Quantitative kinetic analysis in a microfluidic device using frequency-domain fluorescence lifetime imaging

A novel microfluidic approach for the quantification of reaction kinetics is presented. A three-dimensional finite difference numerical simulation was developed in order to extract quantitative kinetic information from fluorescence lifetime imaging experimental data. This approach was first utilized for the study of a fluorescence quenching reaction within a microchannel; the lifetime of a fluorophore was used to map the diffusion of a quencher across the microchannel. The approach was then applied to a more complex chemical reaction between a fluorescent amine and an acid chloride, via numerical simulation the bimolecular rate constant for this reaction was obtained.     

Single-cell fluorescence imaging using metal plasmon-coupled probe 2: single-molecule counting on lifetime image

Multiple Alexa Fluor 647-conjugated concanavalin A (conA) molecules were covalently bound to a single 20 nm silver particle to synthesize metal plasmon-coupled probes (PCPs). The fluorescence images were recorded by scanning confocal microscopy in both intensity and lifetime. The brightness of PCPs was 30-fold brighter than those of free conA and the lifetime of PCPs was shortened dramatically. PCPs were used to label T-lymphocytic ( PM1) cells. The emission spots by PCPs bound on the cell surfaces were separated clearly from the cell images by autofluorescence due to the brighter signal and shorter lifetime of PCPs. The emission spots by PCPs were also scanned in three dimensions to count the distribution of bound fluorophores on the cell surfaces. The metal-associated fluorophores thus are suggested using as novel molecular imaging agents to quantify the components and describe their distributions on the cell surfaces.     

Theoretical investigation of the signal-to-noise ratio in fluorescence lifetime imaging

We deduce the signal-to-noise ratio for fluorescence lifetime imaging when using frequency-domain methods. We assume mono-exponential decay and quantum-noise-limited performance. The results are compared with Monte Carlo simulations with good agreement. We also compare our results with previous investigations of time-domain methods for fluorescence lifetime imaging. For a given number of detected photons, we find that frequency-domain and time-domain methods are equally good. The correct choice of detection technique and its parameters is important for obtaining good results.     

Multispectral fluorescence lifetime imaging of feces-contaminated apples by time-resolved laser-induced fluorescence imaging system with tunable excitation wavelengths

We recently developed a time-resolved multispectral laser-induced fluorescence (LIF) imaging system capable of tunable wavelengths in the visible region for sample excitation and nanosecond-scale characterizations of fluorescence responses (lifetime imaging). Time-dependent fluorescence decay characteristics and fluorescence lifetime imaging of apples artificially contaminated with a range of diluted cow feces were investigated at 670 and 685 nm emission bands obtained by 418, 530, and 630 nm excitations. The results demonstrated that a 670 nm emission with a 418 nm excitation provided the greatest difference in time-dependent fluorescence responses between the apples and feces-treated spots. The versatilities of the time-resolved LIF imaging system, including fluorescence lifetime imaging of a relatively large biological object in a multispectral excitation-emission wavelength domain, were demonstrated.     

Enhanced 3D fluorescence live cell imaging on nanoplasmonic substrate

We have created a randomly distributed nanocone substrate on silicon coated with silver for surface-plasmon-enhanced fluorescence detection and 3D cell imaging. Optical characterization of the nanocone substrate showed it can support several plasmonic modes (in the 300-800 nm wavelength range) that can be coupled to a fluorophore on the surface of the substrate, which gives rise to the enhanced fluorescence. Spectral analysis suggests that a nanocone substrate can create more excitons and shorter lifetime in the model fluorophore Rhodamine 6G (R6G) due to plasmon resonance energy transfer from the nanocone substrate to the nearby fluorophore. We observed three-dimensional fluorescence enhancement on our substrate shown from the confocal fluorescence imaging of chinese hamster ovary (CHO) cells grown on the substrate. The fluorescence intensity from the fluorophores bound on the cell membrane was amplified more than 100-fold as compared to that on a glass substrate. We believe that strong scattering within the nanostructured area coupled with random scattering inside the cell resulted in the observed three-dimensional enhancement in fluorescence with higher photostability on the substrate surface.     

3D atomic imaging of SiGe system by X-ray fluorescence holography

X-ray fluorescence holography (XFH) provides three-dimensional atomic images around specific elements without any assumption of the structural model. Six X-ray holograms of Si_0.8Ge_0.2/Si at different energies were measured at the synchrotron radiation facility of SPring-8. Si and/or Ge atoms within 0.7 nm of a radius were clearly visible in the atomic images reconstructed from the holograms. From these images, slight displacements of the images at each shell in between Si_0.8Ge_0.2/Si and the Ge bulk were distinctly revealed. This demonstrated that the XFH method has a great potential to quantitatively analyze a three-dimensional local lattice structure in epitaxial crystals.     

High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy

Conventional fluorescence microscopy can be used to determine the positions of objects in space when those objects are separated by distances greater than several hundred nanometers, as restricted by the diffraction limit of light. Fluorescence microscopy/spectroscopy based on fluorescence resonance energy-transfer techniques can be used to measure separation distances below approximately 10 nm. To fill the gap between these fundamental limits, we have developed an alternative technique for high-resolution colocalization of fluorescent dyes. The technique is based on fluorescence lifetime imaging. Under favorable conditions, the method can be used to distinguish, and to measure the distance between, two dye molecules that are less than 30 nm apart. To demonstrate the method, lifetime images of a mixture of Cy5 and JF9 (rhodamine derivative) molecules statistically adsorbed on a glass surface were acquired and analyzed. Since these two molecular species differ in fluorescence lifetime (for Cy5, tau(f) = 2.0 ns, and for JF9, tau(f) = 4.0 ns), it is possible to assign the contribution of fluorescence of the two dye types to each image pixel using a pattern recognition technique. Since both dye types can be excited using the same laser wavelength, the measurement is free of chromatic aberrations. The results presented demonstrate the first high-precision distance measurements between single conventional fluorescent dyes based solely on fluorescence lifetime.     

An approach to architecture 3D scaffold with interconnective microchannel networks inducing angiogenesis for tissue engineering

The angiogenesis of 3D scaffold is one of the major current limitations in clinical practice tissue engineering. The new strategy of construction 3D scaffold with microchannel circulation network may improve angiogenesis. In this study, 3D poly(d,l-lactic acid) scaffolds with controllable microchannel structures were fabricated using sacrificial sugar structures. Melt drawing sugar-fiber network produced by a modified filament spiral winding method was used to form the microchannel with adjustable diameters and porosity. This fabrication process was rapid, inexpensive, and highly scalable. The porosity, microchannel diameter, interconnectivity and surface topographies of the scaffold were characterized by scanning electron microscopy. Mechanical properties were evaluated by compression tests. The mean porosity values of the scaffolds were in the 65–78% and the scaffold exhibited microchannel structure with diameter in the 100–200 μm range. The results showed that the scaffolds exhibited an adequate porosity, interconnective microchannel network, and mechanical properties. The cell culture studies with endothelial cells (ECs) demonstrated that the scaffold allowed cells to proliferate and penetrate into the volume of the entire scaffold. Overall, these findings suggest that the fabrication process offers significant advantages and flexibility in generating a variety of non-cytotoxic tissue engineering scaffolds with controllable distributions of porosity and physical properties that could provide the necessary physical cues for ECs and further improve angiogenesis for tissue engineering.     

3D mapping of water in oolithic limestone at atmospheric and vacuum saturation using X-ray micro-CT differential imaging

Determining the distribution of fluids in porous sedimentary rocks is of great importance in many geological fields. However, this is not straightforward, especially in the case of complex sedimentary rocks like limestone, where a multidisciplinary approach is often needed to capture its broad, multimodal pore size distribution and complex pore geometries. This paper focuses on the porosity and fluid distribution in two varieties of Massangis limestone, a widely used natural building stone from the southeast part of the Paris basin (France). The Massangis limestone shows locally varying post-depositional alterations, resulting in different types of pore networks and very different water distributions within the limestone. Traditional techniques for characterizing the porosity and pore size distribution are compared with state-of-the-art neutron radiography and X-ray computed microtomography to visualize the distribution of water inside the limestone at different imbibition conditions. X-ray computed microtomography images have the great advantage to non-destructively visualize and analyze the pore space inside of a rock, but are often limited to the larger macropores in the rock due to resolution limitations. In this paper, differential imaging is successfully applied to the X-ray computed microtomography images to obtain sub-resolution information about fluid occupancy and to map the fluid distribution in three dimensions inside the scanned limestone samples. The detailed study of the pore space with differential imaging allows understanding the difference in the water uptake behavior of the limestone, a primary factor that affects the weathering of the rock.     

An approach to 3D dose mapping using Gafchromic film

The tissue equivalent composition of the Gafchromic films makes them particularly suitable for the mapping of 2D and 3D treatment fields. This paper presents the results obtained using MD-55 film for the verification of real radiotherapy treatments through proton beam irradiation of suitable phantoms at the Clatterbridge Centre for Oncology (CCO) in Bebington (UK). After exposure, the variation in optical density of the films was measured using a CCD100 Microdensitometer (source at 665 nm). Holes of calibrated diameter, made during the assembly phase of the phantom, are identified by the MIRA software, used for data analysis, and allow the rendering of the films. The surface dose distributions were obtained from the variation in optical density of each of the films making up the phantom. Their elaboration to duplicate their position within the phantom, performed by 3D-doctor software, allows the volumetric reconstruction of the dose distribution.     

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.     

Theoretical investigation of the photon efficiency in frequency-domain fluorescence lifetime imaging microscopy

We investigate the photon efficiency of frequency-domain fluorescence lifetime imaging microscopy, using both theoretical and Monte Carlo methods. Our analysis differs from previous work in that it incorporates the data fitting process used in real experiments, allows for the arbitrary choice of excitation and gain waveforms, and calculates lifetimes as well as associated F-values from higher harmonics in the data. Using our analysis, we found different photon efficiencies to those previously reported and were able to propose optimal excitation and gain waveforms. Additionally, we suggest measurement protocols that lead to further improvement in photon efficiency. We compare our results to other techniques for lifetime imaging and consider the implications of our higher-harmonic analysis for multi-exponential lifetime determination.     

3D reconstruction of concave surfaces using polarisation imaging

This paper presents a novel algorithm for improved shape recovery using polarisation-based photometric stereo. The majority of previous research using photometric stereo involves 3D reconstruction using both the diffuse and specular components of light; however, this paper suggests the use of the specular component only as it is the only form of light that comes directly off the surface without subsurface scattering or interreflections. Experiments were carried out on both real and synthetic surfaces. Real images were obtained using a polarisation-based photometric stereo device while synthetic images were generated using PovRay® software. The results clearly demonstrate that the proposed method can extract three-dimensional (3D) surface information effectively even for concave surfaces with complex texture and surface reflectance.     

Time-resolved fluorescence imaging of slab gels for lifetime base-calling in DNA sequencing applications

A compact time-resolved near-IR fluorescence imager was constructed to obtain lifetime and intensity images of DNA sequencing slab gels. The scanner consisted of a microscope body with f/1.2 relay optics onto which was mounted a pulsed diode laser (repetition rate 80 MHz, lasing wavelength 680 nm, average power 5 mW), filtering optics, and a large photoactive area (diameter 500 microns) single-photon avalanche diode that was actively quenched to provide a large dynamic operating range. The time-resolved data were processed using electronics configured in a conventional time-correlated single-photon-counting format with all of the counting hardware situated on a PC card resident on the computer bus. The microscope head produced a timing response of 450 ps (fwhm) in a scanning mode, allowing the measurement of subnano-second lifetimes. The time-resolved microscope head was placed in an automated DNA sequencer and translated across a 21-cm-wide gel plate in approximately 6 s (scan rate 3.5 cm/s) with an accumulation time per pixel of 10 ms. The sampling frequency was 0.17 Hz (duty cycle 0.0017), sufficient to prevent signal aliasing during the electrophoresis separation. Software (written in Visual Basic) allowed acquisition of both the intensity image and lifetime analysis of DNA bands migrating through the gel in real time. Using a dual-labeling (IRD700 and Cy5.5 labeling dyes)/two-lane sequencing strategy, we successfully read 670 bases of a control M13mp18 ssDNA template using lifetime identification. Comparison of the reconstructed sequence with the known sequence of the phage indicated the number of miscalls was only 2, producing an error rate of approximately 0.3% (identification accuracy 99.7%). The lifetimes were calculated using maximum likelihood estimators and allowed on-line determinations with high precision, even when short integration times were used to construct the decay profiles. Comparison of the lifetime base calling to a single-dye/four-lane sequencing strategy indicated similar results in terms of miscalls, but reduced insertion and deletion errors using lifetime identification methods, improving the overall read accuracy.     

3D mapping of microfluidic flow in laboratory-on-a-chip structures using optical tweezers

Accurate measurement of flow in microfluidic systems is both challenging and important, providing information that can be used to better understand flow fields within laboratory-on-a-chip devices and validate computational simulations. Here, we use optical tweezers within a microfluidic system to measure the velocity vectors of flow fields in two and three dimensions around a microstructures including both molded features within channels and cells. The experimental results are compared to a complex fluid dynamics model showing an agreement between the two of better than 3 microm/s. This measurement is highly reproducible and minimally invasive, which in the future could be used to provided more in-depth studies of the rheological properties of biological cells and microstructures in laboratory-on-a-chip devices.     

Quantitative evaluation on 3D fetus morphology via X-ray grating based imaging technique

Synchrotron-based X-ray phase sensitive micro-tomography techniques enable to visualize detailed three-dimensional (3D) insight into nondestructive inner-structure of biomedical samples. Different phase sensitive mechanisms have been employed for discrimination of tissue's tiny density variations in biomedical research. We effectively visualized and analyzed the phase-contrast experimental results of X-ray grating-based imaging, based on grating interferometry with phase stepping, by using transgenic mouse fetus. We quantitatively measured and evaluated the contrast-to-noise ratio or the mass density resolution, spatial resolution, radiation dose, and figure of merit of X-ray grating-based imaging technique in biomedical research respectively. Moreover, the complex coherent degrees of light source were duly taken into account in the analysis of spatial resolution. In addition, the mass density distribution of soft biomedical specimens can be estimated using our presented method preliminarily. For most soft tissue and organ observation, this work provides explicit guidelines to help future synchrotron users obtain the quantitative image information, suitable for their specific biomedical research.     

Label-free detection of single protein molecules using deep UV fluorescence lifetime microscopy

We present the detection of single beta-galactosidase molecules from Escherichia coli (Ecbeta Gal) using deep UV laser-based fluorescence lifetime microscopy. The native fluorescence from intrinsic tryptophan emission has been observed after one-photon excitation at 266 nm. Applying the time-resolved single-photon counting method, we investigated the fluorescence lifetime distribution and the bursts of autofluorescence photons from tryptophan residues in Ecbeta Gal protein as well as fluorescence correlation spectroscopy of Ecbeta Gal. The results demonstrate that deep UV laser-based fluorescence lifetime microscopy is useful for identification of biological macromolecules at the single-molecule level using intrinsic fluorescence.     

3D strain mapping in rocks using digital volumetric speckle photography technique

Speckle photography technique is a versatile displacement/deformation mapping technique that can be applied to almost any material. It has its genesis in the laser speckle interferometry technique whereby the natural speckles created by illuminating an optically rough surface using a coherent laser beam are used as displacement gauging elements. It evolves into the white light speckle photography technique whereby a random pattern of any type is used as a displacement measurement transducer. With the advent of digital cameras and ubiquitous usage of computers, the digital version of the technique is developed. Up to now, the technique is essentially limited to 2D applications. Recently, we extended the technique into the 3D domain by using the volumetric imaging capabilities of CT or MRI. In this paper, we apply this technique to measuring the internal deformation of rocks. It so happens that certain rocks have natural density variation at various places such that when imaged by CT these impurities can be treated as 3D speckles. The elements of volumetric speckle photography technique are as follows. A reference volumetric image of the rock is recorded by a micro-CT scan and stored as a reference. Under load, the deformed CT image of the rock is also recorded. Both volume images are divided into subsets of certain voxel arrays. Each corresponding pair of the subsets is “compared” via a two-step 3D Fourier transform analysis. The result is a 3D map of displacement vectors representing the collective displacement experienced by all the speckles within the subset of voxels. The strain distribution of the entire rock specimen can then be calculated using appropriate displacement strain relations. The application of this technique to strain mapping of red sandstone and argillite rocks is presented.     

Study of miscible and immiscible flows in a microchannel using magnetic resonance imaging

Magnetic resonance imaging (MRI) is a noninvasive technique that can be used to visualize mixing processes in optically opaque systems in up to three dimensions. Here, MRI has been used for the first time to obtain both cross-sectional velocity and concentration maps of flow through an optically opaque Y-shaped microfluidic sensor. Images of 23 micromx23 microm resolution were obtained for a channel of rectangular cross section (250 micromx500 microm) fed by two square inlets (250 micromx250 microm). Both miscible and immiscible liquid systems have been studied. These include a system in which the coupling of flow and mass transfer has been observed, as the diffusion of the analyte perturbs local hydrodynamics. MRI has been shown to be a versatile tool for the study of mixing processes in a microfluidic system via the multidimensional spatial resolution of flow and mass transfer.