Two-dimensional imaging of dense tissue-simulating turbid media by use of sonoluminescence

An optical imaging technique that is believed to be novel was developed for noninvasive cross-sectional imaging of tissuelike turbid media. By use of a sonoluminescence signal generated internally in the media with a 1-MHz continuous-wave ultrasound, two-dimensional images were produced for objects embedded in turbid media by a raster scan of the media. Multiple objects of different shapes were resolved with this imaging technique. The images showed a high contrast and good spatial resolution. The spatial resolution was limited by the focal size of the ultrasonic focus.     

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.     

Influence of the emission-reception geometry in laser-induced fluorescence spectra from turbid media

Routine clinical detection of precancerous lesions by laser-inducedautofluorescence was recently demonstrated in several medicalfields. This technique is based on the analysis of complex spectrawith overlapping broad structures. However, in biological tissues, scattering and absorption are wavelength dependent, and the observedfluorescence signals are distorted when the illumination and detectiongeometry varies, making comparison of results from different groupsdifficult. We study this phenomenon experimentally in human tissuein a simple experiment: A fiber is used for the excitation and anidentical fiber is used for reception of the signal; both fibers aremaintained in contact with the tissue. We study the distortion ofthe spectra as a function of the distance between the twofibers. For correction of the spectra we show that it is possibleto use a fast and accurate ab initio Monte Carlo simulationwhen the spectral variations of the optical properties of the mediumare known. The main advantage of this simulation is itsapplicability even for complex boundary conditions or when the sampleconsists of several layers.     

Ultrasound-modulated optical tomography of absorbing objects buried in dense tissue-simulating turbid media

Continuous-wave ultrasonic modulation of scattered laser light was used to image objects buried in tissue-simulating turbid media. The buried object had an absorption coefficient greater than the background turbid medium. The ultrasonic wave that was focused into the turbid media modulated the laser light that passed through the ultrasonic field. The modulated laser light that was collected by a photomultiplier tube reflected the local mechanical and optical properties in the zone of ultrasonic modulation. Objects buried in the middle plane of 5-cm-thick dense turbid media were imaged with millimeter resolution through the scanning and detecting alterations of the ultrasound-modulated optical signal. The optical properties of the dense turbid media included an absorption coefficient of 0.1 cm(-1) and a reduced scattering coefficient of 10 cm(-1) and were comparable with those of biological tissues in the visible and near-IR ranges. The dependence of the ultrasound-modulated optical signal on the off-axis distance of the detector from the optic axis and the area of the detector was studied as well.     

Three-dimensional imaging of objects embedded in turbid media with fluorescence and Raman spectroscopy

We present a single-ended technique for three-dimensional imaging of objects embedded in a turbid medium by the use of time-resolved fluorescence emission or Raman scattering. The technique uses the earliest arriving photons, which we show are not sensitive to the relatively long fluorescence lifetime, and thus can be used to extract the desired spatial information accurately, even at a distance equivalent to 100 mean free paths. The results also demonstrate the feasibility and the potential of one's combining time-resolved optical tomography with fluorescence or Raman spectroscopy to localize and identify the embedded objects. This technique may be valuable for the diagnosis of disease in highly scattering human tissue because it can provide spatial and biochemical information about the composition of embedded lesions.     

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.     

Quantification of fluorophore concentration in tissue-simulating media by fluorescence measurements with a single optical fiber

Quantifying fluorescent compounds in turbid media such as tissue is made difficult by the effects of multiple scattering and absorption of the excitation and emission light. The approach that we used was to measure fluorescence using a single 200-microm optical fiber as both the illumination source and the detector. Fluorescence of aluminum phthalocyanine tetrasulfonate (AlPcS4) was measured over a wide range of fluorophore concentrations and optical properties in tissue-simulating phantoms. A root-mean-square accuracy of 10.6% in AlPcS4 concentration was attainable when fluorescence was measured either interstitially or at the phantom surface. The individual effects of scattering, absorption, and the scattering phase function on the fluorescence signal were also studied by experiments and Monte Carlo simulations.     

Ensemble-averaged Shack-Hartmann wave-front sensing for imaging through turbid media

A technique is described for ensemble-averaging the light wave emerging from a turbid medium, enabling the recovery of optical information that is otherwise lost in a speckle pattern. The technique recovers both an amplitude and a phase function for a wave that has been corrupted by severe scattering, without the use of holography. With the phase estimated, an ensemble-averaged field is constructed that can be backprojected to form an image of the object obscured by the scattering medium. Experimental results suggest that the technique can resolve two object points whose signals are unresolved on the exit surface of a diffuser.     

Two-dimensional Kerr-Fourier imaging of translucent phantoms in thick turbid media

Translucent scattering phantoms hidden inside a 5.5-cm-thick Intralipid solution were imaged as a function of phantom scattering coefficients by the use of a picosecond time- and space-gated Kerr-Fourier imaging system. A 2-mm-thick translucent phantom with a 0.1% concentration (scattering coefficient) difference from the 55-mm-thick surrounding scattering host can be distinguished at a signal level of ~10(-10) of the incidence illumination intensity.     

Confocal fluorescence polarization microscopy in turbid media: effects of scattering-induced depolarization

We present an experimental and theoretical study of confocal fluorescence polarization microscopy in turbid media. We have performed an experimental study using a fluorophore-embedded polymer rod immersed in aqueous suspensions of 0.1 and 0.5 microm diameter polystyrene microspheres. A Monte Carlo approach to simulate confocal fluorescence polarization imaging in scattering media is also presented. It incorporates a detailed model of polarized fluorescence generation that includes sampling of elliptical polarization, excited-state molecular rotational Brownian motion, and dipole fluorescence emission. Using both approaches, we determine the effects of the number of scattering events, target depth, photon scattering statistics, objective numerical aperture, and pinhole size on confocal anisotropy imaging. From this detailed analysis and comparison of experiment with simulation, we determine that fluorescence polarization is maintained to depths at which meaningful intensity images can be acquired.     

Adjoint time domain method for fluorescent imaging in turbid media

Application of adjoint time domain methods to the inverse problem in 3D fluorescence imaging is a novel approach. We demonstrate the feasibility of this approach experimentally on the basis of a time gating technique completely in the time domain by using a small number of time windows. The evolution of the fluorescence energy density function inside a highly scattering cylinder was reconstructed together with optical parameters. Reconstructed energy density was used in localizing two fluorescent tubes. Relatively accurate reconstruction demonstrates the effectiveness and the potential of the proposed technique.     

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..     

Polarization-degree imaging contrast in turbid media: a quantitative study

Scattering in biological tissue can degrade imaging contrast and reduce the probe depth. Polarization-based measurement has shown its advantages in overcoming such drawbacks. Here, linear and circular polarization degree imaging is applied to a comblike metal target submerged in Intralipid solutions. Contrasts of the metal bars are measured quantitatively as functions of the Intralipid concentration and the submersion depths. Different behaviors in contrast for linear and circular polarizations are compared. Contributions to the background of circular polarization degree images by backscattering, snake, and diffusive photons are examined carefully.     

Monte Carlo modeling of optical coherence tomography imaging through turbid media

We combine a Monte Carlo technique with Mie theory to develop a method for simulating optical coherence tomography (OCT) imaging through homogeneous turbid media. In our model the propagating light is represented by a plane wavelet; its line propagation direction and path length in the turbid medium are determined by the Monte Carlo technique, and the process of scattering by small particles is computed according to Mie theory. Incorporated into the model is the numerical phase function obtained with Mie theory. The effect of phase function on simulation is also illustrated. Based on this improved Monte Carlo technique, OCT imaging is directly simulated and phase information is recorded. Speckles, resolution, and coherence gating are discussed. The simulation results show that axial and transversal resolutions decrease as probing depth increases. Adapting a light source with a low coherence improves the resolution. The selection of an appropriate coherence length involves a trade-off between intensity and resolution.     

Intrinsic fluorescence spectroscopy in turbid media: disentangling effects of scattering and absorption

The fluorescence from a turbid medium such as biologic tissue contains information about scattering and absorption, as well as the intrinsic fluorescence, i.e., the fluorescence from an optically thin sample of pure fluorophores. The interplay of scattering and absorption can result in severe distortion of the intrinsic spectral features. These distortions can be removed by use of a photon-migration-based picture and information from simultaneously acquired fluorescence and reflectance spectra. We present experimental evidence demonstrating the validity of such an approach for extracting the intrinsic fluorescence for a wide range of scatterer and absorber concentrations in tissue models, ex vivo and in vivo tissues. We show that variations in line shape and intensity in intrinsic tissue fluorescence are significantly reduced compared with the corresponding measured fluorescence.     

Experimental and simulated angular profiles of fluorescence and diffuse reflectance emission from turbid media

Given the wavelength dependence of sample optical properties and the selective sampling of surface emission angles by noncontact imaging systems, differences in angular profiles due to excitation angle and optical properties can distort relative emission intensities acquired at different wavelengths. To investigate this potentiality, angular profiles of diffuse reflectance and fluorescence emission from turbid media were evaluated experimentally and by Monte Carlo simulation for a range of incident excitation angles and sample optical properties. For emission collected within the limits of a semi-infinite excitation region, normalized angular emission profiles are symmetric, roughly Lambertian, and only weakly dependent on sample optical properties for fluorescence at all excitation angles and for diffuse reflectance at small excitation angles relative to the surface normal. Fluorescence and diffuse reflectance within the emission plane orthogonal to the oblique component of the excitation also possess this symmetric form. Diffuse reflectance within the incidence plane is biased away from the excitation source for large excitation angles. The degree of bias depends on the scattering anisotropy and albedo of the sample and results from the correlation between photon directions upon entrance and emission. Given the strong dependence of the diffuse reflectance angular emission profile shape on incident excitation angle and sample optical properties, excitation and collection geometry has the potential to induce distortions within diffuse reflectance spectra unrelated to tissue characteristics.     

Technique for enhancing signal in conventional backscattering fluorescence and Raman spectroscopy of turbid media

We demonstrate experimentally, for the first time, the feasibility of passively enhancing fluorescence and Raman signals from diffusely scattering media in a conventional backscattering collection geometry. The method employs transmission of the collimated excitation laser beam through a "unidirectional" dielectric mirror placed directly in front of the sample. This permits laser light that escapes from the sample surface to be reflected back into the sample where it can be more usefully employed in generating Raman and fluorescence signals. This leads to improved Raman signal, higher signal-to-noise ratio, and shorter acquisition times. Feasibility studies performed on standard pharmaceutical tablets and on sheets of Teflon, using a single enhancing element, demonstrate signal enhancement factors of 6 (fluorescence) and 3 (Raman). Potential applications of this simple device include improving quality control of pharmaceutical products, disease diagnosis of biological tissue, forensics, and security screening.     

Effects of turbid media optical properties on object visibility in subsurface polarization imaging

We studied the effectiveness of using polarized illumination and detection to enhance the visibility of targets buried in highly scattering media. The effects of background optical properties including scattering coefficient, absorption coefficient, and anisotropy on image visibility were examined. Both linearly and circularly polarized light were used in the imaging. Three different types of target were investigated: scattering, absorption, and reflection. The experimental results indicate that target visibility improvement achieved by a specific polarization method depends on both the background optical properties and the target type. By analyzing all polarization images, it is possible to reveal certain information about target or the scattering background.     

Statistical detection and imaging of objects hidden in turbid media using ballistic photons

We exploit recent advances in active high-resolution imaging through scattering media with ballistic photons. We derive the fundamental limits on the accuracy of the estimated parameters of a mathematical model that describes such an imaging scenario and compare the performance of ballistic and conventional imaging systems. This model is later used to derive optimal single-pixel statistical tests for detecting objects hidden in turbid media. To improve the detection rate of the aforementioned single-pixel detectors, we develop a multiscale algorithm based on the generalized likelihood ratio test framework. Moreover, considering the effect of diffraction, we derive a lower bound on the achievable spatial resolution of the proposed imaging systems. Furthermore, we present the first experimental ballistic scanner that directly takes advantage of novel adaptive sampling and reconstruction techniques.