Monte carlo analysis of two-photon fluorescence imaging through a scattering medium

The behavior of two-photon fluorescence imaging through a scattering medium is analyzed by use of the Monte Carlo technique. The axial and transverse distributions of the excitation photons in the focused Gaussian beam are derived for both isotropic and anisotropic scatterers at different numerical apertures and at various ratios of the scattering depth with the mean free path. The two-photon fluorescence profiles of the sample are determined from the square of the normalized excitation intensity distributions. For the same lens aperture and scattering medium, two-photon fluorescence imaging offers a sharper and less aberrated axial response than that of single-photon confocal fluorescence imaging. The contrast in the corresponding transverse fluorescence profile is also significantly higher. Also presented are results comparing the effects of isotropic and anisotropic scattering media in confocal reflection imaging. The convergence properties of the Monte Carlo simulation are also discussed.     

Three-dimensional imaging analysis of confocal and conventional polarization microscopes by use of Mie scattering theory

We present a three-dimensional imaging analysis of confocal and conventional polarization microscopes by using the extended Mie scattering theory. In the analysis, we calculate the images of a Mie particle whose diameter is comparable with the wavelength of confocal and conventional microscopes. It was found that, when we observe a Mie particle, polarization confocal microscopy is not affected by the polarization distortion that is due to focusing with high-numerical-aperture lenses and does not produce pseudopeaks in the images in comparison with conventional polarization microscopy. The three-dimensional resolution of the polarization microscope and the verification of the proposed analysis method are also discussed.     

Near-infrared laser tomographic imaging with right-angled scattered coherent light using an optical heterodyne-detection-based confocal scanning system

We demonstrate, what is to the best of our knowledge, a novel optical tomographic method for the visualization of the inner structure of scattering media such as biological tissue in the near-infrared region. We constructed a scanning confocal imaging system with a cross-axes arrangement using optical fibers. This system is based on the optical heterodyne technique and enables the detection of very weak coherence photons that are generated in the spatially restricted confocal region and scattered laterally (90 degrees ) against an incident beam. To evaluate the fundamental imaging capabilities of the system, we assessed measurements from scattering phantoms composed of an Intralipid suspension with varying volume concentrations. The results of this study demonstrate that the right-angled scattered light adheres to the Lambert-Beer law and that the present system can detect light propagating through a distance of approximately 31l of the mean free path. An inclusion as small as 100 microm can be discriminated in a scattering media with an optical thickness of 4. We investigated the potential of the proposed system for imaging biological tissues in preliminary experiments using samples of chicken breast tissue.     

Polarization-rich continuous wave direct imaging: modeling and visualization

We report a study and comparison of continuous-wave, optical polarization difference imaging (PDI) and polarization modulation imaging (PMI) for imaging through scattering media. The problem is cast in the framework of a theoretical estimation, and the comparison is based on three visualization parameters, namely, the magnitude, the degree, and the orientation of the polarization. We show that PDI is superior in estimating the first two parameters in active imaging under specific conditions, while the PMI is suitable for passive imaging and is the only way to estimate polarization orientation. We also propose new schemes for rendering polarization information as a color image and for applying the newly introduced polarization-orientation imaging for segmentation. Simulation and experimental results verify the theoretical conclusions.     

Two-color excitation fluorescence microscopy through highly scattering media

We study the performance of two-color excitation (2CE) fluorescence microscopy [Opt. Lett. 24, 1505 (1999)] in turbid media of different densities and anisotropy. Excitation is achieved with two confocal excitation beams of wavelengths lambda(1) and lambda(2), which are separated by an angular displacement theta, where lambda(1) not equal lambda(2), 1/lambda(e) = 1/lambda(1) + 1/lambda(2), and lambda(e) is the single-photon excitation wavelength of the sample. 2CE fluorescence is generated only in regions of the sample where the two excitation beams overlap. The 2CE fluorescence intensity is proportional to the product of the two excitation intensities and could be detected with a large-area photodetector. The requirement of spatiotemporal simultaneity for the two excitation beams makes 2CE fluorescence imaging a promising tool for observing microscopic objects in a highly scattering medium. Optical scattering asymmetrically broadens the excitation point-spread function and toward the side of the focusing lens that leads to the contrast deterioration of the fluorescence image in single- or two-photon (lambda(1) = lambda(2)) excitation. Image degradation is caused by the decrease in the excitation energy density at the geometrical focus and by the increase in background fluorescence from the out-of-focus planes. In a beam configuration with theta not equal 0, 2CE fluorescence imaging is robust against the deleterious effects of scattering on the excitation-beam distribution. Scattering only decreases the available energy density at the geometrical focus and does not increase the background noise. For both isotropic and anisotropic scattering media the performance of 2CE imaging is studied with a Monte Carlo simulation for theta = 0, pi/2, and pi, and at different h/d(s) values where h is the scattering depth and d(s) is the mean-free path of the scattering medium.     

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.     

混浊介质荧光显微成像的层析能力研究

采用蒙特卡罗方法给出了混浊介质显微成像中,共焦荧光、双光子激发荧光的空间分布。该分布表明双光子激发荧光显微成像具有内存的轴向层析能力,而共焦荧光成像的轴向层析能力依赖于共焦针孔的采用。进一步的研究则表明共焦针孔对双光子激发显微成像也同样具有提高层析能力的特点。     

Three-dimensional imaging of microspheres with confocal and conventional polarization microscopes

We experimentally studied the three-dimensional imaging of the microspheres by using confocal and conventional scanning polarization microscopes. Because of the field amplitude averaging effect of the confocal system, the polarization of the detected signals is mainly parallel to the initial polarization. As a result, the signal intensity from the microspheres in the confocal polarization microscope with a crossed analyzer was found to be weaker than that in the conventional system. Based on a vector approach that takes the polarization into account and on the image formations of the two systems, theoretical expressions are given that agree well with the experimental results.     

Adaptive algorithms for two-channel polarization sensing under various polarization statistics with nonuniform distributions

The polarization of light carries much useful information about the environment. Biological studies have shown that some animal species use polarization information for navigation and other purposes. It has been previously shown that a bioinspired polarization-difference imaging (PDI) technique can facilitate detection and feature extraction of targets in scattering media. It has also been established [J. Opt. Soc. Am. A 15, 359 (1998)] that polarization sum and polarization difference are the optimum pair of linear combinations of images taken through two orthogonally oriented linear polarizers of a scene having a uniform distribution of polarization directions. However, in many real environments the scene has a nonuniform distribution of polarization directions. Using principal component analysis of the polarization statistics of the scene, we develop a method to determine the two optimum information channels with unequal weighting coefficients that can be formed as linear combinations of the images of a scene taken through a pair of linear polarizers not constrained to the horizontal and vertical directions of the scene. We determine the optimal orientations of linear polarization filters that enhance separation of a target from the background, where the target is defined as an area with distinct polarization characteristics as compared to the background. Experimental results confirm that in most situations adaptive PDI outperforms conventional PDI with fixed channels.     

Theory for confocal and conventional microscopes imaging small dielectric scatterers

Abstract

In this paper we develop the theory of confocal microscopes imaging small scatterers. Since scattering is a polarization dependent phenomenon we employ a full vectorial theory to treat this problem. This approach permits us to consider both imaging in high aperture systems as well as image formation in polarized light microscopy. We extend previous theories by including effects of the finite sized detector apertures. Numerical examples are presented for the most important cases. The results of the full vectorial theory are compared with those obtained from low aperture paraxial theory.     

Fluorescence lifetime optical tomography in weakly scattering media in the presence of highly scattering inclusions

We consider the problem of fluorescence lifetime optical tomographic imaging in a weakly scattering medium in the presence of highly scattering inclusions. We suggest an approximation to the radiative transfer equation, which results from the assumption that the transport coefficient of the scattering media differs by an order of magnitude for weakly and highly scattering regions. The image reconstruction algorithm is based on the variational framework and employs angularly selective intensity measurements. We present numerical simulation of light scattering in a weakly scattering medium that embeds highly scattering objects. Our reconstruction algorithm is verified by recovering optical and fluorescent parameters from numerically simulated datasets.     

Discrimination of globally unpolarized fields through Stokes vector element correlations

In a previous publication [J. Opt. Soc. Am. A 21, 988 (2004)], we examined theoretically joint probability distributions of Stokes vector elements and suggested the existence of various types of globally unpolarized light that could be discriminated through measurement of the Stokes vector element correlations. We now study the joint distribution of the degree of polarization and the three Stokes parameters as it relates to material properties in highly scattering, depolarizing random media. We describe numerical and experimental results of second-order Stokes vector element correlations, demonstrating the existence of various types of nonclassical, globally unpolarized light, and we suggest experimental means for discriminating between such field distributions. We also discuss the usefulness of the Stokes vector element correlations as an experimental tool for discriminating between different globally unpolarized fields and for verifying the assumption of Gaussian statistics usually invoked in the context of multiple light scattering.     

Polarization asymmetry in waves backscattering from highly absorbant random media

Within the range in which light penetration depth is approximately the same as or less than the diameter of the particles in the medium, particulate media with considerable absorption behave as two-dimensional, rough-surface structures. As penetration depth increases, a complicated transition between volume and surface effects is seen. For these media, low-order scattering sequences have small spatial extent, making observation of polarization characteristics difficult. We present an experimental technique to access the low-order scattered photons by artificially reinjecting them through total internal reflections. Using a dielectric layer in contact with the high-absorption medium, we are able to observe fourfold polarization asymmetry in backscattering from highly absorbant media. We discuss the origin of the polarization patterns in a ray-optics approximation and suggest possibilities for solving practical problems encountered in characterizing composites with appreciable absorption.     

Circularly symmetric light scattering from nanoplasmonic spirals

In this paper, we combine experimental dark-field imaging, scattering, and fluorescence spectroscopy with rigorous electrodynamics calculations in order to investigate light scattering from planar arrays of Au nanoparticles arranged in aperiodic spirals with diffuse, circularly symmetric Fourier space. In particular, by studying the three main types of Vogel's spirals fabricated by electron-beam lithography on quartz substrates, we demonstrate polarization-insensitive planar light diffraction in the visible spectral range. Moreover, by combining dark-field imaging with analytical multiparticle calculations in the framework of the generalized Mie theory, we show that plasmonic spirals support distinctive structural resonances with circular symmetry carrying orbital angular momentum. The engineering of light scattering phenomena in deterministic structures with circular Fourier space provides a novel strategy for the realization of optical devices that fully leverage on enhanced, polarization-insensitive light-matter coupling over planar surfaces, such as thin-film plasmonic solar cells, plasmonic polarization devices, and optical biosensors.     

Improved restoration from multiple images of a single object: application to fluorescence microscopy

We present an approach for the combined restoration of multiple different images of a single object. A linear Tikhonov filter adapted for this purpose is derived in detail. Nonlinear constrained algorithms can also be adapted, and we illustrate this possibility for an iterative constrained Tikhonov algorithm. Both the linear and the iterative constrained Tikhonov algorithms were used to analyze performance in fluorescence confocal imaging by use of simulated and experimental data. One can improve the quality of restored confocal images significantly if the signal that normally is rejected by the detection pinhole of a confocal laser scanning microscope is also recorded on a separate detector such that the two recorded signals are used together for image restoration according to the proposed algorithms.     

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.     

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.     

Fluorescence anisotropy based single liposome assay to measure molecule-membrane interactions

Nanometer-scaled liposomes are used frequently for research, therapeutic, and analytical applications as carriers for water-soluble molecules. Recent technical advances allow the monitoring of single liposomes, which provides information on heterogeneous properties that were otherwise hidden due to ensemble averaging. Recent observations demonstrated that the efficiency of entrapping water-soluble molecules increases with decreasing vesicle size. The molecular mechanism behind this observation is not clear, but enhanced molecule-membrane interactions due to the increase of the surface area-to-volume ratio could play an important role. To investigate this hypothesis, we extended our single liposome assay based on confocal fluorescence imaging by implementation of fluorescence anisotropy. This combination has not been widely exploited, and confocal fluorescence anisotropy imaging in particular has seldom been used. We investigated different small dye molecules and were able to determine if these molecules interact or not with the liposome membrane. We confirm the liposome size-dependent entrapment of molecules whereas the molecule-membrane interactions appear to be independent of liposome size. Our fluorescence anisotropy assay can be used as a general method to investigate molecule-membrane interactions or molecule-molecule interactions in a high-throughput manner in nanometer-scaled containers like liposomes.     

Enhanced 3D fluorescence live cell imaging on nanoplasmonic substrate

We have created a randomly distributed nanocone substrate on silicon coated with silver for surface-plasmon-enhanced fluorescence detection and 3D cell imaging. Optical characterization of the nanocone substrate showed it can support several plasmonic modes (in the 300-800 nm wavelength range) that can be coupled to a fluorophore on the surface of the substrate, which gives rise to the enhanced fluorescence. Spectral analysis suggests that a nanocone substrate can create more excitons and shorter lifetime in the model fluorophore Rhodamine 6G (R6G) due to plasmon resonance energy transfer from the nanocone substrate to the nearby fluorophore. We observed three-dimensional fluorescence enhancement on our substrate shown from the confocal fluorescence imaging of chinese hamster ovary (CHO) cells grown on the substrate. The fluorescence intensity from the fluorophores bound on the cell membrane was amplified more than 100-fold as compared to that on a glass substrate. We believe that strong scattering within the nanostructured area coupled with random scattering inside the cell resulted in the observed three-dimensional enhancement in fluorescence with higher photostability on the substrate surface.     

Precision and accuracy of the analog mean-delay method for high-speed fluorescence lifetime measurement

The analog mean-delay (AMD) method is a new alternative method to measure the lifetime of a fluorescence molecule. Because of its powerful advantages of accurate lifetime determination, good photon economy, and a high photon detection rate, the AMD method is considered to be very suitable for high-speed confocal fluorescence lifetime imaging microscopy (FLIM). For the practical usage of the AMD method in FLIM (AMD-FLIM), detailed study on various experimental conditions and parameters that affect the precision and the accuracy of the AMD method is required. In this paper, we present the relation between the precision and accuracy of the lifetime versus iteration number in the AMD method, the best cutoff frequency of a low-pass filter used in the AMD-FLIM system for a given fluorophore, and the optimum position and width of the integration window by using Monte Carlo simulations and a series of AMD-FLIM experiments.