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..     

Resolution improvement in two-photon fluorescence microscopy with a single-mode fiber

The dependence of spectral broadening of an ultrashort-pulsed laser beam on the fiber length and the illumination power is experimentally characterized in order to deliver the laser for two-photon fluorescence microscopy. It is found that not only the spectral width but also the spectral blue shift increases with the fiber length and illumination power, owing to the nonlinear response in the fiber. For an illumination power of 400 mW in a 3-m-long single-mode fiber, the spectral blue shift is as large as 15 nm. Such a spectral blue shift enhances the contribution from the short-wavelength components within the pulsed beam and leads to an improvement in resolution under two-photon excitation, whereas the efficiency of two-photon excitation is slightly reduced because of the temporal broadening of the pulsed beam. The experimental measurement of the axial response to a two-photon fluorescence polymer block confirms this feature.     

Simultaneous two-photon spectral and lifetime fluorescence microscopy

When a fluorescence photon is emitted from a molecule within a living cell it carries a signature that can potentially identify the molecule and provide information on the microenvironment in which it resides, thereby providing insights into the physiology of the cell. To unambiguously identify fluorescent probes and monitor their physiological environment within living specimens by their fluorescent signatures, one must exploit as much of this information as possible. We describe the development and implementation of a combined two-photon spectral and lifetime microscope. Fluorescence lifetime images from 16 individual wavelength components of the emission spectrum can be acquired with 10-nm resolution on a pixel-by-pixel basis. The instrument provides a unique visualization of cellular structures and processes through spectrally and temporally resolved information and may ultimately find applications in live cell and tissue imaging.     

三苯胺-PAMAM树枝分子的单／双光子荧光增强

合成了新的发光分子-4-N,N-二苯基氛基苯甲酸琉角酸亚胺(简称TBA-SCM ),利用其琉拍跳亚胺基与PAMAM所含的氛基之间高度反应活性,将三苯胺甲统基组装键合在树枝分子PAMAM (0～3G)表面,构筑新的树枝分子TBA-PAMAM.研究表明,在稀溶液(10-6)下未观测到TBA-PAMAM的荧光增强现象;当浓度...     

Three-dimensional optical-transfer-function analysis of fiber-optical two-photon fluorescence microscopy

The three-dimensional optical transfer function is derived for analyzing the imaging performance in fiber-optical two-photon fluorescence microscopy. Two types of fiber-optical geometry are considered: The first involves a single-mode fiber for delivering a laser beam for illumination, and the second is based on the use of a single-mode fiber coupler for both illumination delivery and signal collection. It is found that in the former case the transverse and axial cutoff spatial frequencies of the three-dimensional optical transfer function are the same as those in conventional two-photon fluorescence microscopy without the use of a pinhole.However, the transverse and axial cutoff spatial frequencies in the latter case are 1.7 times as large as those in the former case. Accordingly, this feature leads to an enhanced optical sectioning effect when a fiber coupler is used, which is consistent with our recent experimental observation.     

Adaptively controlled supercontinuum pulse from a microstructure fiber for two-photon excited fluorescence microscopy

Selective fluorescence excitation of specific molecular species is demonstrated by using coherent control of two-photon excitation with supercontinuum pulses generated with a microstructure fiber. Pulse shaping prior to pulse propagation through the fiber is controlled by a self-learning optimization loop so that the highest fluorescence signal contrast between two fluorescent proteins is obtainable. The self-learning optimization loop successfully controls both the optical nonlinarity of the microstructure fiber and the two-photon excitation of the fluorescent proteins.     

Analytical solutions for time-resolved fluorescence lifetime imaging in a turbid medium such as tissue.

An analytical solution is developed to quantify a site-specific fluorophore lifetime perturbation that occurs, for example, when the local metabolic status is different from that of surrounding tissue. This solution may be used when fluorophores are distributed throughout a highly turbid media and the site of interest is embedded many mean scattering distances from the source and the detector. The perturbation in lifetime is differentiated from photon transit delays by random walk theory. This analytical solution requires a priori knowledge of the tissue-scattering and absorption properties at the excitation and emission wavelengths that may be obtained from concurrent time-resolved reflection measurements. Additionally, the solution has been compared with the exact, numerically solved solution. Thus the presented solution forms the basis for practical lifetime imaging in turbid media such as tissue.     

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.     

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.     

New modal wave-front sensor: application to adaptive confocal fluorescence microscopy and two-photon excitation fluorescence microscopy

Confocal and multiphoton microscopes are particularly sensitive to specimen- or system-induced aberrations, which result in decreased resolution and signal-to-noise ratio. The inclusion of an adaptive optics correction system could help overcome this limitation and restore diffraction-limited performance, but such a system requires a suitable method of wave-front measurement. By extending the concept of a modal wave-front sensor previously described by Neil et al. [J. Opt. Soc. Am. A 17, 1098-1107 (2000)], we present a new sensor capable of measuring directly the Zernike aberration modes introduced by a specimen. This modal sensor is particularly suited to applications in three-dimensional microscopy because of its inherent axial selectivity; only those wave fronts originating in the focal region contribute to the measured signal. Four wave-front sensor configurations are presented and their input response is characterized. Sensitivity matrices and axial responses are presented.     

Gold nanoparticle-mediated fluorescence enhancement by two-photon polymerized 3D microstructures

Fluorescence enhancement achieved by functionalized microstructures made by two-photon polymerization (TPP) is reported for the first time. Microstructures of various shapes made of SU-8 photoresist were prepared and coated with gold nanoparticles (NP) of 80 nm. Localized fluorescence enhancement was demonstrated by microstructures equipped with tips of sub-micron dimensions. The enhancement was realized by positioning the NP-coated structures over fluorescent protein layers. Two fluorophores with their absorption in the red and in the green region of the VIS spectrum were used. Laser scanning confocal microscopy was used to quantify the enhancement. The enhancement factor was as high as 6 in areas of several square-micrometers and more than 3 in the case of local enhancement, comparable with literature values for similar nanoparticles. The structured pattern of the observed fluorescence intensity indicates a classic enhancement mechanism realized by standing waves over reflecting surfaces. With further development mobile microtools made by TPP and functionalized by metal NPs can be actuated by optical tweezers and position to any fluorescent micro-object, such as single cells to realize localized, targeted fluorescence enhancement.     

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.     

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.     

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.     

High-density optical data storage with one-photon and two-photon near-field fluorescence microscopy

A spatially localized photochemical reaction induced by near-field femtosecond laser pulses is demonstrated on a nanometer scale and used for high-density optical data storage. Recorded domains down to 120 and 70 nm are obtained with one-photon and two-photon excitation, respectively. It is shown that the local-field confinement that is due to the quadratic dependence of two-photon excitation on light intensity has the potential to increase the near-field optical storage density.     

Spatial coherence of forward-scattered light in a turbid medium

We study spatially coherent forward-scattered light propagating in a turbid medium of moderate optical depth (0-9 mean free paths). Coherent detection was achieved by using a tilted heterodyne geometry, which desensitizes coherent detection of the attenuated incident light. We show that the degree of spatial coherence is significantly higher for light scattered only once in comparison with that for multiply scattered light and that it approaches a small constant value for large numbers of scattering events.     

Epifluorescence collection in two-photon microscopy

We present a simple model to describe epifluorescence collection in two-photon microscopy when one images in a turbid slab with an objective. Bulk and surface scattering determine the spatial and angular distributions of the outgoing fluorescence photons at the slab surface, and geometrical optics determines how efficiently the photons are collected. The collection optics are parameterized by the objective's numerical aperture and working distance and by an effective collection field of view. We identify the roles of each of these parameters and provide simple rules of thumb for the optimization of the epifluorescence collection efficiency. Analytical results are corroborated by Monte Carlo simulation.     

Green functions for diffuse light in a medium comprising two turbid half-spaces

A review of Green functions for diffuse light in two semi-infinite scattering and absorbing half-spaces separated by a plane interface is presented. The frequency-domain Green functions for an intensity-modulated point source are derived within the diffusion approximation by the Hankel transform with respect to the variable in the plane of the interface. Green functions for a line source and a plane source parallel to the interface are obtained from the three-dimensional Green functions by the method of descent. Green functions for a steady state are obtained as a limit of zero modulation frequency. Connection of the frequency-domain Green functions with the time-domain Green functions is shown by use of the Fourier transform in time. The influence of the relative optical parameters, namely, the ratios of diffusion coefficients, absorption coefficients, and refractive indices of the two media on the shape of the contour lines of the specific intensity, is shown for the continuous and intensity-modulated point sources.     

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.     

Vertex/propagator model for least-scattered photons traversing a turbid medium

The least-scattered photons that arrive at a detector through highly scattering tissues have the potential to image internal structures, functions, and status with high imaging resolution. In contrast, optical diffusing tomography is based on the use of the late-arriving photons, which have been diffusely scattered, leading to very low imaging resolution. A good model of the early-arriving photons, i.e., the least-scattered photons, may have a significant effect on the development of imaging algorithms and a further understanding of imaging mechanisms within current high-resolution optical-imaging techniques. We describe a vertex/propagator approach that attempts to find the probabilities for least-scattered photons traversing a scattering medium, based on analytical expressions for photon histories. The basic mathematical derivations for the model are outlined, and the results are discussed and found to be in very good agreement with those from the Monte Carlo simulations.