Two-photon near- and far-field fluorescence microscopy with continuous-wave excitation

We report on scanning far- and near-field two-photon microscopy of cell nuclei stained with DAPI and bisbenzimidazole Hoechst 33342 (BBI-342) with the 647-nm laser line of a cw ArKr mixed-gas laser. Two-photon-excited fluorescence images are obtained for 50-200 mW of average power at the sample. A nearly quadratic dependence of fluorescence intensity on laser power confirmed the two-photon effect. The nonlinearity was further supported by evidence of three-dimensional sectioning in a scanning far-field microscope. We find that the cw two-photon irradiation sufficient for imaging within typically 5 s does not significantly impair cell cycling of BBI-342-labeled live cells. Finally, high-resolution imaging in scanning near-field microscopy with good contrast is demonstrated.     

Two-photon resonance fluorescence

The excitation spectrum of a two-level atom interacting with a strong electromagnetic field is considered when the atomic transition frequency is nearly twice the frequency of the laser field. The spectral function consists of four Lorentzian lines describing: the central line peaked near the two-photon resonance, two sidebands peaked at the high and low frequency regions respectively and the one-photon frequency mode. In the limit of high photon densities, the low frequency photon mode is induced by the pump field which removes the singularity occurring at the two-photon resonance frequency.     

Two-photon standing-wave fluorescence correlation spectroscopy

A fluorescence correlation spectroscopy experiment that combines two-photon excitation and a standing-wave interference pattern is presented. The experimental correlation function can be analyzed using a simple expression involving (1) an exponential decay with time constant tau(f), which reflects diffusion across the interference fringes, and (2) a longer-lived decay with time constant tau(omega), which reflects diffusion in and out of the focal spot. The diffusion of Rhodamine 110 in water and ethylene glycol is measured using this method. The ability to simultaneously measure diffusion on two different time and lengthscales makes this experiment especially useful in environments where anomalous diffusion is suspected.     

Two-photon fluorescence scanning near-field microscopy based on a focused evanescent field under total internal reflection

We present two-photon fluorescence near-field microscopy based on an evanescent field focus produced by a ring beam under total internal reflection. The evanescent field produced by this method is focused by a high-numerical-aperture objective, producing a tightly confined volume that can effectively induce two-photon excitation. The imaging system is characterized by the two-photon-excited images of the nanocrystals, which show that the focused evanescent field is split into two lobes because of the enhancement of the longitudinal polarization component at the focus. This feature is confirmed by the theoretical prediction. Unlike other two-photon near-field probes, this method does not have the heating effect and requires no control mechanism of the distance between a sample and the probe.     

Two-photon excited fluorescence from biological aerosol particles

We used a 40 MHz mode-locked 524 nm laser source to evaluate the utility of sub-picosecond excitation of fluorescence from 2-photon absorption in biological aerosols. Individual particles of biological composition, as well as other calibration particles, suspended in an inlet air flow were illuminated and measured as they passed through an optical chamber. To our knowledge, this was the first demonstration of 2-photon excited fluorescence from micron-sized particles composed of micro-organisms. We also observed a high fluorescence signal at visible wavelengths, which was not present with single-photon excitation.     

Two-photon resonance fluorescence. II numerical results

Some numerical results and graphs are presented for the excitation spectrum of a two level atom whose transition frequency ω₂₁ is in resonance with twice the frequency of a strong laser field. These results are computed directly from the Green function and are in agreement with earlier theoretical predictions.     

Two-photon fluorescence measurements in a hexagonal CdS crystal

Measurements of two-photon fluorescence (TPF) in a CdS crystal using a dye laser pumped by a Q-switched ruby laser are reported. The TPF is measured as a function of source intensity and wavelength. The total fluorescence shows linear and non-linear dependence on the source intensity; from these measurements the TPF part is determined. The measured transition of the crystal's fundamental gap is 2.5 ± 0.02 eV and another forbidden one-photon but allowed two-photon transition is found at 3.24 ± 0.02 eV.     

Two-photon fluorescence endoscopy with a micro-optic scanning head

A major obstacle in the race to develop two-photon fluorescence endoscopy is the use of complicated bulk optics to transmit an ultrashort-pulsed laser beam and return the emitted fluorescence signal. We describe an all-fiber two-photon fluorescence microendoscope based on a single-mode optical fiber coupler, a microprism, and a gradient-index rod lens. It is found that the new endoscope exhibits an axial resolution of 3.2 microm and is capable of imaging transverse cross sections of internal cylindrical structures as small as approximately 3.0 mm in diameter. This device demonstrates the potential for developing a real-time diagnostic tool for biomedical research without the need for surgical biopsy and may find applications in photodynamic therapy, microsurgery, and early cancer detection.     

Three-dimensional holographic fluorescence microscopy

Most commonly used methods for three-dimensional (3D) fluorescence microscopy make use of sectioning techniques that require that the object be physically scanned in a series of two-dimensional (2D) sections along the z axis. The main drawback in these approaches is the need for these sequential 2D scans. An alternative approach to fluorescence imaging in three dimensions has been developed that is based on optical scanning holography. This novel approach requires only a 2D scan to record 3D information. Holograms of 15-microm fluorescent latex beads with longitinal separation of ~2 mm have been recorded and reconstructed. To our knowledge, this is the first time holograms of fluorescent specimens have been recorded by an optical holographic technique.     

Phase and fluorescence imaging by combination of digital holographic microscopy and fluorescence microscopy

Optical Review - Hybrid digital holographic microscopy that combines fluorescence microscopy and digital holographic microscopy into a single system for biological applications is proposed. In the...     

Two-photon fluorescence measurements of reversible photodegradation in a dye-doped polymer

We report on the dynamics of photodegradation and subsequent recovery of two-photon fluorescence in a dye-doped polymer. The energy dependence suggests that photodegradation is a linear process, while recovery is entropic. Such recovery could be useful to high-intensity devices such as two-photon absorbers, which can be used in many applications.     

Two-photon fluorescence spectrum in an atom + dielectric microsphere system

A strong resonant interaction of a two-level atom with a dielectric microsphere is studied on the basis of quantum electrodynamics. The initial condition considered is one in which the atom is initially excited and the resonant mode of the microsphere has been excited by a single photon. The spectrum of two emitted photons depends strongly on the method used to excite the microsphere, i.e., on the spatial distribution of the photon energy. The most characteristic feature of the two-photon fluorescence spectrum is a strong energy correlation of the emitted photons. This correlation is expressed in the fact that the energies of the emitted photons are related by the equation of an ellipse (ω+ω ₂−2ω _vA )²+3(ω ₁−ω ₂)²= 4Ω _Rabi ² . The relation between the results obtained and the predictions of the theory of dressed states is discussed. Pis’ma Zh. éksp. Teor. Fiz. 70, No. 3, 192–197 (10 August 1999)     

Two-photon fluorescence bioimaging with an all-semiconductor laser picosecond pulse source

We have demonstrated successful two-photon excitation fluorescence bioimaging using a high-power pulsed all-semiconductor laser. Toward this purpose, we developed a pulsed light source consisting of a mode-locked laser diode and a two-stage diode laser amplifier. This pulsed light source provided optical pulses of 5 ps duration and having a maximum peak power of over 100 W at a wavelength of 800 nm and a repetition frequency of 500 MHz.     

Femtosecond Kerr-gated wide-field fluorescence microscopy

We present a Kerr-gated microscope capable of collecting diffraction-limited 2D fluorescence images with sub-100 fs time resolution. The concept is based on the insertion of a solid-state optical Kerr gate into a wide-field microscope. In addition to the considerably improved temporal resolution, the wide-field design allows for simultaneous tracking of several objects and ultrafast fluorescence lifetime imaging of doped and heterogeneous surfaces. The ultrafast fluorescence dynamics of gold nanoparticles is presented as an example of the capabilities of the instrument.     

Two-photon spectroscopy and microscopy of II–VI semiconductor nanocrystals

We review our recent nonlinear spectroscopies of nanocrystals and synthetic efforts to improve their luminescence properties. A two-photon spectroscopic study of CdSe nanocrystals as a function of size is presented and compared with predictions from the effective mass model with spherical confinement. We also detail our efforts at improving the luminescence properties of nanocrystals which have culminated in a 50% fluorescence quantum yield for inorganic capping of some sizes of CdSe nanocrystals. Finally, we present the application of two-photon microscopy to resolve fluorescence from single nanocrystals at room temperature and cryogenic temperatures.     

有机染料CSPI的双光子吸收和上转换荧光性质

报道了一种有机染料Trans 4 [p carbazol 9 ylstyryl] ] N methylpyridiniumiodide的线性和非线性光学性质。当用 830nm皮秒激光激发时 ,可以得到强烈的黄绿色上转换荧光 ,中心波长在 538nm附近。该染料的双光子荧光寿命为 95 .6ps。测试了该染料在 72 0～ 1 0 50nm的非线性透过率 ,并求出了该染料在上述波长时的双光子吸收截面。双光子吸收最强在 80 0nm ,吸收截面为 9.2× 1 0 - 48cm4 ·s/ photon。     

Two-photon excited fluorescence of the lens for the diagnosis of presbyopia

Presbyopia is a wide spread phenomenon in elder people and is caused by the hardening of the lens in human eyes. Research is performed to make such lenses again more flexible by application of geometrically optimised cuts through the lens with a femtosecond-laser. Different protein agglomerations are responsible for the flexibility reduction of the lens. Two-photon excited fluorescence of the lens can be used as a diagnostic tool to localise such protein accumulations. In in-vitro experiments with human cataract lenses and also lenses of the Philly-mouse it could be demonstrated that with age the fluorescence increases as presbyopia proceeds. The distribution of the fluorescing compounds are not homogeneous but rather cloudy. Discrimination of the compounds by fluorescence lifetime measurements in relation of the depth in the lens is possible.     

Two-photon confocal microscopy in the study of the volume characteristics of semiconductors

Zn–Se crystals are used to analyze prospects for application of two-photon confocal microscopy in the study of plane and volume interband and impurity luminescence in semiconductors. Such maps can be formed with a depth step and planar spatial resolution of several micrometers at distances of up to 1 mm from the surface. The method is used to detect luminescence-active inhomogeneities in crystals and study their structure and luminescence characteristics. Prospects for the application of the two-photon confocal microscopy in the study of direct-band-semiconductors and materials of the fourth group are discussed.     

双色荧光辐射差分超分辨显微系统研究

为了拓展荧光辐射差分(Fluorescence Emission Difference,FED)显微术的应用,使得该方法可以同时对生物样品的不同组织结构进行超分辨成像,本文对双色FED显微系统展开了研究。FED的基本原理是将实心光斑扫描得到的共焦显微图像减去空心光斑扫描得到的负共焦图像,以此获得超分辨显微图像。在对单色FED显微系统进行研究后,本文提出了一种可行的双色FED显微成像系统方案。实验结果表明,在488 nm和640 nm激发光下,该系统在荧光颗粒上分别实现了135 nm和160 nm的空间分辨率,另外也能对生物样品的不同组织进行多色同时超分辨显微成像,满足了实际应用的要求。     

Laser-assisted fluorescence microscopy and biomedical screening

An overview of recent developments and applications of the Biophotonics Group at Hochschule Aalen is given. In view of in vitro diagnostics and biomedical screening, some advanced methods of laser-assisted microscopy and micromanipulation, as well as fluorescence reader technology, have been established. Present applications are concentrated on energy metabolism of the cell, membrane dynamics, membrane associated fluorescent proteins, laser-assisted optoporation, and photodynamic therapy.