Excitation-and-collection geometry insensitive fluorescence imaging of tissue-simulating turbid media

We present an imaging technique for the correction of geometrical effects in fluorescence measurement of optically thick, turbid media such as human tissue. Specifically, we use the cross-polarization method to reject specular reflection and enhance the diffusive backscattering of polarized fluorescence excitation light from the turbid media. We correct the nonuniformity of the image field caused by the excitation-and-collection geometry of a fluorescence imaging system by normalizing the fluorescence image to the cross-polarized reflection image. The ratio image provides a map of relative fluorescence yield, defined as the ratio of emerging fluorescence power to incident excitation, over the surface of an imaged homogeneous turbid medium when fluorescence excitation-and-collection geometries vary in a wide range. We investigate the mechanism of ratio imaging by using Monte Carlo modeling. Our findings show that this technique could have a potential use in the detection of early cancer, which usually starts from a superficial layer of tissue, based on the contrast in the tissue fluorescence of an early lesion and of the surrounding normal tissue.     

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.     

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.     

Optical cross-sectional imaging with pulse ultrasound wave assistance.

We have developed an optical cross-sectional imaging method for turbid media with the aid of a pulse ultrasound wave. Observation of deep regions in turbid media, such as tissue samples, is difficult owing to the rapid dispersion of an incoming laser beam by scattering. A pulse ultrasound wave, which is less scattered in tissues, can indicate the measuring point on the basis of the change of the optical scattering properties in a localized region. A depth-resolving capability can be achieved from the time-dependent measurement of the scattered-light intensity as the pulse ultrasound wave propagates in the sample. We verified the method by observing absorptive objects embedded in silicone rubber and by obtaining the cross-sectional image of an absorbing object surrounded by a strong scattering medium.     

Determination of quantum efficiency in fluorescing turbid media

A method is proposed to estimate the optical parameters in a fluorescing turbid medium with strong absorption for which traditional Kubelka-Munk theory is not applicable, using a model for the radiative properties of optically thick fluorescent turbid media of finite thickness proposed in 2009 [J. Opt. Soc. Am. A26, 1896 (2009)]. The method is successfully applied to uncoated papers with different thicknesses. It is found that the quantum efficiency of fluorescent whitening agents (FWAs) is nearly independent of the fiber type, FWA type, FWA concentration, and filler additive concentration used in this study. The results enable an estimation of the model parameters as function of the FWA concentration and substrate composition. This is necessary in order to use the model for optimizing fluorescence in the paper and textile industries.     

Fluorescence spectroscopy of tissue: recovery of intrinsic fluorescence from measured fluorescence

We present a method for recovering the intrinsic fluorescence coefficient, defined as the product of the fluorophore absorption coefficient and the fluorescence energy yield, of an optically thick, homogeneous, turbid medium from a surface measurement of fluorescence and from knowledge of medium optical properties. The measured fluorescence signal is related to the intrinsic fluorescence coefficient by an optical property dependent path-length factor. A simple expression was developed for the path-length factor, which characterizes the penetration of excitation light and the escape of fluorescence from the medium. Experiments with fluorescent tissue phantoms demonstrated that intrinsic fluorescence line shape could be recovered and that fluorophore concentration could be estimated within ±15%, over a wide range of optical properties.     

Deconvolution-based spatial resolution in optical diffusion tomography

The role that deconvolution plays in the achievable spatial resolution in optical diffusion tomography is examined for the case of imaging an inhomogeneity in an otherwise homogeneous medium. It is shown that, in the measured data, it is the shape of the turbid medium modulation transfer function that determines the maximum spatial resolution. When the turbid medium transfer function is deconvolved from the measured data, it is the signal-to-noise ratio properties of the Fourier-transformed measured data that determine the maximum spatial resolution. It is shown that deconvolution-based methods can improve the spatial resolution in measured data up to a factor of 5 for realistic noise levels. These results are demonstrated with computer-simulated data.     

Optoacoustic tomography: time-gated measurement of pressure distributions and image reconstruction

Optoacoustic imaging is a potential novel medical imaging technology to image structures in turbid media to depths of several millimeters with a resolution of some tens of micrometers. Thereby short laser pulses generate thermoelastic pressure waves inside a tissue, which are detected on the surface with a wideband ultrasonic transducer. Image reconstruction has the goal of calculating the distribution of the absorbing structures in the tissue. We present a method in which the acoustic field distribution is captured as a two-dimensional snapshot at the sample surface, using an optical-reflectance-based detection principle with a detection resolution of 20 mum. A new image reconstruction is accomplished by backprojection of the detected two-dimensional pressure distributions into the sample volume by use of the delay between the laser pulse and the time the snapshot was taken. Two-dimensional pressure-wave distribution and image reconstruction are demonstrated by simulations and experiments, in which small objects are irradiated with laser pulses of 6-ns duration. The method opens the possibility to irradiate the sample hidden in a light-scattering medium directly through the detector plane, thus enabling front-surface detection of the optoacoustic signals, which is especially important if structures close to the tissue surface have to be imaged. Reconstructed tomography images with a depth resolution of 20 mum and a lateral resolution of 200 mum are presented.     

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.     

Nanoscale Ultrasound‐Switchable FRET‐Based Liposomes for Near‐Infrared Fluorescence Imaging in Optically Turbid Media

A new approach for fluorescence imaging in optically turbid media centered on the use of nanoscale ultrasound‐switchable FRET‐based liposome contrast agents is reported. Liposomes containing lipophilic carbocyanine dyes as FRET pairs with emission wavelengths located in the near‐infrared window are prepared. The efficacy of FRET and self‐quenching for liposomes with a range of fluorophore concentrations is first calculated from measurement of the liposome emission spectra. Exposure of the liposomes to ultrasound results in changes in the detected fluorescent signal, the nature of which depends on the fluorophores used, detection wavelength, and the fluorophore concentration. Line scanning of a tube containing the contrast agents with 1 mm inner diameter buried at a depth of 1 cm in a heavily scattering tissue phantom demonstrates an improvement in image spatial resolution by a factor of 6.3 as compared with images obtained in the absence of ultrasound. Improvements are also seen in image contrast with the highest obtained being 9% for a liposome system containing FRET pairs. Overall the results obtained provide evidence of the potential the nanoscale ultrasound‐switchable FRET‐based liposomes studied here have for in vivo fluorescence imaging.     

Continuous-wave terahertz phase imaging using a far-infrared laser interferometer

Terahertz phase imaging can reveal the depth information of an optically opaque object and provide much better contrast for weak-absorption materials. We demonstrate a continuous-wave terahertz interferometric imaging method in which a far-infrared laser interferometer is used to measure the phase distribution with diffraction-limited lateral resolution and subwavelength axial resolution. An improved four-step phase-shifting algorithm is introduced to retrieve the phase map with very high accuracy and low distortion. The relative depth profiles of two transparent samples are successfully extracted by using this method. Experimental results verify that terahertz interferometric imaging in combination with the phase-shifting technique enables effective reconstruction of the phase image of the object under test.     

Improvement of image quality in diffuse optical tomography by use of full time-resolved data

In the field of diffuse optical tomography (DOT), it is widely accepted that time-resolved (TR) measurement can provide the richest information on photon migration in a turbid medium, such as biological tissue. However, the currently available image reconstruction algorithms for TR DOT are based mostly on the cw component or some featured data types of original temporal profiles, which are related to the solution of a time-independent diffusion equation. Although this methodology can greatly simplify the reconstruction process, it suffers from low spatial resolution and poor quantitativeness owing to the limitation of effectively applicable data types. To improve image quality, it has been argued that exploiting the full TR data is essential. We propose implementation of a DOT algorithm by using full TR data and furthermore a variant algorithm with time slices of TR data to alleviate the computational complexity and enhance noise robustness. Compared with those algorithms where the featured data types are used, our evaluations on the spatial resolution and quantitativeness show that a significant improvement in imaging quality can be achieved when full TR data are used, which convinces the DOT community of the potential advantage of the TR domain over cw and frequency domains.     

Influence of size parameter and refractive index of the scatterer on polarization-gated optical imaging through turbid media

The influence of incident polarized light, refractive index, and size parameter of the scatterer on achievable resolution and contrast (image quality) of polarization-gated transillumination imaging in turbid media is reported here. Differential polarization detection led to significant improvement of image quality of an object embedded in a medium of small-sized scatterers (diameter Dor=lambda,g>or=0.7), the improvement in image quality was less pronounced using either linear or circular polarization gating when the refractive index of the scatterer was high (ns=1.59), but for a lower value of refractive index (ns=1.37), image quality improved with the differential circular polarization gating. We offer a plausible explanation for these observations.     

Full-color imaging through a turbid medium by use of photorefractive coherence gating and a technique to separate the recording spaces

We demonstrate full-color imaging through a turbid medium by use of photorefractive coherence gating and a technique to separate the recording space of each color from those of the other colors. We found that the recording spaces must be separate when a multicolor image is recorded in a photorefractive crystal to prevent the interference of the holograms with one another. For full-color imaging we used a He-Cd white-light laser, which is compact and useful for full-color holography. Full-color-image retrieval is demonstrated through five mean free paths of a turbid medium.     

Depth profiling in diffusely scattering media using Raman spectroscopy and picosecond Kerr gating

We demonstrate how pulsed laser Raman excitation (approximately 1 ps) followed by fast optical Kerr gating (approximately 4 ps) can be used to effectively separate Raman signals originating from different depths in heterogeneous diffusely scattering media. The diffuse scattering slows down photon propagation through turbid samples enabling higher depth resolution than would be obtained for a given instrumental time resolution in an optically transparent medium. Two types of experiments on two-layer systems demonstrate the ability to differentiate between surface and sub-surface Raman signals. A Raman spectrum was obtained of stilbene powder buried beneath a 1 mm over-layer of PMMA (poly(methyl methacrylate)) powder. The signal contrasts of the lower stilbene layer and upper PMMA layer were improved by factors >or=5 and >or=180, respectively, by rejecting the Raman component of the counterpart layer. The ability to select the Raman signal of a thin top surface layer in preference to those from an underlying diffusely scattering substrate was demonstrated using a 100 mum thick optically transparent film of PET (poly(ethylene terephthalate)) on top of stilbene powder. The gating resulted in the suppression of the underlying stilbene Raman signal by a factor of 1200. The experiments were performed in back-scattering geometry using 400 nm excitation wavelength. The experimental technique should be well suited to biomedical applications such as disease diagnosis.     

Optical tomographic image reconstruction from ultrafast time-sliced transmission measurements

Optical imaging and localization of objects inside a highly scattering medium, such as a tumor in the breast, is a challenging problem with many practical applications. Conventional imaging methods generally provide only two-dimensional (2-D) images of limited spatial resolution with little diagnostic ability. Here we present an inversion algorithm that uses time-resolved transillumination measurements in the form of a sequence of picosecond-duration intensity patterns of transmitted ultrashort light pulses to reconstruct three-dimensional (3-D) images of an absorbing object located inside a slab of a highly scattering medium. The experimental arrangement used a 3-mm-diameter collimated beam of 800-nm, 150-fs, 1-kHz repetition rate light pulses from a Ti:sapphire laser and amplifier system to illuminate one side of the slab sample. An ultrafast gated intensified camera system that provides a minimum FWHM gate width of 80 ps recorded the 2-D intensity patterns of the light transmitted through the opposite side of the slab. The gate position was varied in steps of 100 ps over a 5-ns range to obtain a sequence of 2-D transmitted light intensity patterns of both less-scattered and multiple-scattered light for image reconstruction. The inversion algorithm is based on the diffusion approximation of the radiative transfer theory for photon transport in a turbid medium. It uses a Green s function perturbative approach under the Rytov approximation and combines a 2-D matrix inversion with a one-dimensional Fourier-transform inversion to achieve speedy 3-D image reconstruction. In addition to the lateral position, the method provides information about the axial position of the object as well, whereas the 2-D reconstruction methods yield only lateral position.     

Autocorrelation of scattered laser light for ultrasound-modulated optical tomography in dense turbid media

Based on measurement of the intensity autocorrelation function, a new method to determine the modulation depth of scattered laser light modulated by an ultrasonic wave in turbid media was applied to ultrasound-modulated optical tomography. Good signal-to-noise ratios and high sensitivities were demonstrated. Images of double optically absorbing objects buried in a highly optically scattering gel sample were obtained. The contrast was more than 10%, and the spatial resolution was approximately 2 mm.     

Spatial distribution of single-photon and two-photon fluorescence light in scattering media: Monte Carlo simulation

Three-dimensional fluorescence spatial distributions under single-photon and two-photon excitation within a turbid medium are studied with Monte Carlo simulation. It is demonstrated that two-photon excitation has an advantage of producing much less fluorescence light outside the focal region compared with single-photon excitation. With the increase of the concentration of scattering particles in a turbid medium, the position of the maximum fluorescence intensity point shifts from the geometric focal region toward the medium surface. Further studies show that the optical sectioning property of two-photon fluorescence microscopy is degraded in thick turbid media or when the numerical aperture of an objective becomes low.     

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.     

Time-resolved optoacoustic measurement of absorption of light by inhomogeneous media

A method for measuring the absorption of light with optically turbid media is considered. The method is based on the registration of the temporal shape (leading-edge slope) of the developing optoacoustic signal in a medium that is absorbing a short laser pulse. Results of experiments with a Nd:YAG laser (10 ns) demonstrate the effectiveness of the method for both homogeneous and inhomogeneous optical media.