We propose a straightforward implementation of two-photon image scanning microscopy (2PE-ISM) that, by leveraging our recently introduced single-photon avalanche diode (SPAD) array detector and a novel blind image reconstruction algorithm is shown to dramatically improve the optical resolution of two-photon imaging, in various test samples. We show how our computational ISM approach is able to adapt to changing imaging conditions, thus ensuring optimal image quality. We also show how our recently introduced blind deconvolution approaches can be integrated into the image reconstruction workflow to further improve the image quality.
Worldwide, outdoor air pollution is responsible for 4.2 million premature deaths per year. Both chronic and acute exposure to particulate matter air pollution is a risk factor for heart and lung diseases. One of the atmospheric pollutant particles is represented by soot or carbonaceous particles (CPs), which are produced during the incomplete combustion of fuels. To evaluate human CP exposure, a direct and label-free approach for detecting such particles in body fluids and tissues was still lacking. We present a novel technique to finally close the diagnostic gap. We report for the first time white-light generation by CPs under femtosecond pulsed near-infrared light illumination in aqueous environments and demonstrate the potential of this approach in biomedical and diagnostic context. In fact, it was shown that urinary carbon loading can serve as an exposure matrix to carbon-based air pollution, reflecting the passage of soot particles from circulation into urine. The novel method is straightforward, fast and flexible without the need of sample pretreatment. Moreover, the technique offers several other advantages such as inherent 3D sectioning and high imaging depths making it possible to screen at the cellular and tissue level. In conclusion, this novel diagnostic technique allows to quantify exposure at the personal level including different scenarios like occupational exposure, smog, forest fires, etc.. Additionally, this approach paves the way to unravel the complexity of soot-related health effects.
The adverse health effects of particulate matter exposure are a generally accepted concern. Dramatic statistical figures suggest that fine dust is a main environmental risk in Europe and can be held accountable for hundreds of thousands of deaths per year [1]. Locating and tracking these nanometer sized particles, however, is not straight forward: In epidemiological and toxicology research only measurements based on labels [2] such as radionuclide markers have been applied.
In this paper we present a direct, label-free optical contrast mechanism to detect carbon nanoparticles immersed in aqueous environments [3]. The virtue of this technique is its ability to perform in body fluids such as urine but also in cells and tissues. The mechanism is based on white light (WL) generation upon illumination with femtosecond pulsed near-infrared and is therefore non-incandescence related. We demonstrate the technique in various biological settings with dry and suspended carbon black particles (CB), a widely used model compound for soot [4]. Our approach allows for the unequivocal localization of CB alongside of common fluorophores and markers and can be performed on multiphoton laser-scanning microscopy platforms, a system commonly available in research laboratories.
[1] European Environment Agency (2015). Press release.
[2] Kong et al. Int. J. Mol. Sci. 2013, 14, (11), 22529-22543
[3] Bové and Steuwe et al. Nano letters, 2016, (16) , pages 3173-3178
[4] Arnal et al. Combust. Sci. Technol. 2012, 184, (7-8), 1191-1206.
Although structural changes on the sarcomere level of skeletal muscle are known to occur due to various pathologies, rigorous studies of the reduced sarcomere quality remain scarce. This can possibly be explained by the lack of an objective tool for analyzing and comparing sarcomere images across biological conditions. Recent developments in second harmonic generation (SHG) microscopy and increasing insight into the interpretation of sarcomere SHG intensity profiles have made SHG microscopy a valuable tool to study microstructural properties of sarcomeres. Typically, sarcomere integrity is analyzed by fitting a set of manually selected, one-dimensional SHG intensity profiles with a supramolecular SHG model. To circumvent this tedious manual selection step, we developed a fully automated image analysis procedure to map the sarcomere disorder for the entire image at once. The algorithm relies on a single-frequency wavelet-based Gabor approach and includes a newly developed normalization procedure allowing for unambiguous data interpretation. The method was validated by showing the correlation between the sarcomere disorder, quantified by the M-band size obtained from manually selected profiles, and the normalized Gabor value ranging from 0 to 1 for decreasing disorder. Finally, to elucidate the applicability of our newly developed protocol, Gabor analysis was used to study the effect of experimental autoimmune encephalomyelitis on the sarcomere regularity. We believe that the technique developed in this work holds great promise for high-throughput, unbiased, and automated image analysis to study sarcomere integrity by SHG microscopy.
Recently, a supramolecular model was developed for predicting striated skeletal muscle intensity profiles obtained by label-free second harmonic generation (SHG) microscopy. This model allows for a quantitative determination of the length of the thick filament antiparallel range or M-band (M), and results in M=0.12 μm for single-band intensity profiles when fixing the A-band length (A) to A=1.6 μm, a value originating from electron microscopy (EM) observations. Using simulations and experimental data sets, we showed that the objective numerical aperture (NA) and the refractive index (RI) mismatch (Δn=n2ω−nω) between the illumination wave (ω) and the second harmonic wave (2ω) severely affect the simulated sarcomere intensity profiles. Therefore, our recovered filament lengths did not match with those observed by EM. For an RI mismatch of Δn=0.02 and a moderate illumination NA of 0.8, analysis of single-band SHG intensity profiles with freely adjustable A- and M-band sizes yielded A=1.40±0.04 μm and M=0.07±0.05 μm for skeletal muscle. These lower than expected values were rationalized in terms of the myosin density distribution along the myosin thick filament axis. Our data provided new and practical insights for the application of the supramolecular model to study SHG intensity profiles in striated muscle.
A microcavity-based deoxyribonucleic acid (DNA) optical biosensor is demonstrated for the first time using synthetic sapphire for the optical cavity. Transmitted and elastic scattering intensity at 1510 nm are analyzed from a sapphire microsphere (radius 500 μm, refractive index 1.77) on an optical fiber half coupler. The 0.43 nm angular mode spacing of the resonances correlates well with the optical size of the sapphire sphere. Probe DNA consisting of a 36-mer fragment was covalently immobilized on a sapphire microsphere and hybridized with a 29-mer target DNA. Whispering gallery modes (WGMs) were monitored before the sapphire was functionalized with DNA and after it was functionalized with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). The shift in WGMs from the surface modification with DNA was measured and correlated well with the estimated thickness of the add-on DNA layer. It is shown that ssDNA is more uniformly oriented on the sapphire surface than dsDNA. In addition, it is shown that functionalization of the sapphire spherical surface with DNA does not affect the quality factor (Q≈104) of the sapphire microspheres. The use of sapphire is especially interesting because this material is chemically resilient, biocompatible, and widely used for medical implants.
KEYWORDS: Diffusion, Luminescence, Point spread functions, Particles, Data modeling, Proteins, Monte Carlo methods, Image resolution, Biomedical optics, Medical research
Fluorescence recovery after photobleaching (FRAP) is a common technique to probe mobility of fluorescently labeled proteins in biological membranes by monitoring the time-dependence of the spatially integrated fluorescence signals after a bleaching pulse. Discrimination by FRAP between free diffusion with an immobile fraction (FDIM) and the phenomenological model for anomalous diffusion based on the time-dependent diffusion coefficient (TDDC) is a challenging problem, requiring extremely long observation times for differentiation. Recently, rectangular FRAP (rFRAP) has been introduced for normal diffusion by considering not only the temporal but also spatial information, taking the effective point spread function of the optical system into account. In this work we provide an extension of rFRAP toward anomalous diffusion according to the continuous time random walk (CTRW). We explore whether the spatial information in rFRAP allows for enhanced discrimination between FDIM, TDDC, and CTRW in a single experiment within a feasible time window. Simulations indicate that rFRAP can indeed differentiate the different models by evaluating the spatial autocorrelation of the differences between the measured and fitted pixel values. Hence, rFRAP offers a tool that is capable of discriminating different types of diffusion at shorter time scales than in the case where spatial information is discarded.
Second harmonic generation microscopy has recently become an important tool for studying materials. In this article, we use a recently developed analytical method, for second-harmonic generation microscopy, to determine the point group symmetry of micro crystals of enantiomerically pure 1,1’-bi-2-naphtol.
We report our latest results on second harmonic generation (SHG) microscopy from arrays of G-shaped
chiral gold nanostructures. The nanostructures are arranged in unit cells composed of four Gs, each rotated
at 90° with respect to its neighbors. As it has already been demonstrated, for linearly polarized light, these
unit cells yield a pattern of four SHG hotspots. However, upon increasing the pitch of the nanostructured
arrays, extra hotspots can be observed at the edges of the unit cells. While the origin of these extra hotspots
remains to be elucidated, their position indicates a relationship to coupling behavior between the unit cells.
Scanning second harmonic generation (SHG) microscopy is becoming an important tool for characterizing
nanopatterned metal surfaces and mapping plasmonic local field enhancements. Here we study G-shaped
and mirror-G-shaped gold nanostructures and test the robustness of the experimental results versus the
direction of scanning, the numerical aperture of the objective, the magnification, and the size of the laser
spot on the sample. We find that none of these parameters has a significant influence on the experimental
results.
In an effort to complement cellular two-photon excited fluorescence (TPEF) microscopy with structural information
from second-harmonic generation (SHG) imaging, we investigated the applicability of fluorescent proteins for SHG
imaging. In the first stage, the first hyperpolarizability β, a measure for the second-order nonlinear optical properties of
a molecule, was determined for several fluorescent proteins. In a second stage, an established HeLa cell line expressing
a membrane protein labeled with a fluorescent protein, was adapted and imaged using simultaneous TPEF and SHG
microscopy. The contour of stretched cells observed in these experiments was proven to be originating in microtubules
instead of the fluorescent proteins.
Fluorescence recovery after photobleaching (FRAP) carried out on a confocal laser-scanning microscope (CLSM) performs well for photobleached disks that are large compared to the resolution of the bleaching beam. For smaller disks approaching this resolution, current FRAP models providing a closed-form solution do not allow one to extract the diffusion coefficient accurately. The new generalized disk model we present addresses this shortcoming by bringing into account the bleaching resolution and the total confocal imaging resolution. A closed-form solution is obtained under the assumption of linear photobleaching. Furthermore, simultaneous analysis of FRAP data collected at various disk sizes allows for the intrinsic determination of the instrumental resolution parameters, thereby obviating the need for an extrinsic calibration. A new method to estimate the variance of FRAP data is introduced to allow for proper weighting in this global analysis approach by nonlinear least squares. Experiments are performed on two independent CLSMs on homogeneous samples providing validation over a large range of diffusion coefficients.
Within the framework of the General Support Technology Program of the European Space Agency (ESA), a compact dedicated confocal laser scanning microscope has been developed for 3D fluorescence imaging of biological samples. The microscope permits normal confocal mode operation with excitation at 488nm and fluorescence lifetime imaging (FLIM) with excitation at 630nm and a time resolution of 200ps. Each fluorescence signal is detected by a dedicated photomultipier tube. Proper optical signal separation and filtering is performed by a set of optical filters and dichroics. The software and hardware further include the specific imaging modes allowing for fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). In addition to this dual wavelength fluorescence imaging mode, the microscope includes transmission imaging capabilities via differential imaging contrast (DIC). Both fluorescence and DIC imaging can be acquired simultaneously. The source for the DIC is a near infrared LED. This choice permits the decoupling of DIC and fluorescence signals by a dichroic cold mirror. The opto-mechanical assembly is constructed on the two sides of a rigid 16mm thick aluminum base plate of dimensions 389 mm by 575.5 mm. The total volume under light and dust shielding removable covers is just 53 dm3, excluding PC and control electronics. In this paper, the design, performance and limitations of this compact confocal microscope are discussed. Illustrative examples of applications on biological samples are shown.
A proper interpretation of the signals from fluorescent indicators in imaging technology implies a knowledge of the processes in the excited state. This work focuses on the pH probe C.SNAFL-1. The kinetics of the excited-state processes are investigated by global compartmental analyses of fluorescence decay surfaces obtained by time-correlated single photon counting. Within the pH range 5-12 only two species have to be considered in the ground and the excited state. The two fluorescent decay times do not depend on pH. Therefore, the process of protonation in the excited state is very slow as compared to the deactivation rate of the excited states. A proper identifiability study has been performed to determine the rate constant of the deprotonation process. The rate of deprotonation is also small and its upper value is estimated to be 0.05 ns-1. It can be concluded that there is negligible interference of the excited-state reaction on the determination of intracellular pH by C.SNAFL-1.
The fluorescent indicators Fura-2 and PBFI are widely used for the determination of intracellular concentrations of Ca2+ and K+, respectively. To investigate the complex forming reaction between Fura-2 and Ca2+, and between PBFI and K+ in the ground and excited states, steady-state and time-resolved measurements were performed. The fluorescence decay surfaces were analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution: (1) for Fura-2: k01 equals 1.2 X 109 s-1, k21 equals 1.0 X 1011 M-1x-1, k02 equals 5.5 X 108s-1, k12 equals 2.2 X 107s-1 (2) for PBFI: k01 equals 1.1 X 109s-1, k21 equals 2.7 X 108M-1s-1, k02 equals 1.8 X 109s-1, k12 equals 1.4 X 109s-1 k01 and k02 denote the deactivation rate constants of the free and bound forms of the indicator, respectively k21 represents the bimolecular rate constant of binding of the cation by the indicator whereas k12 is the rate constant of dissociation of the cation:indicator complex. For both probes the effect of the excited-state reaction can be neglected in the determination of Kd and/or the ion concentration.
This report gives evidence that the biexponential fluorescence decay of tryptophan zwitterion in H2O solution is due to the occurrence of a reversible two-state excited-state process whereby the corresponding ground-state species are excited. The rate constants are within the intervals: 0<k01<0.57(ns)-1, 0.76(ns)-1<k21<1.33(ns)-1, 0<k02<0.58(ns)-1, 0.77(ns)-1<k12<1.35(ns)-1. These limits were calculated using the values for S1 equals k01 + k21 (1.33+/- 0.01(ns)-1), S2 equals k02 + k12(1.35+/- 0.01(ns)-1), and P equals k21k12(1.03+/- 0.01(ns)-2). The emission spectra of the two excited-state species can be uniquely determined and are different from those associated with the decay times. These results were obtained by repetitive global compartmental analyses of the fluorescence decay surface of tryptophan zwitterion measured over the entire emission spectrum as a function of quencher concentration. This new and powerful analysis method is applicable to all biexponential protein fluorescence decays.
The fluorescence decay analysis of intramolecular two-state excited-state processes with added quencher is discussed in terms of compartments. The kinetic equations specifying the fluorescence decay and the time-course of the two excited-state species concentrations are expressed in terms of the rate constants and the spectroscopic parameters b1 and c1. b1 and c1 are respectively the relative absorbance and the normalized spectral emission weighting factor of species 1. The report investigates what has to be known beforehand to determine all relevant parameters. The results of this identifiability study indicate that the following conditions have to be satisfied in order to make an intramolecular two-state excited-state system with added quencher identifiable. First, at least three different quencher concentrations must be used. Second, the two rate constants of quenching must be different. Third, at least one parameter must be known. This parameter can be (1) one rate constant which is not a rate constant of quenching, (2) one b1 value or, (3) one c1 value. In each of these cases an alternative set of system parameters is mathematically possible. A unique solution is guaranteed when the fluorescence decays of a quenched model compound are included in the compartmental analysis.
fluorescence decay surfaces of excited state reactions can be globally analyzed directly in terms of reaction rates and species associated spectra [Beechem et al., Chem. Phys. Letters 120 (1985) 466]. The identifiability of two-state excited state reactions has been investigated for properly normalized decay curves assuming that the ratio of the ground state absorbances is known [Ameloot et al, Chem. Phys. Letters 129 [1986] 21 1]. It is demonstrated in this paper that the condition of proper normalization is not always required. In addition, it is shown that the ratio of the absorbances of the species in the ground state can be obtained from fluorescence decay surfaces. The required experimental design is indicated.
The performance of global (simultaneous) analysis of multiexponential fluorescence decay surfaces using reference convolution is investigated in a systematic way using simulated and experimental data sets. It is shown that the increased model discrimination ability and the more accurate parameter recovery by global analysis as compared to single curve analysis originate from combining decay traces with differing contributions of the decay components. Simultaneous analysis of decay traces in which the contributions of the components are changed as much as possible is the most beneficial. Consequently, including more decay traces in the global decay surface does not necessarily lead to a better model distinction capability. For decay surfaces collected as a function of the emission (excitation) wavelength, the decays with minimal overlap between the emission (absorption) spectra associated with the decay components will contribute the most to the improved model discrimination and parameter recovery.
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