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.     

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.     

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.     

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.     

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.     

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.     

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.     

Random access three-dimensional two-photon microscopy

We propose a two-photon microscope scheme capable of real-time, three-dimensional investigation of the electric activity pattern of neural networks or signal summation rules of individual neurons in a 0.6 mm x 0.6 mm x 0.2 mm volume of the sample. The points of measurement are chosen according to a conventional scanning two-photon image, and they are addressed by separately adjustable optical fibers. This allows scanning at kilohertz repetition rates of as many as 100 data points. Submicrometer spatial resolution is maintained during the measurement similarly to conventional two-photon microscopy.     

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.     

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.     

Fast aerosol analysis by fourier transform imaging fluorescence microscopy

Fourier transform imaging spectroscopy was combined with fluorescence microscopy and a cooled CCD detector for fast analysis of aerosols contaminated with polycyclic aromatic hydrocarbons (PAHs). Aerosols were collected on glass fiber filters and inspected, for the first time, by this imaging technique, which provides a full fluorescence spectrum at each pixel. Mapping of PAH contamination was carried out and used for identification and quantification of the compounds. Quantification limits (based on 95% confidence intervals of calibration plots) in the 10 ng cm(-)(2) range on filter are reported, which corresponds to 20 ng m(-)(3) in air, integrated in 1 min. The absolute detection limit (on filter) is estimated as low as 0.25 pg, corresponding to an air concentration of 0.5 pg m(-)(3), integrated in 1 min. The method is examined for analysis of monocomponent contamination and for simple mixtures. After a proper automation, this method has the potential to provide in situ and on-line results regarding particulate airborne PAH contaminations.     

Excitation saturation in two-photon fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) has become a powerful and sensitive research tool for the study of molecular dynamics at the single-molecule level. Because photophysical dynamics often dramatically influence FCS measurements, the role of various photophysical processes in FCS measurements must be understood to accurately interpret FCS data. We describe the role of excitation saturation in two-photon fluorescence correlation measurements. We introduce a physical model that characterizes the effects of excitation saturation on the size and shape of the two-photon fluorescence observation volume and derive a new analytical expression for fluorescence correlation functions that includes the influence of saturation. With this model, we can accurately describe both the temporal decay and the amplitude of measured fluorescence correlation functions over a wide range of illumination powers.     

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 confocal microscopy of colloids

Confocal microscopy is used in the study of colloidal gels, glasses, and binary fluids. We measure the three-dimensional positions of colloidal particles with a precision of approximately 50 nm (a small fraction of each particle's radius) and with a time resolution sufficient for tracking the thermal motions of several thousand particles at once. This information allows us to characterize the structure and the dynamics of these materials in qualitatively new ways, for example, by quantifying the topology of chains and clusters of particles as well as by measuring the spatial correlations between particles with high mobilities. We describe our experimental technique and describe measurements that complement the results of light scattering.     

Three-dimensional time-resolved fluorescence imaging by multifocal multiphoton microscopy for a photosensitizer study in living cells

Two-photon fluorescence microscopy is widely applied to biology and medicine to study both the structure and dynamic processes in living cells. The main issue is the slow acquisition rate due to the point scanning approach limiting the multimodal detection (x, y, z, t). To extend the performances of this powerful technique, we present a time-resolved multifocal multiphoton microscope (MMM) based on laser amplitude splitting. An array of 8 x 8 foci is created on the sample that gives a direct insight of the fluorescence localization. Four-dimensional (4D) imaging is obtained by combining simultaneous foci scanning, time-gated detection, and z displacement. We illustrate time-resolved MMM capabilities for 4D imaging of a photosensitizer inside living colon cancer cells. The aim of this study is to have a better understanding of the photophysical processes implied in the photosensitizer reactivity.     

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.     

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.     

Three-dimensional autofluorescence spectroscopy of rat skeletal muscle tissue under two-photon excitation

We report on three-dimensional autofluorescence spectroscopy obtained from rat skeletal muscle tissue under two-photon excitation by an ultrashort pulsed-laser beam. It is demonstrated that two types of fluorophores within the skeletal muscle tissue can be simultaneously excited with the laser beam at a wavelength of 800 nm. The two fluorophores exhibited unique fluorescence spectral peaks at wavelengths of 450 and 550 nm. These spectroscopic signals can be used to form a three-dimensional image, giving the information about the biochemical makeup of the skeletal muscle tissue.     

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.