Precision and accuracy of the analog mean-delay method for high-speed fluorescence lifetime measurement

The analog mean-delay (AMD) method is a new alternative method to measure the lifetime of a fluorescence molecule. Because of its powerful advantages of accurate lifetime determination, good photon economy, and a high photon detection rate, the AMD method is considered to be very suitable for high-speed confocal fluorescence lifetime imaging microscopy (FLIM). For the practical usage of the AMD method in FLIM (AMD-FLIM), detailed study on various experimental conditions and parameters that affect the precision and the accuracy of the AMD method is required. In this paper, we present the relation between the precision and accuracy of the lifetime versus iteration number in the AMD method, the best cutoff frequency of a low-pass filter used in the AMD-FLIM system for a given fluorophore, and the optimum position and width of the integration window by using Monte Carlo simulations and a series of AMD-FLIM experiments.     

Visualizing Oxidative Cellular Stress Induced by Nanoparticles in the Subcytotoxic Range Using Fluorescence Lifetime Imaging

Nanoparticles hold a great promise in biomedical science. However, due to their unique physical and chemical properties they can lead to overproduction of intracellular reactive oxygen species (ROS). As an important mechanism of nanotoxicity, there is a great need for sensitive and high‐throughput adaptable single‐cell ROS detection methods. Here, fluorescence lifetime imaging microscopy (FLIM) is employed for single‐cell ROS detection (FLIM‐ROX) providing increased sensitivity and enabling high‐throughput analysis in fixed and live cells. FLIM‐ROX owes its sensitivity to the discrimination of autofluorescence from the unique fluorescence lifetime of the ROS reporter dye. The effect of subcytotoxic amounts of cationic gold nanoparticles in J774A.1 cells and primary human macrophages on ROS generation is investigated. FLIM‐ROX measures very low ROS levels upon gold nanoparticle exposure, which is undetectable by the conventional method. It is demonstrated that cellular morphology changes, elevated senescence, and DNA damage link the resulting low‐level oxidative stress to cellular adverse effects and thus nanotoxicity. Multiphoton FLIM‐ROX enables the quantification of spatial ROS distribution in vivo, which is shown for skin tissue as a target for nanoparticle exposure. Thus, this innovative method allows identifying of low‐level ROS in vitro and in vivo and, subsequently, promotes understanding of ROS‐associated nanotoxicity.     

One-pot synthesis of near-infrared fluorescent gold clusters for cellular fluorescence lifetime imaging

A facile strategy to synthesize water-soluble fluorescent gold nanoclusters (Au NCs) stabilized with the bidentate ligand dihydrolipoic acid (DHLA) is reported. The DHLA-capped Au NCs are characterized by UV-vis absorption spectroscopy, fluorescence spectroscopy, transmission electron microscopy, and X-ray photoelectron spectroscopy. The Au NCs possess many attractive features including ultrasmall size, bright near-infrared luminescence, high colloidal stability, and good biocompatibility, making them promising imaging agents for biomedical and cellular imaging applications. Moreover, their long fluorescence lifetime (>100 ns) makes them attractive as labels in fluorescence lifetime imaging (FLIM) applications. As an example, the internalization of Au NCs by live HeLa cells is visualized using the FLIM technique.     

Time-resolved Hadamard fluorescence imaging

We present a new concept for fluorescence lifetime imaging (FLIM) based on time-resolved Hadamard imaging (HI). HI allows image acquisition by use of one single-point detector without requiring a moving scanning stage. Moreover, it reduces the influence of detector noise compared with raster scanning. By use of Monte Carlo simulations it could be confirmed that Hadamard transformation may decrease the error in lifetime estimation and in general in fluorescence parameter estimation when the signal-to-noise ratio is low and detector dark noise is high. This concept may find applications whenever the performance of FLIM or similar methods is limited by high dark-count rates and when the use of a single-point detector is preferable.     

Fluorescence Lifetime Imaging and FRET‐Induced Intracellular Redistribution of Tat‐Conjugated Quantum Dot Nanoparticles through Interaction with a Phthalocyanine Photosensitiser

The interaction of Tat‐conjugated PEGylated CdSe/ZnS quantum dots (QD) with the amphiphilic disulfonated aluminium phthalocyanine photosensitiser is investigated in aqueous solution and in a human breast cancer cell line. In aqueous solution, the QDs and phthalocyanine form stable nanocomposites. Using steady‐state and time‐resolved fluorescence measurements combined with singlet oxygen detection, efficient Förster resonance energy transfer (FRET) is observed with the QDs acting as donors, and the phthalocyanine photosensitiser, which mediates production of singlet oxygen, as acceptors. In cells, the Tat‐conjugated QDs localise in lysosomes and the QD fluorescence lifetimes are close to values observed in aqueous solution. Strong FRET‐induced quenching of the QD lifetime is observed in cells incubated with the nanocomposites using fluorescence lifetime imaging microscopy (FLIM). Using excitation of the QDs at wavelengths where phthalocyanine absorption is negligible, FRET‐induced release of QDs from endo/lysosomes is confirmed using confocal imaging and FLIM, which is attributed to photooxidative damage to the endo/lysosomal membranes mediated by the phthalocyanine acceptor.     

Studying biological tissue with fluorescence lifetime imaging: microscopy,endoscopy, and complex decay profiles

We have applied fluorescence lifetime imaging (FLIM) to the autofluorescence of different kinds of biological tissue in vitro, including animal tissue sections and knee joints as well as human teeth, obtaining two-dimensional maps with functional contrast. We find that fluorescence decay profiles of biological tissue are well described by the stretched exponential function (StrEF), which can represent the complex nature of tissue. The StrEF yields a continuous distribution of fluorescence lifetimes, which can be extracted with an inverse Laplace transformation, and additional information is provided by the width of the distribution. Our experimental results from FLIM microscopy in combination with the StrEF analysis indicate that this technique is ready for clinical deployment, including portability that is through the use of a compact picosecond diode laser as the excitation source. The results obtained with our FLIM endoscope successfully demonstrated the viability of this modality, though they need further optimization. We expect a custom-designed endoscope with optimized illumination and detection efficiencies to provide significantly improved performance.     

Scene-based method for spatial misregistration detection in hyperspectral imagery

Hyperspectral imaging (HSI) sensors suffer from spatial misregistration, an artifact that prevents the accurate acquisition of the spectra. Physical considerations let us assume that the influence of the spatial misregistration on the acquired data depends both on the wavelength and on the across-track position. A scene-based method, based on edge detection, is therefore proposed. Such a procedure measures the variation on the spatial location of an edge between its various monochromatic projections, giving an estimation for spatial misregistration, and also allowing identification of misalignments. The method has been applied to several hyperspectral sensors, either prism, or grating-based designs. The results confirm the dependence assumptions on lambda and theta, spectral wavelength and across-track pixel, respectively. Suggestions are also given to correct for spatial misregistration.     

Liquid-crystal tunable filter spectral imaging for brain tumor demarcation

Past studies have demonstrated that combined fluorescence and diffuse reflectance spectroscopy can successfully discriminate between normal, tumor core, and tumor margin tissues in the brain. To achieve efficient, real-time surgical resection guidance with optical biopsy, probe-based spectroscopy must be extended to spectral imaging to spatially demarcate the tumor margins. We describe the design and characterization of a combined fluorescence and diffuse reflectance imaging system that uses liquid-crystal tunable filter technology. Experiments were conducted to quantitatively determine the linearity, field of view, spatial and spectral resolution, and wavelength sensitivity of the imaging system. Spectral images were acquired from tissue phantoms, mouse brain in vitro, and human cortex in vivo for functional testing of the system. The spectral imaging system produces measured intensities that are linear with sample emission intensity and integration time and possesses a 1 in. (2.54 cm) field of view for a 7 in. (18 cm) object distance. The spectral resolution is linear with wavelength, and the spatial resolution is pixel-limited. The sensitivity spectra for the imaging system provide a guide for the distribution of total image integration time between wavelengths. Functional tests in vitro demonstrate the capability to spectrally discriminate between brain tissues based on exogenous fluorescence contrast or endogenous tissue composition. In vivo imaging captures adequate fluorescence and diffuse reflectance intensities within a clinically viable 2 min imaging time frame and demonstrates the importance of hemostasis to acquired signal strengths and imaging speed.     

Visualizing resonance energy transfer in supramolecular surface patterns of β-CD-functionalized quantum dot hosts and organic dye guests by fluorescence lifetime imaging

Detection of an analyte via supramolecular host-guest binding and quantum dot (QD)-based fluorescence resonance energy transfer (FRET) signal transduction mechanism is demonstrated. Surface patterns consisting of CdSe/ZnS QDs functionalized at their periphery with β-cyclodextrin (β-CD) were obtained by immobilization of the QDs from solution onto glass substrates patterned with adamantyl-terminated poly(propylene imine) dendrimeric "glue." Subsequent formation of host-guest complexes between vacant β-CD on the QD surface and an adamantyl-functionalized lissamine rhodamine resulting in FRET was confirmed by fluorescence microscopy, spectroscopy, and fluorescence lifetime imaging microscopy (FLIM).     

Verification of antiparallel G-quadruplex structure in human telomeres by using two-photon excitation fluorescence lifetime imaging microscopy of the 3,6-Bis(1-methyl-4-vinylpyridinium)carbazole diiodide molecule

Different G-quadruplex structures for the human telomeric sequence d(T2AG3)4 in vitro have been documented in the presence of sodium and potassium. Verification of the G-quadruplex structures in human telomeres in vivo is the main issue in establishing the biological function of the G-quadruplex structures in telomeres as well as the development of anticancer agents. Here we have applied two-photon excitation fluorescence lifetime imaging microscopy to measure the fluorescence lifetime of the BMVC molecule upon interaction with various DNA structures. The distinction in lifetime measured with submicrometer spatial resolution in two-photon excitation fluorescence lifetime imaging microscopy provides a powerful approach not only to verify the existence of the antiparallel G-quadruplex structure in human telomeres but also to map its localizations in metaphase chromosomes.     

High-speed spectral imager for imaging transient fluorescence phenomena

We describe fluorescence spectral imaging results with the microscope computed-tomography imaging spectrometer (muCTIS). This imaging spectrometer is capable of recording spatial and spectral data simultaneously. Consequently, muCTIS can be used to image dynamic phenomena. The results presented consist of proof-of-concept imaging results with static targets composed of 6-mum fluorescing microspheres. Image data were collected with integration times of 16 ms, comparable with video-frame-rate integration times. Conversion of raw data acquired by the muCTIS to spatial and spectral data requires postprocessing. The emission spectra were sampled at 10-nm intervals between 420 and 710 nm. The smallest spatial sampling interval presented is 1.7 mum.     

On-chip, time-correlated, fluorescence lifetime extraction algorithms and error analysis

A new, simple, and hardware-only fluorescence-lifetime-imaging microscopy (FLIM) is proposed to implement on-chip lifetime extractions, and their signal-to-noise-ratio based on statistics theory is also deduced. The results are compared with Monte Carlo simulations, giving good agreement. Compared with the commonly used iterative least-squares method or the maximum-likelihood-estimation- (MLE-) based, general purpose FLIM analysis software, our algorithm offers direct calculation of fluorescence lifetime based on the collected photon counts stored in on-chip counters and therefore delivers faster analysis for real-time applications, such as clinical diagnosis. Error analysis considering timing jitter based on statistics theory is carried out for the proposed algorithms and is also compared with MLE to obtain optimized channel width or measurement window and bit resolution of the time-to-digital converters for a given accuracy. A multi-exponential, pipelined fluorescence lifetime method based on the proposed algorithms is also introduced. The performance of the proposed methods has been tested on mono-exponential and four-exponential decay experimental data.     

Fluorescence lifetime imaging with near-field scanning optical microscopy

Near-field scanning optical microscopy (NSOM) is a high-resolution scanning probe technique capable of obtaining simultaneous optical and topographic images with spatial resolution of tens of nanometers. We have integrated time-correlated single-photon counting and NSOM to obtain images of fluorescence lifetimes with high spatial resolution. The technique can be used to measure either full fluorescence lifetime decays at individual spots with a spatial resolution of <100 nm or NSOM fluorescence images using fluorescence lifetime as a contrast mechanism. For imaging, a pulsed Ti:sapphire laser was used for sample excitation and fluorescent photons were time correlated and sorted into two time delay bins. The intensity in these bins can be used to estimate the fluorescence lifetime at each pixel in the image. The technique is demonstrated on thin films of poly(9,9'-dioctylfluorene) (PDOF). The fluorescence of PDOF is the results of both inter- and intrapolymer emitting species that can be easily distinguished in the time domain. Fluorescence lifetime imaging with near-field scanning optical microscopy demonstrates how photochemical degradation of the polymer leads to a quenching of short-delay intrachain emission and an increase in the long-delay photons associated with interpolymer emitting species. The images also show how intra- and interpolymer species are uniformly distributed in the films.     

Intravital Multiphoton Imaging of the Selective Uptake of Water‐Dispersible Quantum Dots into Sinusoidal Liver Cells

Although many studies reporting the organ‐level biodistribution of nanoparticles (NPs) in animals, very few have addressed the fate of NPs in organs at the cellular level. The liver appears to be the main organ for accumulation of NPs after intravenous injection. In this study, for the first time, the in vivo spatiotemporal disposition of recently developed mercaptosuccinic acid (MSA)‐capped cadmium telluride/cadmium sulfide (CdTe/CdS) quantum dots (QDs) is explored in rat liver using multiphoton microscopy (MPM) coupled with fluorescence lifetime imaging (FLIM), with subcellular resolution (～1 μm). With high fluorescence efficiency and largely improved stability in the biological environment, these QDs show a distinct distribution pattern in the liver compared to organic dyes, rhodamine 123 and fluorescein. After intravenous injection, fluorescent molecules are taken up by hepatocytes and excreted into the bile, while negatively charged QDs are retained in the sinusoids and selectively taken up by sinusoidal cells (Kupffer cells and liver sinusoidal endothelial cells), but not by hepatocytes within 3 h. The results could help design NPs targeting the specific types of liver cells and choose the fluorescent markers for appropriate cellular imaging.     

Energy transfer: Visualizing Resonance Energy Transfer in Supramolecular Surface Patterns of β‐CD‐Functionalized Quantum Dot Hosts and Organic Dye Guests by Fluorescence Lifetime Imaging (Small 24/2010)

Detection of an analyte via supramolecular host–guest binding and quantum dot (QD)‐based fluorescence resonance energy transfer (FRET) signal transduction mechanism is demonstrated. Surface patterns consisting of CdSe/ZnS QDs functionalized at their periphery with β‐cyclodextrin (β‐CD) were obtained by immobilization of the QDs from solution onto glass substrates patterned with adamantyl‐terminated poly(propylene imine) dendrimeric “glue.” Subsequent formation of host–guest complexes between vacant β‐CD on the QD surface and an adamantyl‐functionalized lissamine rhodamine resulting in FRET was confirmed by fluorescence microscopy, spectroscopy, and fluorescence lifetime imaging microscopy (FLIM).     

The Spatial LASSO With Applications to Unmixing Hyperspectral Biomedical Images

Hyperspectral imaging (HSI) is a spectroscopic method that uses densely sampled measurements along the electromagnetic spectrum to identify the unique molecular composition of an object. Traditionally HSI has been associated with remote sensing-type applications, but recently has found increased use in biomedicine, from investigations at the cellular to the tissue level. One of the main challenges in the analysis of HSI is estimating the proportions, also called abundance fractions of each of the molecular signatures. While there is great promise for HSI in the area of biomedicine, large variability in the measurements and artifacts related to the instrumentation has slow adoption into more widespread practice. In this article, we propose a novel regularization and variable selection method called the spatial LASSO (SPLASSO). The SPLASSO incorporates spatial information via a graph Laplacian-based penalty to help improve the model estimation process for multivariate response data. We show the strong performance of this approach on a benchmark HSI dataset with considerable improvement in predictive accuracy over the standard LASSO. Supplementary materials for this article are available online.     

Wide-field fluorescence lifetime imaging with optical sectioning and spectral resolution applied to biological samples

Abstract. Wide-field fluorescence lifetime imaging with spectral resolution and optical sectioning has been performed to achieve five-dimensional fluorescence microscopy. Spectral filtering has been shown to have the potential to provide functional information about biological tissue by simultaneously measuring the spectral/lifetime signature of the sample. The potential to use multispectral imaging to separate cellular components spatially by their different emission wavelengths has also been demonstrated thus reducing artefacts in the calculated lifetime maps. The instrument is based on diode-pumped solid-state laser technology and an ultrafast gated optical image intensifier. Also reported is the use of a picosecond blue laser diode as the excitation source to produce a fluorescence lifetime microscope with a footprint of less than 0.25m².     

Solid-state digital micro-mirror array spectrometer for Hadamard transform measurements of glucose and lactate in aqueous solutions

A novel solid-state near-infrared spectrometer is presented based on a digital micro-mirror array device (DMD) that is well designed for Hadamard transform spectroscopy. This spectrometer is designed for the collection of transmission spectra over the C-H first overtone region of the near-infrared spectrum (6500-5500 cm(-1)). A spectral resolution of 2.2 nm (~11 cm(-1)) is realized by using a 25 μm diameter linear tungsten filament as the source. Such a thin filament reduces imaging aberrations into the micro-mirror array, thereby enhancing spectral resolution. After passing through the sample, the transmitted radiation is dispersed with a grating before being imaged onto the surface of the DMD. Hadamard transform masks are implemented through the DMD and the reflected light is monitored by a single-element photodiode detector. The analytical utility of this approach is demonstrated through the multivariate quantification of glucose and lactate in binary mixtures composed in an aqueous buffer solution. A signal-to-noise ratio of 35,000 is achieved through these aqueous samples, and the resulting quantitative measurements provide a standard error of prediction of 1.4 and 0.9 mM for glucose and lactate, respectively. The selectivity of the resulting calibration models is established by using both a pure component selectivity analysis as well as analysis of the net analyte signal for each component. These quantitative results from the DMD Hadamard transform spectrometer compare favorably to similar measurements performed with a commercial Fourier transform spectrometer.     

Automated Deep Learning Driven Crop Classification on Hyperspectral Remote Sensing Images

Hyperspectral remote sensing/imaging spectroscopy is a novel approach to reaching a spectrum from all the places of a huge array of spatial places so that several spectral wavelengths are utilized for making coherent images. Hyperspectral remote sensing contains acquisition of digital images from several narrow, contiguous spectral bands throughout the visible, Thermal Infrared (TIR), Near Infrared (NIR), and Mid-Infrared (MIR) regions of the electromagnetic spectrum. In order to the application of agricultural regions, remote sensing approaches are studied and executed to their benefit of continuous and quantitative monitoring. Particularly, hyperspectral images (HSI) are considered the precise for agriculture as they can offer chemical and physical data on vegetation. With this motivation, this article presents a novel Hurricane Optimization Algorithm with Deep Transfer Learning Driven Crop Classification (HOADTL-CC) model on Hyperspectral Remote Sensing Images. The presented HOADTL-CC model focuses on the identification and categorization of crops on hyperspectral remote sensing images. To accomplish this, the presented HOADTL-CC model involves the design of HOA with capsule network (CapsNet) model for generating a set of useful feature vectors. Besides, Elman neural network (ENN) model is applied to allot proper class labels into the input HSI. Finally, glowworm swarm optimization (GSO) algorithm is exploited to fine tune the ENN parameters involved in this article. The experimental result scrutiny of the HOADTL-CC method can be tested with the help of benchmark dataset and the results are assessed under distinct aspects. Extensive comparative studies stated the enhanced performance of the HOADTL-CC model over recent approaches with maximum accuracy of 99.51%.     

Multimodal bioimaging based on gold nanorod and carbon dot nanohybrids as a novel tool for atherosclerosis detection

Advanced biocompatible and robust platforms equipped with diverse properties are highly required in biomedical imaging applications for the early detection of atherosclerotic vascular disease and cancers. Designing nanohybrids composed of noble metals and fluorescent materials is a new way to perform multimodal imaging to overcome the limitations of single-modality counterparts. Herein, we propose the novel design of a multimodal contrast agent; namely, an enhanced nanohybrid comprising gold nanorods (GNRs) and carbon dots (CDs) with silica (SiO₂) as a bridge. The nanohybrid (GNR@SiO₂@CD) construction is based on covalent bonding between SiO₂ and the silane-functionalized CDs, which links the GNRs with the CDs to form typical core–shell units. The novel structure not only retains and even highly improves the optical properties of the GNRs and CDs, but also possesses superior imaging performance in both diffusion reflection (DR) and fluorescence lifetime imaging microscopy (FLIM) measurements compared with bare GNRs or fluorescence dyes and CDs. The superior bioimaging properties of the GNR@SiO₂@CD nanohybrids were successfully exploited for in vitro DR and FLIM measurements of macrophages within tissue-like phantoms, paving the way toward a theranostic contrast agent for atherosclerosis and cancer.

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