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.     

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.     

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.     

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.     

Biorthogonally accessed three-dimensional two-photon memory for relational database operations

Memory bandwidth is a bottleneck for very large database machines. Parallel-access three-dimensional two-photon memories have the potential of achieving enormous throughput (>100 Gbit/s) and capacity (1 Tbit/cm(3)) [Appl. Opt. 29, 2058 (1990)] and, consequently, are well suited for this application. Our analysis shows that some operations can be completed more than 2 orders of magnitude faster with this type of memory than with a system based on serial-access storage. These particular memories have a further feature of being accessible in orthogonal directions. We show that this property, used in conjunction with a three-dimensional data-organization scheme designed for this approach, leads to improved performance by permitting the user a choice of accessing strategies for a given operation.     

Nanoshells for in vivo imaging using two-photon excitation microscopy

Gold nanoshells have been intensively investigated and applied to various biomedical fields because of their flexible optical tunability and biological compatibility. They hold great potential to serve as luminescent contrast agents excitable with near-infrared (NIR) lasers. In this paper, we describe the development of nanoshells with a peak of plasmon resonance at 800 nm and their subsequent use for in vivo blood vessel imaging using two-photon excitation microscopy at an excitation wavelength of 750 nm. We were able to image single nanoshell particles in blood vessels and generate optical contrast for blood vessel structure using luminescent signals. These results confirm the feasibility of engineering nanoshells with controlled optical properties for single-particle-based in vivo imaging.     

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.     

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.     

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.     

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.     

Scanning holographic microscopy of three-dimensional fluorescent specimens

We demonstrate experimentally the three-dimensional reconstructions of fluorescent biological specimens using scanning holographic microscopy. Three-dimensional reconstructions with transverse resolution below about 1 microm of transmission and fluorescence emission images are presented and analyzed. The limitations of the method are discussed.     

Depth-variant maximum-likelihood restoration for three-dimensional fluorescence microscopy

We derive an algorithm for maximum-likelihood image estimation on the basis of the expectation-maximization (EM) formalism by using a new approximate model for depth-varying image formation for optical sectioning microscopy. This new strata-based model incorporates spherical aberration that worsens as the microscope is focused deeper under the cover slip and is the result of the refractive-index mismatch between the immersion medium and the mounting medium of the specimen. Images of a specimen with known geometry and refractive index show that the model captures the main features of the image. We analyze the performance of the depth-variant EM algorithm with simulations, which show that the algorithm can compensate for image degradation changing with depth.     

High-resolution ab initio three-dimensional x-ray diffraction microscopy

Coherent x-ray diffraction microscopy is a method of imaging nonperiodic isolated objects at resolutions limited, in principle, by only the wavelength and largest scattering angles recorded. We demonstrate x-ray diffraction imaging with high resolution in all three dimensions, as determined by a quantitative analysis of the reconstructed volume images. These images are retrieved from the three-dimensional diffraction data using no a priori knowledge about the shape or composition of the object, which has never before been demonstrated on a nonperiodic object. We also construct two-dimensional images of thick objects with greatly increased depth of focus (without loss of transverse spatial resolution). These methods can be used to image biological and materials science samples at high resolution with x-ray undulator radiation and establishes the techniques to be used in atomic-resolution ultrafast imaging at x-ray free-electron laser sources.     

Radiation dosimetry using three-dimensional optical random access memories

This and twelve previous Symposia reflect the evolution of microdosimetry, a field of research that has determined major new developments in radiation research, radiation protection, and radiology during the past four decades. The concepts of microdosimetry and its techniques were developed almost single handedly by H. H. Rossi. This memorial lecture outlines some of the ideas and some of the work of Harald Rossi that led to microdosimetry. It describes its major impact on radiobiology and, especially, its impact on studies with fast neutrons and on risk assessment. Microdosimetry was primarily designed as a tool for the elucidation of basic mechanisms of radiation action, but it has found its most important applications in the dosimetric measurement techniques that have become indispensable in radiation protection and in the dosimetry for radiation therapy. The advances of molecular biology are now providing new possibilities for a quantitative application of microdosimetry to radiobiology along the lines that Harald Rossi defined.     

Fast maximum-likelihood image-restoration algorithms for three-dimensional fluorescence microscopy

We have evaluated three constrained, iterative restoration algorithms to find a fast, reliable algorithm for maximum-likelihood estimation of fluorescence microscopic images. Two algorithms used a Gaussian approximation to Poisson statistics, with variances computed assuming Poisson noise for the images. The third method used Csiszar's information-divergence (I-divergence) discrepancy measure. Each method included a nonnegativity constraint and a penalty term for regularization; optimization was performed with a conjugate gradient method. Performance of the methods was analyzed with simulated as well as biological images and the results compared with those obtained with the expectation-maximization-maximum-likelihood (EM-ML) algorithm. The I-divergence-based algorithm converged fastest and produced images similar to those restored by EM-ML as measured by several metrics. For a noiseless simulated specimen, the number of iterations required for the EM-ML method to reach a given log-likelihood value was approximately the square of the number required for the I-divergence-based method to reach the same value.     

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.     

Influence of refractive-index mismatch in high-resolution three-dimensional confocal microscopy

The effects of the refractive-index mismatch in confocal laser scanning microscopy were extensively studied. The axial aberration induced in the case of fluorescent microspheres was measured. The data were used to take into account the mismatch-induced aberrations and to consider object-size influence. Then we focused on the effect of refractive-index mismatch on the effective system's point-spread function under different mismatch conditions and on depth of focusing. We experimentally verified that the peak of the point-spread function intensity profile decreases and the point-spread function itself progressively broadens as a function of the combined effect of the refractive-index mismatch and of the penetration depth, leading to a worsening of the system's overall performances. We also performed these same measurements by embedding subresolution beads in an oocyte's cytoplasm, which can be considered a turbid medium. We found evidence consistent with the previously developed theoretical model; in particular we found a strong dependence of the intensity peak on the focusing depth.     

Effects of refractive-index mismatch on three-dimensional optical data-storage density in a two-photon bleaching polymer

Reported is an investigation into the effect of spherical aberration caused by the mismatch of the refractive indices between the recording material and its immersion medium on the three-dimensional optical data-storage density in a two-photon bleaching polymer. It is found both theoretically and experimentally that spherical aberration can be compensated for by a change in the tube length at which a microscope objective is operated in recording and reading processes. After compensation for the spherical aberration it is possible to achieve a three-dimensional recording density of 3.5 Tbits/cm(3) for a commercial objective with a numerical aperture of 1.4.     

Complex-shaped three-dimensional microstructures and photonic crystals generated in a polysiloxane polymer by two-photon microstereolithography

Two-photon photopolymerization of inorganic–organic hybrid materials permits the generation of complex-shaped three-dimensional microstructures at submicrometer resolution of structural elements. Due to their favorable optical, chemical and thermal properties these materials are particularly useful for photonic microdevice fabrication. Focussing ultrashort pulsed visible light into a modified commercially available polysiloxane polymer a Sydney Opera House design and a series of woodpile-type photonic crystals were fabricated. Fourier transform infrared spectroscopy revealed photonic stop gaps in the stacking direction at wavelengths varying from 6 to 4 μm upon reduction of the woodpile rod size. The structures allowed for the observation of higher-order stop gaps.