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.     

Reduced effects of spherical aberration on penetration depth under two-photon excitation

We compare the effects of spherical aberration on the penetration depth of single-photon and two-photon excitation for instances in which the aberration is caused by the refractive-index mismatch when a beam is focused through an interface. It is shown both theoretically and experimentally that two-photon fluorescence imaging experiences less spherical aberration and can thus propagate to a deeper depth within a thick medium.     

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.     

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.     

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.     

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..     

Excitation with a focused, pulsed optical beam in scattering media: diffraction effects

To gain a better understanding of the spatiotemporal problems that are encountered in two-photon excitation fluorescence imaging through highly scattering media, we investigate how diffraction affects the three-dimensional intensity distribution of a focused, pulsed optical beam propagating inside a scattering medium. In practice, the full potential of the two-photon excitation fluorescence imaging is unrealized at long scattering depths, owing to the unwanted temporal and spatial broadening of the femtosecond excitation light pulse that reduces the energy density at the geometric focus while it increases the excitation energy density in the out-of-focus regions. To analyze the excitation intensity distribution, we modify the Monte Carlo-based photon-transport model to a semi-quantum-mechanical representation that combines the wave properties of light with the particle behavior of the propagating photons. In our model the propagating photon is represented by a plane wave with its propagation direction in the scattering medium determined by the Monte Carlo technique. The intensity distribution in the focal region is given by the square of the linear superposition of the various plane waves that arrive at different incident angles and optical path lengths. In the absence of scattering, the propagation model yields the intensity distribution that is predicted by the Huygens-Fresnel principle. We quantify the decrease of the energy density delivered at the geometric focus as a function of the optical depth to the mean-free-path ratio that yields the average number of scattering events that a photon encounters as it propagates toward the focus. Both isotropic and anisotropic scattering media are considered. Three values for the numerical aperture (NA) of the focusing lens are considered: NA = 0.25, 0.5, 0.75.     

Two-dimensional imaging without scanning by multifocal multiphoton microscopy

We describe multifocal multiphoton microscopy giving images without laser scanning. A multitude of 8 x 8 laser beams is focused into a sample yielding two-photon excitation in a plane. The focal spots are arranged in a rectangular array with close spacing between individual points (approximately 0.5 microm). The fluorescence emission from the sample is recorded with a CCD camera, but, owing to the close distance between the beams, they can no longer be regarded as individual points but rather as an illumination of the plane that is covered by the array of focal points. The axial sectioning capability is comparable with an ordinary single-beam two-photon microscope. Interference between the beams that could compromise the axial sectioning capability does not occur in our setup owing to small temporal delays between the individual beams. The axial sectioning capability of the setup is discussed in detail by means of the step response in which the foci are scanned axially into a uniformly fluorescent medium.     

Two-photon nitric oxide laser-induced fluorescence measurements in a diesel engine

A two-photon nitric oxide (NO) laser-induced fluorescence (LIF) technique was developed and applied to study in-cylinder diesel combustion. The technique prevents many problems associated with in-cylinder, single-photon NO planar-laser-induced fluorescence measurements, including fluorescence interference from the Schumann-Runge bands of hot O2, absorption of a UV excitation beam by in-cylinder gases, and difficulty in rejecting scattered laser light while simultaneously attempting to maximize fluorescence signal collection. Verification that the signal resulted from NO was provided by tuning of the laser to a vibrational off-resonance wavelength that showed near-zero signal levels, which resulted from either fluorescence or interference at in-cylinder pressures of as much as 20 bar. The two-photon NO LIF signal showed good qualitative agreement with NO exhaust-gas measurements obtained over a wide range of engine loads.     

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.     

Frequency upconversion and two-photon absorption of salicylaldehyde azine 1

Two-photon absorption and two-photon excitation fluorescence of salicylaldehyde azine 1 crystals were investigated. It was observed an intense visible fluorescence when this material was excited with a laser tuned at the near infrared region. Varying the laser intensity we characterized this phenomenon as a simultaneous two-photon laser absorption process. Using open aperture Z-scan measurements we characterized this two-photon absorption phenomenon and measured the value of the two-photon absorption cross-section of this molecule to be equal to 87 GM. Our results indicate that this is a promising organic material aiming nonlinear photonics applications.     

Effect of scattering on nonlinear optical scanning microscopy imaging of highly scattering media

The intensity of two-photon excited fluorescence (TPF) generated by ultrashort laser pulses was measured as a function of the depth of a focal point inside highly scattering media. The purpose was to investigate the spatial location of TPF in a scattering medium. Owing to the scattering, the intensity of the incident beam as well as the generated TPF signal was attenuated exponentially as the focal point was scanned into the medium. As the scattering strength of the medium was increased, the TPF was not confined to the focal region and had a wider distribution. These observations show that the scattering will result in the degradation of the ability of optical depth sectioning of nonlinear optical scanning microscopy.     

Two-photon fluorescence microscopy with a diode-pumped Cr:LiSAF laser

We demonstrate the acquisition of high-quality two-photon fluorescence microscopy images using an all-solid-state self-mode-locked Cr:LiSAF laser. We contrast the performance of the two-photon technique with single-photon confocal fluorescence microscopy images taken with an argon-ion laser. Examples of improved depth penetration and reduced dye bleaching are presented.     

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.     

Primary spherical aberration in two-color (two-photon) excitation fluorescence microscopy with two confocal excitation beams

We study the effects of primary spherical aberration on the three-dimensional point spread function (PSF) of the two-color (two-photon) excitation (2CE) (2PE) fluorescence microscope with two confocal excitation beams that are separated by an angle theta. The two excitation wavelengths lambda1 and lambda2 are related to the single-photon excitation wavelength lambda(e) by: 1/lambda(e) = 1/lambda1 + 1/lambda2. The general case is considered where both focused beams independently suffer from spherical aberration. For theta = 0, pi/2, and pi, the resulting deterioration of the PSF structure is evaluated for different values of the spherical aberration coefficients via the Linfoot's criteria of fidelity, structural content, and correlation quality. The corresponding degradation of the peak 2CE fluorescence intensity is also determined. Our findings are compared with that of the 2PE fluorescence (lambda1 = lambda2) under the same aberration conditions. We found that the 2CE microscope is more robust against spherical aberration than its 2PE counterpart, with the pi/2 configuration providing the clearest advantage. The prospect of aberration correction in the two-beam 2CE microscope is also discussed.     

Homogeneous immunoassay based on two-photon excitation fluorescence resonance energy transfer

A two-photon excitable small organic molecule (abbreviated as TP-NH 2) with large two-photon absorption cross section and competitive fluorescence quantum yield was prepared, which emitted fluorescence in the visible region upon excitation at 800 nm. Using the TP-NH 2 molecule as an energy donor, a two-photon excitation fluorescence resonance energy-transfer (TPE-FRET) based homogeneous immunoassay method was proposed. The donor and the acceptor (DABS-Cl, a dark quencher) were labeled to bovine serum albumin (BSA) separately, and anti-BSA protein was determined by employing an antibody bridging assay scheme. Rabbit anti-BSA serum containing other biomolecules was intentionally used as the sample to introduce interference. A parallel assay was performed using the traditional one-photon excitation FRET model, which failed to carry out quantitative determination due to the serious background luminescence arising from those biomolecules in the sample. The TPE-FRET model showed its strong ability to overcome the problem of autofluorescence and provided satisfying analytical performance. Quite good sensitivity and wide linear range (0.05-2.5 nM) for anti-BSA protein was obtained. The results of this work suggest that TPE-FRET could be a promising technique for homogeneous assays excluding separation steps, especially in complicated biological sample matrixes.     

Two-photon excited coherence gratings in inhomogeneously broadened organic solid

For the first time a frequency-domain grating is observed in the visible region as a result of coherence excited by two-photon absorption of time-delayed near-IR femtosecond pulses in an inhomogeneously broadened organic solid-state system. The grating appears in chlorin (2,3-dihydroporphyrin)-doped polymer film at low temperature and is detected in two different ways. First, modulation is observed in the pure electronic fluorescence band upon 'resonant' two-photon excitation with laser carrier frequency equal to one-half of the fluorescence band maximum. Second, upon long exposure time at half of the transition frequency, a persistent spectral hole with periodic structure is observed. The two-photon absorption spectrum of the chlorin molecule in the spectral range 1130–1310nm is also presented for the first time.     

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.     

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.     

Penetration depth of single-, two-, and three-photon fluorescence microscopic imaging through human cortex structures: Monte Carlo simulation

Penetration depth is investigated in terms of the performance of transverse image resolution and signal level in human cortex under single-, two-, and three-photon fluorescence microscopy. Simulation results show that, in a double-layer human cortex structure consisting of gray and white matter media, the signal level is strongly affected by the existence of the white matter medium under three-photon excitation. Compared with three-photon excitation, two-photon excitation keeps a better signal level and sacrifices a slight degradation in image resolution. In a thick gray matter medium, a penetration depth of 1500 microm with a near-diffraction-limited resolution is obtainable under three-photon excitation. It is also demonstrated that the numerical aperture has a slight influence on image resolution and signal level under two- and three-photon excitation because of the nonlinear nature in the excitation process.