High-resolution imaging in a deep turbid medium based on an ultrasound-switchable fluorescence technique

The spatial resolution of fluorescence imaging techniques in deep optically turbid media such as tissues is limited by photon diffusion. To break the diffusion limit and achieve high-resolution and deep-tissue fluorescence imaging, a fundamentally different method was demonstrated based on a concept of ultrasound-switchable fluorescence. The results showed that a small fluorescent tube with a diameter of ～180?μm at a depth of ～20?mm in an optical scattering medium ([Formula: see text] and [Formula: see text] cm(-1)) can be clearly imaged with a size of ～260?μm. The depth-to-resolution ratio is shown to be about one order of magnitude better than other deep-tissue fluorescence imaging techniques.     

Analytical method for localizing a fluorescent inclusion in a turbid medium

We describe a novel method for localizing a fluorescent inclusion in a homogeneous turbid medium through the use of time-resolved techniques. Based on the calculation of the mean time of the fluorescence curves, the method does not require a priori knowledge of either the fluorescence lifetime or the mean time of the instrument response function since it adopts a differential processing approach. Theoretical expressions were validated and experiments for assessing the accuracy of localization were carried out on liquid optical phantoms with a small fluorescent inclusion. The illumination and detection optical fibers were immersed in the medium to achieve infinite medium geometry as required by the model used. The experimental setup consisted of a time-correlated single-photon counting system. Submillimeter accuracy was achieved for the localization of the inclusion.     

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.     

Image contrast enhancement for two-photon fluorescence microscopy in a turbid medium

Image contrast enhancement is investigated for two-photon excitation fluorescence images of a microscopic sample that is buried underneath a turbid medium. The image contrast, which deteriorates rapidly with sample depth because of scattering loss, is enhanced by an increase in the average excitation power of the focused Gaussian (the TEM(00) mode) beam according to a compensation relation that has been derived by use of a Monte Carlo analysis of the scattering problem. A correct increase in the excitation power results in a detected fluorescence signal that remains invariant with sample depth. The scheme is demonstrated on images of DAPI-stained nuclei cells viewed underneath a suspension of 0.105-mum-diameter polystyrene spheres.     

Digital micromirror device as a spatial illuminator for fluorescence lifetime and hyperspectral imaging

Time-domain fluorescence lifetime imaging (FLIM) and hyper-spectral imaging (HSI) are two advanced microscopy techniques widely used in biological studies. Typically both FLIM and HSI are performed with either a whole-field or raster-scanning approach, which often prove to be technically complex and expensive, requiring the user to accept a compromise among precision, speed, and spatial resolution. We propose the use of a digital micromirror device (DMD) as a spatial illuminator for time-domain FLIM and HSI with a laser diode excitation source. The rather unique features of the DMD allow both random and parallel access to regions of interest (ROIs) on the sample, in a very rapid and repeatable fashion. As a consequence both spectral and lifetime images can be acquired with a precision normally associated with single-point systems but with a high degree of flexibility in their spatial construction. In addition, the DMD system offers a very efficient way of implementing a global analysis approach for FLIM, where average fluorescence decay parameters are first acquired for a ROI and then used as initial estimates in determining their spatial distribution within the ROI. Experimental results obtained on phantoms employing fluorescent dyes clearly show how the DMD method supports both spectral and temporal separation for target identification in HSI and FLIM, respectively.     

Ultrafast optical Kerr gate imaging in a poly-disperse turbid medium

The influence of the size parameter of the scatterers on ultrafast optical Kerr gate (OKG) imaging is investigated in highly scattering poly-disperse turbid media. The results show that in a poly-disperse turbid medium, which in our case, is a suspension of two different sized mono-disperse microspheres, the temporal and spatial behaviors of the light pulses transmitted through it are dominated by the smaller microspheres. The contrasts of the OKG images for the poly-disperse microsphere sample are closer to the contrasts of the OKG images for the smaller sized mono-disperse microsphere sample.     

Fast algorithm to determine optical properties of a turbid medium from time-resolved measurements

After analytical expressions for the time-resolved reflectance are introduced from the diffusion approximation under the three most commonly used boundary conditions, a novel algorithm is demonstrated for determining the reduced scattering and the absorption coefficients from time-resolved reflectance (or backscatter) measurements at two positions on the surface of biotissue. The algorithm is straightforward and fast and involves only some simple mathematical operations, avoiding complicated iterative nonlinear fitting to the time-resolved curve. The derived reduced scattering coefficient is not affected by whatever boundary condition is applied. The algorithm was verified with time-resolved data from the Monte Carlo model. Both a semi-infinite medium and a turbid slab medium were tested. In contrast to the nonlinear fitting method, this algorithm allows both the scattering and the absorption coefficients to be determined to a high accuracy.     

Closed-form expressions for obtaining the absorption and scattering coefficients of a turbid medium with time-resolved spectroscopy

A simple closed-form relation for extracting the absorption coefficient of a multiply scattering medium from a measured temporal impulse response is pointed out. Such a relation eliminates the need for time-consuming iterative curve-fitting techniques and may also be of use for calculating the regional sensitivity of the time-resolved spectroscopy technique. Equations for three types of measurement geometry (reflection from an infinite half-space, transmission through an infinite slab, and reflection from an infinite slab) are described. The effects of detector temporal resolution and detector intensity linearity are also discussed.     

Two-photon fluorescence imaging of microspheres embedded in turbid media

Abstract

Fluorescent microspheres of diameter 10μm embedded in a turbid medium consisting of polystyrene beads suspended in water are imaged under two-photon and single-photon excitation. A comparison of two-photon and single-photon fluorescence images shows that multiple scattering leads to a dominant limiting factor of the signal-to-noise ratio in the former case, while it results in a dominant limiting factor of resolution in the latter case. These results are qualitatively consistent with the predication by the Monte-Carlo simulation based on Mie scattering theory.     

Microscopic imaging through a turbid medium by use of annular objectives for angle gating

We report a new method for microscopic imaging of an object embedded in a turbid medium. The new method is based on the angle-gating mechanism achieved by the use of polarized annular objectives in the illumination and collection paths of a microscopic imaging system. A detailed experimental study is presented of the effects of the size of annular obstructions on image quality when turbid media, including polystyrene microspheres and milk suspensions, are imaged. Images of 22-mum polystyrene microspheres embedded in the turbid media show that misinterpretation can occur when circular objectives are used, because of the detection of mainly multiply scattered photons (i.e., diffusing photons). However, when annular objectives are employed, diffusing photons from a turbid medium can be efficiently suppressed; thus image contrast appears correctly, and image resolution is increased.     

Referencing techniques for the analog mean-delay method in fluorescence lifetime imaging

The analog mean-delay (AMD) method is a new powerful alternative method in determining the lifetime of a fluorescence molecule for high-speed confocal fluorescence lifetime imaging microscopy. Even though the photon economy and the lifetime precision of the AMD method are proven to be as good as those of the state-of-the-art time-correlated single photon counting method, there have been some speculations and concerns about the accuracy of this method with respect to the absolute lifetime value of a fluorescence probe. In the AMD method, the temporal waveform of an emitted fluorescence signal is directly recorded with a slow digitizer whose bandwidth is much lower than the temporal resolution of the lifetime to be measured. We have found that the drifts and the fluctuations of the absolute zero position in a measured temporal waveform are the major problems in the AMD method. We have proposed electrical and optical referencing techniques that may suppress these errors. It is shown that there may exist more than 2 ns drift in a measured temporal waveform during the period of the first 12 min after electronic components are turned on. The standard deviation of a measured lifetime after this warm-up period can be as large as 51 ps without any referencing technique. We have shown that this error can be reduced to 9 ps with our electronic referencing technique. It is demonstrated that this can be further reduced to 4 ps by the optical referencing technique we have introduced.     

Temperature-modulated fluorescence tomography in a turbid media

High scattering in biological tissues makes fluorescence tomography inverse problem very challenging in thick medium. We describe an approach termed "temperature-modulated fluorescence tomography" that can acquire fluorescence images at focused ultrasound resolution. By utilizing recently emerged temperature sensitive fluorescence contrast agents, this technique provides fluorescence images with high resolution prior to any reconstruction process. We demonstrate that this technique is well suited to resolve small fluorescence targets located several centimeters deep in tissue.     

Influence of optical properties on two-photon fluorescence imaging in turbid samples

A numerical model was developed to simulate the effects of tissue optical properties, objective numerical aperture (N.A.), and instrument performance on two-photon-excited fluorescence imaging of turbid samples. Model data are compared with measurements of fluorescent microspheres in a tissuelike scattering phantom. Our results show that the measured two-photon-excited signal decays exponentially with increasing focal depth. The overall decay constant is a function of absorption and scattering parameters at both excitation and emission wavelengths. The generation of two-photon fluorescence is shown to be independent of the scattering anisotropy, g, except for g > 0.95. The N.A. for which the maximum signal is collected varies with depth, although this effect is not seen until the focal plane is greater than two scattering mean free paths into the sample. Overall, measurements and model results indicate that resolution in two-photon microscopy is dependent solely on the ability to deliver sufficient ballistic photon density to the focal volume. As a result we show that lateral resolution in two-photon microscopy is largely unaffected by tissue optical properties in the range typically encountered in soft tissues, although the maximum imaging depth is strongly dependent on absorption and scattering coefficients, scattering anisotropy, and objective N.A..     

Three-dimensional time-resolved fluorescence imaging by multifocal multiphoton microscopy for a photosensitizer study in living cells

Two-photon fluorescence microscopy is widely applied to biology and medicine to study both the structure and dynamic processes in living cells. The main issue is the slow acquisition rate due to the point scanning approach limiting the multimodal detection (x, y, z, t). To extend the performances of this powerful technique, we present a time-resolved multifocal multiphoton microscope (MMM) based on laser amplitude splitting. An array of 8 x 8 foci is created on the sample that gives a direct insight of the fluorescence localization. Four-dimensional (4D) imaging is obtained by combining simultaneous foci scanning, time-gated detection, and z displacement. We illustrate time-resolved MMM capabilities for 4D imaging of a photosensitizer inside living colon cancer cells. The aim of this study is to have a better understanding of the photophysical processes implied in the photosensitizer reactivity.     

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.     

Multichannel time-resolved tissue oximeter for functional imaging of the brain

This paper reports the development and characterization of a novel multichannel time-resolved (TR) instrument for functional brain imaging studies. The instrument is based on picosecond diode lasers, fiber optics for light injection and delivery, a compact multianode photomultiplier, and a personal computer (PC) board for time-correlated single photon counting (TCSPC). The instrument has been characterized in terms of reproducibility among the nine sources and the 12 collection channels, linearity in the determination of optical properties (absorption and reduced scattering), and stability. Preliminary in vivo measurements were performed on volunteers to monitor the optical response to stimuli following a motor task (finger opposition, 5 Hz).     

Phosphorescence lifetime imaging in turbid media: the inverse problem and experimental image reconstruction

Three-dimensional phosphorescence lifetime imaging is a novel method for the mapping of oxygen concentration in biological tissues. We present reconstruction techniques for recovering phosphorescent objects in highly scattering media based on the telegraph equation and two regularization methods, i.e., the Tikhonov-Phillips regularization and the maximum entropy method. Theoretical results are experimentally validated, and the reconstructed images of phosphorescent objects rendering oxygen maps in a layer are presented.     

Sensitivity studies for imaging a spherical object embedded in a spherically symmetric, two-layer turbid medium with photon-density waves

We present analytic expressions for the amplitude and phase of photon-density waves in strongly scattering, spherically symmetric, two-layer media containing a spherical object. This layered structure is a crude model of multilayered tissues whose absorption and scattering coefficients lie within a range reported in the literature for most tissue types. The embedded object simulates a pathology, such as a tumor. The normal-mode-series method is employed to solve the inhomogeneous Helmholtz equation in spherical coordinates, with suitable boundary conditions. By comparing the total field at points in the outer layer at a fixed distance from the origin when the object is present and when it is absent, we evaluate the potential sensitivity of an optical imaging system to inhomogeneities in absorption and scattering. For four types of background media with different absorption and scattering properties, we determine the modulation frequency that achieves an optimal compromise between signal-detection reliability and sensitivity to the presence of an object, the minimum detectable object radius, and the smallest detectable change in the absorption and scattering coefficients for a fixed object size. Our results indicate that (l) enhanced sensitivity to the object is achieved when the outer layer is more absorbing or scattering than the inner layer; (2) sensitivity to the object increases with the modulation frequency, except when the outer layer is the more absorbing; (3) amplitude measurements are proportionally more sensitive to a change in absorption, phase measurements are proportionally more sensitive to a change in scattering, and phase measurements exhibit a much greater capacity for distinguishing an absorption perturbation from a scattering perturbation.