-specific drug-molecular interactions and cell damage. We investigated the fluorescencelifetime changes of Photofrin ® at various intracellular
Molecular organization of a cell is dynamically transformed along the course of cellular physiological processes, pathologic developments or derived from interactions with drugs. The capability to measure and monitor concentrations of macromolecules in a single cell would greatly enhance studies of cellular processes in heterogeneous populations. In this communication, we introduce and experimentally validate a bio-analytical single-cell assay, wherein the overall concentration of macromolecules is es- timated in specific subcellular domains, such as structure-function compartments of the cell nucleus as well as in nucleoplasm. We describe quantitative mapping of local biomolecular concentrations, either intrinsic relating to the functional and physiological state of a cell, or altered by a therapeutic drug ac- tion, using two-photon excited fluorescencelifetime imaging (FLIM). The proposed assay utilizes a correlation between the fluorescencelifetime of fluorophore and the refractive index of its microen- vironment varying due to changes in the concentrations of macromolecules, mainly proteins. Two-photon excitation in Near-Infra Red biological transparency window reduced the photo-toxicity in live cells, as compared with a conventional single-photon approach. Using this new assay, we estimated average concentrations of proteins in the compartments of nuclear speckles and in the nucleoplasm at ~150 mg/ml, and in the nucleolus at ~284 mg/ml. Furthermore, we show a profound influence of pharmaceutical inhibitors of RNA synthesis on intracellular protein density. The approach proposed here will significantly advance theranostics, and studies of drug-cell interactions at the single-cell level, aiding development of personal molecular medicine.
The other two compounds indicated in Figure 1, E-ACA4 and E-DMACA4, and not discussed so far have also been shown to be taken up into live cells as shown by the images in Figure 4. Whilst both E-ACA4 and E-DMACA4 exhibited pronounced non-exponential fluorescence decays in some solvents that remains to be investigated further and is suggestive of excited charge transfer processes, the differences in lifetimes (Figure 4) and emission spectra (Figure 5) of the intracellular fluorescence from these compounds is further confirmation that the fluorescence observed does originate from these combretastatins after cellular uptake. The intensity image for E-DMACA4 in CHO cells (Figure 4a1) shows a cytoplasmic distribution similar to that for E-CA4F, with a few bright regions resembling lipid droplets. This appears consistent with the logP (P is the octanol/water partition coefficient indicative of polarity) for E-DMACA4 being similar to that for E-CA4F (Figure 1). The similarity with E-CA4F extends to the fluorescencelifetime of E-DMACA4 in DMSO (0.36 ns) which increases to a peak in the lifetime image in CHO cells at 1.07 ns (Figures 4a2 and 4a3). For E-ACA4 the logP value is slightly less than for E-CA4F and closer to that for E-CA4. However as a primary amine, it is likely to act as a weak base and protonates to some extent in aqueous solution becoming more polar. Indeed we observe that E-ACA4 is more soluble in water than the other combretastatins studied here. The fluorescencelifetime is longer than those of the other combretastatins (4.8 ns in DMSO) and although reduced to a peak at 2.15 ns in the FLIM image of CHO cells (Figure 4b3) remains longer than the lifetime of the other intracellular combretastatins observed here. The fluorescence images of E-ACA4 in CHO cells also appear rather different, with fluorescence intensity mainly confined to regions that appear to be intracellular vesicles, reminiscent of the distribution of serotonin in mast cells  or propranolol in rat aorta cells . In these instances vesicular uptake is driven by the accumulation of the weak base into acidic compartments in cells such as lysosomes and mitochondria.
Fluorescence based analysis is a fundamental research technique used in the life sciences. However, conventional fluorescence intensity measurements are prone to misinterpretation due to illumination and fluorophore concentration non-uniformities. Thus, there is a growing interest in time-resolved fluorescence detection, whereby the characteristic fluorescence decay time-constant (or lifetime) in response to an impulse excitation source is measured. The sensitivity of a sample’s lifetime properties to the micro-environment provides an extremely powerful analysis tool. However, current fluorescencelifetime analysis equipment tends to be bulky, delicate and expensive, thereby restricting its use to research laboratories. Progress in miniaturization of biological and chemical analysis instrumentation is creating low-cost, robust and portable diagnostic tools capable of high-throughput, with reduced reagent quantities and analysis times. Such devices will enable point-of-care or in-the-field diagnostics. In this paper, we report an integrated fluorescencelifetime analysis system capable of sub-nano second precision with the core of the instrument measuring less than 1 cm 3 , something hitherto impossible with existing approaches. To accomplish this, recent advances in the development of AlInGaN micro-LEDs and high sensitivity CMOS detectors have been exploited [1,2]. CMOS technology is key to both detection and excitation in our system providing compact, low cost, high speed electronic signal -processing circuitry for the photodetectors and vertically integrated drivers for the micro-LEDs. Furthermore, we demonstrate an array of pixellated fluorescence analysis sites with potential for multiplexed, high-throughput sensors, with reduced alignment tolerances. Combined with recent advances in on-chip, real-time lifetime computation [3,4] this work represents as significant step towards practical, micro-scale lifetime sensors, without the need for additional external hardware or sophisticated software post-processing.
For non-repetitive dynamic events in complex flows and combustions, we expect to obtain the fluorescencelifetime images with a single excitation to freeze the instantaneous structures. Conventional time-gated FLI measures the fluorescence delay functions by gating the image intensifier of an ICCD camera with different delays. It usually needs to acquire more than one time-gated shot. Single-shot lifetime measurements can be achieved by replacing the single ICCD camera with two or more ICCD cameras to take time-resolved images simultaneously. Omrane et al. measured phosphorescence images with a high-speed framing camera containing eight independent ICCD image detectors, their system can obtain lifetimes in the order of milliseconds using an exponential fitting procedure . However, this method is not suitable for measuring lifetimes around nanoseconds in a single shot. Ehn. et al. presented a single-shot FLI method with PLIF, using a dual ICCD detection setup with different gating characteristics for the two cameras, and they successfully demonstrated real-time acquisition of formaldehyde in a premixed, laminar methane/oxygen flame. The measured lifetimes range from 1.0 to 4.5ns .
treated with fresh medium containing no colchicine. At 24 hpi, BV of both control and colchicine-treated samples were harvested. The colchicine- treated cells were then washed three times with fresh medium containing 3 g/ml colchicine and then incubated with fresh medium containing 3 g/ml colchicine. The control cells were washed with only fresh medium, and the last wash was replaced with fresh medium without any colchicine. BV was harvested at 5, 15, 30, 60, and 120 min thereafter and titrated by plaque assay as described previously (23). Cells were fixed, stained for microtubules, and processed for confocal laser scanning microscopy (CLSM) as previously described. The experimental data were analyzed by a two-tailed Student t test using a statistical program, SPSS (version 15). Two-photon fluorescencelifetime imaging analysis of transfected insect cells. Two-photon-induced fluorescencelifetime images were ob- tained with multiphoton microscopy apparatus and an Eclipse TE2000 (Nikon) with confocal scanning capability FLIM (4, 5). Briefly, a high- powered titanium sapphire laser (MIRA 900; Coherent Lasers) was pumped by a frequency-doubled neodynium:vanadate laser (Verdi V18; Coherent Lasers) to produce a 920 ⫾ 5-nm laser light of 180-fs pulses at 75 MHz. Specimens on a Nikon TE2000U microscope stage were excited by focusing the near-infrared laser beam to a diffraction-limited spot through a 60 ⫻ water immersion objective (numerical aperture [NA], FIG 1 A schematic representation of recombinant baculovirus generation via homologous recombination. The first step is the generation of a transfer vector-pBacPAK.EGFP-VP39NatP (a), followed by a second step, which is the production of AcEGFP-VP39NatP via homologous recombination (b). VP39NatP, VP39 native promoter; ORF1629NatP, ORF1629 native promoter; EGFP, enhanced greed fluorescent protein; BAC, bacterial artificial chromosome; ChiA, chitinase A; PolP, polyhedrin promoter; del, deletion.
The first example is to study the fluorescence lifetimes of the autofluorescence emitted from daisy pollen. Autofluorescence of biological samples as well as the fluorescencelifetime can be very useful. For example, cellular autofluorescence is used as a good label-free indicator for studying cytotoxicity . The FLIM data are obtained using the MicroTime 200 (PicoQuant), equipped with the standard piezo scanner from Physik Instrumente (100x100µm scan range) and a Hybrid-PMT (PMA Hybrid-40). The TCSPC system used for the acquisition is the HydraHarp 400 with the bin width set to 8ps and each histogram contains 6253 time bins (the equivalent full range = 50ns). Other parameters include: excitation wavelength = 485nm, the laser repetition rate = 20 MHz (LDH-D-C-485 laser head controlled by the PDL 828 “Sepia II” laser driver) and the detection band = 520/35.
This work explores the use of a green-light-emitting copolymer as a chemosen- sor to detect nitroaromatic-based explosive vapors by recording photoluminescence (PL) and time-resolved PL decay. We show successful detection of 10 ppb 1,4- dinitrobenzene (DNB) vapor. Both a conventional time-correlated single photon counting (TCSPC) device and CMOS time-resolved fluorescencelifetime micro- system are used in the DNB detection. An ultra-portable on-site explosive sensor based on the micro-system has also been demonstrated. This gives rise to the poten- tial for real-time, reliable, inexpensive organic/inorganic hybrid explosives detection. Copyright 2011 Author(s). This article is distributed under a Creative Commons Attribution 3.0 Unported License. [doi:10.1063/1.3624456]
To investigate within live mammalian cells the uptake and disposition of combretastatins, fluorescencelifetime imaging was used with two-photon excitation (2PE). Combretastatin A4 (CA4) and analogues are potential anticancer drugs due to their ability to inhibit angiogenesis. E(trans)-combretastatins are considerably less active than the Z(cis)-combretastatins proposed for clinical use. However the E-combretastatins exhibit stronger intrinsic fluorescence with quantum yields and lifetimes that depend markedly on solvent polarity and viscosity. It is proposed that 2PE in the red and near-infrared tissue window may allow in situ isomerization of E- combretastatins to the more active Z-isomer, offering spatial and temporal control of drug activation and constitute a novel form of photodynamic therapy. In the present work we have characterised 2PE of E-CA4 and have used fluorescencelifetime imaging with 2PE to study uptake and intracellular disposition of E-CA4 and an analogue. The results show that these molecules accumulate rapidly in cells and are located mainly in lipidic environments such as lipid droplets. Within the droplets the local concentrations may be up to 2 orders of magnitude higher than that of the drug in the surrounding medium.
Time-correlated single photon counting (TCSPC) systems are widely as the gold standard fluorescencelifetime imaging (FLIM) owing to high temporal resolution. The acquisition speed has been significantly enhanced for the past few years by applying multiple-channel TCSPCs . FLIM image analysis, on the other hand, still heavily rely on iterative based software. Gated time domain (TD) (or frequency domain, FD) intensified CCD based FLIM systems can achieve fast acquisition when the number of gates or the number of phase images (for FD systems), denoted as M for simplicity, is only two . In commercially available systems, however, the number of gates is usually higher than 10 to ensure enough lifetime resolvability within the field of view [2, 3]. A larger M not just slows down the acquisition, but also complicates image analysis. Commercial FLIM software are usually based on nonlinear iterative analysis methods and slow making them impossible for real-time applications.
HER2-specific Affibody fluorescent probe was injected in 24 mice with 6 different tumor models, and the fluorescencelifetime of tumor and contralateral sites were measured in vivo. These cell lines include high level (+ 3) HER2-expressing human tumor carcinoma (BT-474, NCI-N87, and SKOV-3), medium level (+ 2) HER2-expressing human tumor carcinoma (MDA-MB- 361), low level (+ 1) HER2-expressing human tumor carcinoma (MCF-7); and non-HER2-expressing human tumor carcinoma (MDA-MB-468). In all cases, HER2- specific Affibody (His6-ZHER2:GS-Cys) fluorescent probe was injected intravenously and imaging was per- formed 1 h after injection. We chose this time based on our measured fluorescence intensity at the tumor and contra-lateral sites at different time points for 5 h. About 1 h after injection, the fluorescent probe accumulation stabilizes at the tumor site and its intensity at the contra-lateral site is still considerably higher than
Mutated proteins were expressed in HeLa cells. Immuno- detection by Western Blots revealed that none of the point mutations impeded expression of the Vpr fusion proteins (data not shown). The fluorescencelifetime images were recorded and compared with those of the two wild type Vpr fusion proteins. Figure 5 shows the lifetime images of the Vpr-eGFP mutants expressed in the absence (Column A) and in the presence of the corresponding Vpr-mCherry mutant (Column B). The mean values obtained for the entire cell are reported on the right of the figure. Among the eight mutants, four of them, namely Q3R, W54G, R77Q and R90K, showed a staining pattern similar to that of the wild type fusion proteins with an accumulation at the nuclear rim (compare with Figure 4, panel A2). Oli- gomers of these mutant proteins were found in the cyto- plasm, the nucleus and at the nuclear envelope. The transfer efficiency in the whole cell for these mutants was respectively 19%, 16%, 22% and 18%, similar to the value
Unlike the steady state fluorescence measurements, there are not many reports on the fluorescence lifetimes of curcumin. The competing non-radiative processes shortened the fluorescence lifetimes considerably. The fluorescence decay profiles, obtained from time correlated single photon counting (TCSPC) studies, in most of the organic solvents showed multi-exponential f is and the fluorescencelifetime values as well as their relative amplitudes varied significantly with the nature of the solvent [21,22]. Fig. 1 shows the decay curve of curcumin without and with different concentrations of β cyclodextrin.
Another bottleneck for high-speed lifetime imaging is lifetime calculation. Common FLIM systems usually use iterative linear or non-linear least square methods (LSM), such as Marquardt-Levenberg algorithms, to extract the lifetimes. Although this approach is accurate and suitable for analyzing multi-exponential decays, it is computationally time consuming which makes it unsuitable for real-time applications. It is desirable, therefore, to develop non-iterative simple algorithms to speed up the lifetime calculations while maintaining enough imaging quality. Compared with the LSM, iterative-free gating methods only require two time bins for single-exponential decays [9,13,21], four bins for bi-exponential decays [32,33] or eight bins for multi-exponential decays . The hardware complexity is greatly reduced and the speed is much higher. There are different acquisition schemes for the gating methods. Figure 1(a) shows the traditional sequential acquisition in a pixel, where at least two sub-images are recorded sequentially at different delayed windows with respect to the excited laser pulses to extract the lifetime. The block ‘counter’ can contain front-end circuits, analog-to-digital converters and accumulators in conventional imaging systems or simply inverters and digital buffers in the latest CMOS SPAD systems. Chang and Mycek applied four time-gates to analyze single-exponential decay data . This approach is slow and sensitive to motion artifacts unless the samples are stationary, and the recorded sub-images are uncorrelated. If a full fluorescence emission histogram is needed for detailed examinations, it will take a significant amount of time to record a large number of sub-images with different delay times .
The 2B protein of enteroviruses is the viral membrane-active protein that is responsible for the modifications in host cell membrane permeability that take place in enterovirus-infected cells. The 2B protein shows structural similarities to the group of lytic polypeptides, polypeptides that permeate membranes either by forming multimeric membrane-integral pores or, alternatively, by lying parallel to the lipid bilayer and disturbing the curvature and symmetry of the membrane. Our aim is to gain more insight into the molecular architecture of the 2B protein in vivo. In this study, the possible existence of multimers of the coxsackie B3 virus 2B protein in single living cells was explored by fluorescence resonance energy transfer (FRET) micros- copy. FRET between fusion proteins 2B-ECFP and 2B-EYFP (enhanced cyan and yellow fluorescent variants of green fluorescent protein) was monitored by using spectral imaging microscopy (SPIM) and fluorescencelifetime imaging microscopy (FLIM). Both techniques revealed the occurrence of intermolecular FRET be- tween 2B-ECFP and 2B-EYFP, providing evidence for the formation of protein 2B homomultimers. Putative models for the mode of action of the membrane-active 2B protein and the formation of membrane-integral pores by 2B multimers are discussed.
Abstract—Estimation of signal parameters via rotational in- variance techniques is a classical algorithm widely used in array signal processing for direction-of-arrival estimation of emitters. Inspired by this method, a new signal model and new fluorescencelifetime estimation via rotational invariance techniques (FLERIT) were developed for multiexponential fluorescencelifetime imaging (FLIM) experiments. The FLERIT only requires a few time bins of a histogram generated by a time-correlated single-photon count- ing FLIM system, greatly reducing the data throughput from the imager to the signal processing units. As a noniterative method, the FLERIT does not require initial conditions, prior information nor model selection that are usually required by widely used tra- ditional fitting methods, including nonlinear least square methods or maximum-likelihood methods. Moreover, its simplicity means it is suitable for implementations in embedded systems for real-time applications. FLERIT was tested on synthesized and experimental fluorescent cell data showing the potentials to be widely applied in FLIM data analysis.