Fluorescence-lifetime-based tomography for turbid media

We derive a novel algorithm to recover the in vivo distributions of fluorophores based on an asymptotic life-time analysis of time-domain fluorescence measurements with turbid tissue. We experimentally demonstrate the advantage offered by this method in localizing fluorophores with distinct lifetimes. This algorithm has wide applicability for diagnostic fluorescence imaging in the presence of several-centimeter-thick biological tissue, since fluorescence lifetime is a sensitive indicator of local tissue environment and interactions at the molecular level.     

Beyond nanosizing: an approach to shape analysis of fluorescent nanostructures by SMI-microscopy

Using spatially modulated illumination (SMI) light microscopy it is possible to measure the sizes of fluorescent structures that have an extension far below the conventional optical resolution limit (“subresolution size”). Presently, the sizes are determined as the object extension along the optical axis of the SMI microscope. For this, however, “a priori” assumptions on the fluorochrome distribution (“shape”) within the examined fluorescent structure have to be made. Usually it is assumed that the fluorochrome follows a Gauss-distribution or a spherical distribution. In this report we overcome the necessity to make an assumption on the shape of the fluorochrome distribution. We introduce two new experimentally obtained parameters which allow the determination of a shape measure to describe the spatial distribution of the fluorescent dye. This becomes possible by independent measurements with different excitation wavelengths. As an example, we present shape parameter measurements on individual fluorescent microspheres with a nominal geometrical diameter (“size”) of 190 nm. In the case investigated, the experimental shape correlated well with a homogeneous fluorochrome distribution (“spherical shape”) but not with a variety of other “shapes”.     

Would near-infrared fluorescence signals propagate through large human organs for clinical studies?

We predict the capacity of near-infrared fluorescent signals to propagate through human tissue for non-invasive medical imaging. This analysis employs experimental measurements of a biologically relevant local fluorochrome embedded in tissuelike media and predicts the equivalent photon counts expected from breast, lung, brain, and muscle as a function of diameter by use of an analytical solution of the diffusion equation that can take into account large arbitrary geometries. The findings address feasibility issues for clinical studies and are relevant to recent development of near-infrared fluorescent probes and molecular beacons for in vivo applications.     

Quantitative differentiation of dyes with overlapping one-photon spectra by femtosecond pulse-shaping

We demonstrate that DiI and rhodamine B, which are not easily distinguishable to one-photon measurements, can be differentiated and in fact quantified in mixture via tailored two-photon excitation pulses found by a genetic algorithm (GA). A nearly three-fold difference in the ratio of two-photon fluorescence of the two dyes is achieved, without a drop in signal of the favored fluorophore. Implementing an acousto-optic interferometer, we were able to prove that the mechanism of discrimination is second-harmonic tuning by the phase-shaped pulses to the relative maxima and minima of these cross-sections.     

Time-resolved multi-channel optical system for assessment of brain oxygenation and perfusion by monitoring of diffuse reflectance and fluorescence

Time-resolved near-infrared spectroscopy is an optical technique which can be applied in tissue oxygenation assessment. In the last decade this method is extensively tested as a potential clinical tool for noninvasive human brain function monitoring and imaging. In the present paper we show construction of an instrument which allows for: (i) estimation of changes in brain tissue oxygenation using two-wavelength spectroscopy approach and (ii) brain perfusion assessment with the use of single-wavelength reflectometry or fluorescence measurements combined with ICG-bolus tracking. A signal processing algorithm based on statistical moments of measured distributions of times of flight of photons is implemented. This data analysis method allows for separation of signals originating from extra- and intracerebral tissue compartments. In this paper we present compact and easily reconfigurable system which can be applied in different types of time-resolved experiments: two-wavelength measurements at 687 and 832 nm, single wavelength reflectance measurements at 760 nm (which is at maximum of ICG absorption spectrum) or fluorescence measurements with excitation at 760 nm. Details of the instrument construction and results of its technical tests are shown. Furthermore, results of in-vivo measurements obtained for various modes of operation of the system are presented.     

Fluorescence tomography with simulated data based on the equation of radiative transfer

The quantification of a nonuniform quantum yield or fluorophore absorption distribution is of major interest in molecular imaging of biological tissue. We introduce what is believed to be the first fluorescence image reconstruction algorithm based on the equation of radiative transfer that recovers the spatial distribution of light-emitting fluorophores inside a highly scattering medium from measurements made on the surface of the medium. We obtain images of either the quantum yield or the fluorophore absorption.     

Polar Plot Representation for Frequency-Domain Analysis of Fluorescence Lifetimes

We present applications of polar plots for analyzing fluorescence lifetime data acquired in the frequency domain. This graphical, analytical method is especially useful for rapid FLIM measurements. The usual method for sorting out and determining the underlying lifetime components from a complex fluorescence signal is to carry out the measurement at multiple frequencies. When it is not possible to measure at more than one frequency, such as rapid lifetime imaging, specific features of the polar plot analysis yield valuable information, and provide a diagnostic visualization of the participating fluorescent species underlying a complex lifetime distributions. Data are presented where this polar plot presentation is useful to derive valuable, unique information about the underlying component distributions. We also discuss artifacts of photolysis and how this method can also be applied to samples where each fluorescence species shows a continuous distribution of lifetimes. Polar plots of frequency-domain data are commonly used for analysis of dielectric relaxation experiments (Cole–Cole plots), which have proved to be exceptionally useful in that field for decades. We compare this analytical tool that is well developed and extensively used in dielectric relaxation and chemical kinetics to fluorescence measurements.     

Fast, flexible algorithm for calculating photon correlations

We introduce a new algorithm for computing correlations of photon arrival time data acquired in single-molecule fluorescence spectroscopy and fluorescence correlation spectroscopy (FCS). The algorithm is based on rewriting the correlation as a counting operation on photon pairs and can be used with arbitrary bin widths and spacing. The flexibility of the algorithm is demonstrated by use of FCS simulations and single-molecule photon antibunching experiments. Execution speed is comparable to the commonly used multiple-tau correlation technique. Wide bin spacings are possible that allow for real-time software calculation of correlations, even for high count rates.     

Plane-wave fluorescence tomography with adaptive finite elements

We present three-dimensional fluorescence yield tomography of a tissue phantom in a noncontact reflectance imaging setup. The method employs planar illumination with modulated light and frequency domain fluorescence measurements made on the illumination plane. An adaptive finite-element algorithm is used to handle the ill-posed and computationally demanding inverse image reconstruction problem. Tomographic images of fluorescent targets buried at 1-2 cm depths from the illumination surface demonstrate the feasibility of fluorescence tomography from reflectance tomography in clinically relevant tissue volumes.     

Fluorescence spectroscopy of synthetic melanin in solution

We report a detailed investigation of fluorescence properties of synthetic eumelanin pigment in solution. A complete set of fluorescence spectra in the near-UV and visible range is analysed. Excitation spectra at a few selected emission energies are also investigated. Our measurements support the hypothesis that fluorescence in eumelanin is related to chemically distinct oligomeric units that can be selectively excited. Fluorescence due to large oligomer systems is spectrally differentiated from that due to monomers and small oligomer systems. Fluorescence excitation measurements show the contribution of 5,6-dihydroxyndole-2-carboxylic acid and 5,6-dihydroxyndole monomers to the emission of small-size oligomers.     

Photon-counting technique for rapid fluorescence-decay measurement

We report on a novel laser-induced fluorescence triple-integration method (LIFTIME) that is capable of making rapid, continuous fluorescence lifetime measurements by a unique photon-counting technique. The LIFTIME has been convolved with picosecond time-resolved laser-induced fluorescence, which employs a high-repetition-rate mode-locked laser, permitting the eventual monitoring of instantaneous species concentrations in turbulent flames. We verify the technique by application of the LIFTIME to two known fluorescence media, diphenyloxazole (PPO) and quinine sulfate monohydrate (QSM). PPO has a fluorescence lifetime of 1.28 ns, whereas QSM has a fluorescence lifetime that can be varied from 1.0 to 3.0 ns. From these liquid samples we demonstrate that fluorescence lifetime can currently be monitored at a sampling rate of up to 500 Hz with less than 10% uncertainty (1sigma) .     

The Calibration Kit Spectral Fluorescence Standards—A Simple and Certified Tool for the Standardization of the Spectral Characteristics of Fluorescence Instruments

With the Calibration Kit Spectral Fluorescence Standards BAM-F001-BAM-F005, we developed a simple tool for the characterization of the relative spectral responsivity and the long-term stability of the emission channel of fluorescence instruments under routine measurement conditions thereby providing the basis for an improved comparability of fluorescence measurements and eventually standardization. This first set of traceable fluorescence standards, which links fluorescence measurements to the spectral radiance scale in the spectral range of 300-770 nm and has been optimized for spectrofluorometers, can be employed for different measurement geometries and can be adapted to different fluorescence techniques with proper consideration of the underlying measurement principles.     

Advantages of fluorescence over diffuse reflectance measurements tested in phantom experiments with dynamic inflow of ICG

Time-resolved measurements of diffuse reflectance and fluorescence were carried out using phantom with dynamic inflow of indocyanine green (ICG) in tubes located at different depths. Better sensitivity of fluorescence signals related to the inflow of the dye was observed in comparison to simultaneously acquired diffuse reflectance. Obtained results can be referred to results of in-vivo measurements. We have observed much larger amplitude of changes in relative number of detected photons, mean time of flight and variance of the distributions of times of arrival of fluorescence photons than amplitudes of respective parameters measured from diffuse reflectance distributions of times of flight of photons. The constructed phantom allows us to study influence of concentration of the dye in the tube and the surrounding medium as well as temporal relation between appearance of the boli in deeper and superficial tube. Results of the study were used in optimization of the time-resolved multichannel system for simultaneous monitoring of fluorescence and reflectance.     

Frequency-domain fluorescence lifetime imaging for endoscopic clinical cancer photodetection: Apparatus design and preliminary results

We describe a new fluorescence imaging device for clinical cancer photodetection in hollow organs in which the tumor/normal tissue contrast is derived from the fluorescence lifetime of endogenous or exogenous fluorochromes. This fluorescence lifetime contrast gives information about the physicochemical properties of the environment which are different between normal and certain diseased tissues. The excitation light from a CW laser is modulated in amplitude at a radio frequency by an electrooptical modulator and delivered by an optical fiber through an endoscope to the hollow organ. The image of the tissue collected by the endoscope is separated in two spectral windows, one being the backscattered excitation light and the other the fluorescence of the fluorochrome. Each image is then focused on the photocathode of image intensifiers (II) whose optical gain is modulated at the same frequency as the excitation intensity, resulting in homodyne phase-sensitive images. By acquiring stationary phase-sensitive frames at different phases between the excitation and the detection, it is possible to calculate in quasi-real time the apparent fluorescence lifetime of the corresponding tissue region for each pixel. A result obtained by investigating the endogenous fluorochromes present in the mucous membrane of an excised human bladder is presented to illustrate this method and most of the optical parameters which are of major importance for this photodetection modality have been evaluated.     

Depth-resolved fluorescence measurement in a layered turbid medium by polarized fluorescence spectroscopy

We show that, when a turbid medium with a layered fluorophore distribution is excited by linearly polarized light, measurement of angle-resolved polarized fluorescence can provide depth-resolved fluorescence measurements.     

Spatial photoselection of single molecules on the surface of spherical microcavities

We show that ultrasensitive microdroplet-stream fluorescence techniques combined with surfactant forms of Rhodamine dyes can be used to probe single molecules on the surfaces of spherical microcavities. Individual octadecyl Rhodamine B molecules, shown previously by ensemble measurements to be localized and oriented at the surfaces of liquid microspheres, were spatially photoselected primarily along great circles lying perpendicular or parallel to the detection axis by use of polarized laser excitation. A polarization dependence is observed in the distribution of single-molecule fluorescence amplitudes that can be interpreted qualitatively in terms of position-dependent fluorescence-collection efficiencies.     

Temperature measurements in sooting counterflow diffusion flames using laser-induced fluorescence of flame-produced nitric oxide

Laser-based diagnostic methods are often used for non-intrusive studies of delicate processes of soot formation. When soot particles are heated by the laser pulse, their size distribution can be estimated from the cooling rate, provided that the local gas temperature is known. However, strong light absorption, scattering and fluorescence in sooting environment hinder non-intrusive laser-based temperature measurements. Methods based on fitting of laser-induced fluorescence spectra work well in stationary flames but usually require temperature tracer seeded into the flame. We have shown that in counterflow diffusion flames, often used for soot-formation studies, enough nitric oxide is produced for two-dimensional temperature imaging. Measured temperature profiles agree very well with chemical kinetic calculations for a variety of fuels if laser intensity is reduced to keep NO excitation in the linear regime. Gas composition affects line shapes at temperatures below 600 K and should be taken into account for accurate measurements.     

Statistical aging and nonergodicity in the fluorescence of single nanocrystals

The relation between single particle and ensemble measurements is addressed for semiconductor CdSe nanocrystals. We record their fluorescence at the single molecule level and analyze their emission intermittency, which is governed by unusual random processes known as Lévy statistics. We report the observation of statistical aging and ergodicity breaking, both related to the occurrence of Lévy statistics. Our results show that the behavior of ensemble quantities, such as the total fluorescence of an ensemble of nanocrystals, can differ from the time-averaged individual quantities, and must be interpreted with care.     

Focusing astigmatic Gaussian beams through optical systems with a high numerical aperture

We theoretically derive the electric field distribution of an astigmatic Gaussian laser beam after it is focused through a high-aperture objective. We show that astigmatism values that are hard to detect in the collimated laser beam can have a large effect after diffraction-limited focusing. Such astigmatic beams may be frequently encountered in fluorescence correlation measurements and in laser-scanning confocal microscopy. We present experimental measurements of the excitation intensity distribution measured by 3D scanning of single fluorescent molecules immobilized on a glass surface.     

Resonance fluorescence from a coherently driven semiconductor quantum dot in a cavity

We show that resonance fluorescence, i.e., the resonant emission of a coherently driven two-level system, can be realized with a semiconductor quantum dot. The dot is embedded in a planar optical microcavity and excited in a waveguide mode so as to discriminate its emission from residual laser scattering. The transition from the weak to the strong excitation regime is characterized by the emergence of oscillations in the first-order correlation function of the fluorescence, g(tau), as measured by interferometry. The measurements correspond to a Mollow triplet with a Rabi splitting of up to 13.3 microeV. Second-order correlation measurements further confirm nonclassical light emission.