On the fundamental imaging-depth limit in two-photon microscopy

We have analyzed how the maximal imaging depth of two-photon microscopy in scattering samples depends on properties of the sample and the imaging system. We find that the imaging depth increases with increasing numerical aperture and staining inhomogeneity and with decreasing excitation-pulse duration and scattering anisotropy factor, but is ultimately limited by near-surface fluorescence with slight improvements possible using special detection strategies.     

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.     

Two-photon excitation fluorescence microscopy with a high depth of field using an axicon

In conventional two-photon excitation fluorescence microscopy, the numerical aperture of the objective determines the lateral resolution and the depth of field. In some situations, as with functional imaging of dynamic events distributed in live biological tissue, an improved temporal resolution is needed; as a consequence, it is imperative to use optics with a high depth of field to simultaneously image objects at different axial positions. With a conventional microscope objective, increasing the depth of field is achieved at the expense of lateral resolution. To overcome this limitation, we have incorporated an axicon in a two-photon excitation fluorescence microscopy system; measurements have shown that an axicon provides a depth of field in excess of a millimeter, while the lateral resolution is maintained at the micrometer scale. Thus axicon-based two-photon microscopy has been shown to yield a high-resolution projection image of a sample with a single 2D scan of the laser beam while maintaining the improved tissue penetration typical of two-photon microscopy.     

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.     

Characterization of gradient-index lens-fiber spacing toward applications in two-photon fluorescence endoscopy

We report on the experimental investigation into the characterization of two-photon fluorescence microscopy based on the separation distance of a single-mode optical fiber coupler and a gradient-index (GRIN) rod lens. The collected two-photon fluorescence signal exhibits a maximum intensity at a defined separation distance (gap length) where the increasing effective excitation numerical aperture is balanced by the decreasing confocal emission collection. A maximum signal is found at gap lengths of approximately 2, 1.25, and 1.75 mm for GRIN lenses with pitches of 0.23, 0.25, and 0.29 wavelength at 830 nm. The maximum two-photon fluorescence signal collected corresponds to a threefold reduction of axial resolution (38.5 microm at 1.25 mm), compared with the maximum resolution (11.6 microm at 5.5 mm), as shown by the three-dimensional imaging of 10 microm beads. These results demonstrate an intrinsic trade-off between signal collection and axial resolution.     

Spatial distribution of two-photon-excited fluorescence in scattering media

Spatial distribution of two-photon-excited fluorescence (TPF) for dye beneath the surface of a highly scattering medium was investigated with picosecond laser pulses at 1064 nm. The active scattering media consisted of a suspension of polystyrene particles in a solution of Rhodamine 590 Tetrafluoroborate dye. With the increase of scattering strength of the medium, the location of the maximum TPF intensity was found to move closer to the surface away from the focal region; the intensity of TPF was not confined to the focal region as in the case of nonscattering medium but was more evenly distributed. The spatial resolution of nonlinear optical microscopy for probing a scattering medium is degraded. Taking into account the scattering of the medium qualitatively explains the observed TPF spatial distribution.     

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.     

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.     

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.     

Imaging theory of nonlinear Raman confocal microscopy

A novel nonlinear Raman confocal microscopy utilizing Raman induced Kerr effect spectroscopy (RIKES) is presented in this paper. The imaging theory of RIKES confocal microscopy with Gaussian beam is derived. The imaging properties of RIKES confocal microscopy and the impact of different beam waist widths of Gaussian beam on the lateral and axial resolution have been analyzed in detail. It is proved that RIKES confocal microscopy has high sensitivity and high resolution, besides capability to characterize inherent structural features, such as vibration mode, vibration orientation, and optically induced molecular reorientation etc. Therefore, nonlinear Raman confocal microscopy that is based on RIKES has potential to provide a novel characteristic imaging method comparable to the existing imaging techniques based on other nonlinear optical processes, such as two-photon fluorescence, second harmonic generation (SHG) and coherent anti-Stoke Raman scattering (CARS).     

Conversion of the Nikon C1 confocal laser-scanning head for multiphoton excitation on an upright microscope

Most commercial laser-scanning imaging systems used for confocal fluorescence microscopy can be readily adapted for use with two-photon fluorescence excitation. We report here on the details of the conversion of the Nikon C1 (product released November 2001) with two channels of nondescanned detection of two-photon-excited fluorescence. One of the goals of the design was to utilize off-the-shelf components as much as possible to minimize the use of custom machining and electronics assembly. We also give some initial characterization of the imaging properties of the system.     

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.     

CdTe量子点的微波合成及其在双光子显微成像中的应用

采用微波辐射加热的方法,以亚碲酸钠(Na2TeO3)作碲源,以谷胱甘肽(GSH)作稳定剂,在水相中合成出高质量的CdTe量子点。所合成量子点的发射波长从515～630nm可调,荧光量子产率(PLQYs)最高达95%。利用X射线粉末衍射(XRD)、高分辨透射电镜(HRTEM)、紫外-可见吸收光谱(UV-Vis)和荧光发射光谱(PL)等技术表征产物的物相结构和光学性质。用双光子激发荧光法研究CdTe量子点的双光子吸收性质。用双光子激发荧光成像技术,以发红光的CdTe量子点作为双光子荧光探针成功标记了人肺腺癌细胞(A549)。     

Fluorescence of coumarins and xanthenes after two-photon absorption with a pulsed titanium-sapphire laser

Fluorescence emission after two-photon absorption of coumarins and xanthenes in an alcoholic solution was measured in the tuning range of a femtosecond-pulsed titanium-sapphire laser (750-840 nm). Xanthenes, which have a low one-photon absorption in the near UV, show a higher fluorescence signal after two-photon absorption than the UV-excitable coumarins. When fluxes of 10(28) photons/(cm(2) s) are used, the two-photon absorption cross sections for xanthenes are 1 order of magnitude higher than the two-photon absorption cross sections of the coumarins. Absolute cross sections have been estimated for three coumarins and three xanthenes. For the xanthenes a significant wavelength-dependent departure from the expected fluorescence intensity square law was observed. The coumarins follow the square-law dependence. The consequences of the findings are discussed for analytic and diagnostic methods. An especially important result is that the resolution in two-photon microscopy of xanthenes is worse than expected.     

Tip-enhanced optical spectroscopy

Spectroscopic methods with high spatial resolution are essential for understanding the physical and chemical properties of nanoscale materials including biological proteins, quantum structures and nanocomposite materials. In this paper, we describe microscopic techniques which rely on the enhanced electric field near a sharp, laser-irradiated metal tip. This confined light-source can be used for the excitation of various optical interactions such as two-photon excited fluorescence or Raman scattering. We study the properties of the enhanced fields and demonstrate fluorescence and Raman imaging with sub-20 nm resolution.     

Contrast limits of coherence-gated imaging in scattering media

The fundamental difference between time-resolved and coherence-gated imaging modalities in scattering media is analyzed in terms of their optical transfer functions. The effectiveness of coherence gating for multiple-scattering rejection is shown by imaging a 100-mum-thick razor blade hidden in the scattering phantoms (i.e., Intralipid suspensions) with different scattering coefficients. We found that the imaging contrast is limited by multiple scattering and speckle effects in high-scattering media, and the measured effective penetration depth of optical coherence tomography is approximately equal to six mean free paths under the experimental conditions of a numerical aperture of less than 0.1 and a scattering anisotropy of approximately 0.8.     

Third-harmonic generation microscopy in highly scattering media

The performance of third-harmonic generation (THG) microscopy in highly scattering media is analyzed with the Monte Carlo technique. The three-dimensional point-spread function (PSF) of the laser-scanning THG microscope with a pulsed excitation light source is derived for both isotropic and anisotropic scattering media and at different h/d(s) values, where h is the scattering depth as measured from the geometric focus of the objective lens and d(s) is the mean free path of the scattering medium. The generated THG signal is detected by a large-area photodetector. The PSF of the THG microscope is given by the third power of the normalized distribution of the excitation beam near the beam focus. The behavior of the temporal broadening of the excitation pulse on the generated THG signal is also analyzed as a function of h/d(s). The relative advantages and disadvantages of the THG microscope relative to the two-photon fluorescence microscope are discussed thoroughly.     

Scanning total internal reflection fluorescence microscopy under one-photon and two-photon excitation: image formation

We propose a new type of total internal reflection fluorescence microscopy (TIRFM) called scanning TIRFM (STIRFM) that uses a focused ring-beam illumination and a high-numerical-aperture objective (NA = 1.65). The evanescent field produced by the STIRFM is focused laterally, producing a small excitation volume that can induce a nonlinear effect such as two-photon absorption. Experimental images of CdSe quantum dot nanocrystals and Rhodamine 6G-doped microbeads show that good lateral and axial resolutions are achieved with the current setup. The theoretical simulation of the focal spot produced in STIRFM geometry shows that the focused evanescent field is split into two peaks because of the depolarization effect of a high numerical-aperture objective lens. However, the point-spread function analysis of both one-photon and two-photon excitation cases shows that the detection of the focus-splitting effect is dependent on the detection pinhole size. The effect of pinhole size on image formation is theoretically investigated and confirmed experimentally with the nanocrystal images.     

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.