25 nm resolution single molecular fluorescence imaging by scanning near-field optical/atomic force microscopy

High-resolution single molecular near-field fluorescence images were observed by scanning near-field optical/atomic force microscopy (SNOM/AFM). We modified the SNOM/AFM for both high-resolution fluorescence imaging and high-resolution topographic imaging. The imaged fluorophore, Alexa 532, is prepared with a poly-methyl-methacrylate (PMMA) film coating. A fluorescence resolution of 25 nm was obtained with a simultaneous topographic image of a flat surface. A sample prepared with a lower PMMA concentration exhibited a rough surface in the micro area. The results for the flat surface indicated that the fluorescence resolution is worst in the rough surface sample, that the maximum fluorescence intensities for the individual fluorophore are similar, and that the decay rate is faster. Thus, we concluded that the morphological effect is an important factor in fluorescence image resolution and the apparent lifetimes of the fluorescence molecules.     

Characterization of ultra-weak fluorescence using picosecond non-collinear optical parametric amplifier

We report a technique for characterization of ultra-weak fluorescence based on a 355-nm pumped picosecond non-collinear optical parametric amplifier (OPA). In the experiment, we effectively reduced the strong super-fluorescence background by using a series of methods. With the picosecond OPA as the pre-amplifier and the gating pulse, the decay of the fluorescence of Rhodamine 6G dye in ethanol was measured and the fluorescence lifetime was found to be about 941 ps. The gain factor of this parametric fluorescence amplifier was measured to be ∼4.2 × 10⁶, while the energy detection limit was ∼160 aJ per pulse within a 15-ps gating pulse.     

Breaking the diffraction barrier in fluorescence microscopy by optical shelving

We report the breaking of the diffraction resolution barrier in far-field fluorescence microscopy by transiently shelving the fluorophore in a metastable dark state. Using a relatively modest light intensity of several kW/cm(2) in a focal distribution featuring a local zero, we confine the fluorescence emission to a spot whose diameter is a fraction of the wavelength of light. Nanoscale far-field optical resolution down to 50 nm is demonstrated by imaging microtubules in a mammalian cell and proteins on the plasma membrane of a neuron. The presence of dark states in virtually any fluorescent molecule opens up a new venue for far-field microscopy with resolution that is no longer limited by diffraction.     

Measurement of the photon statistics and the noise figure of a fiber-optic parametric amplifier

We report measurement of the noise statistics of spontaneous parametric fluorescence in a fiber parametric amplifier with single-mode, single-photon resolution. We employ optical homodyne tomography for this purpose, which also provides a self-calibrating measurement of the noise figure of the amplifier. The measured photon statistics agree with quantum-mechanical predictions, and the amplifier's noise figure is found to be almost quantum limited.     

基于荧光非共线光参量放大光谱技术的溶剂化动力学研究中的光谱矫正

利用荧光非共线光参量放大光谱技术测量了DCM染料乙醇溶液的溶剂化动力学过程. 实验结果表明,瞬态荧光光谱经过光谱矫正后,可以产生准确的溶剂化相关函数以及溶剂化过程中瞬态光谱峰值频率移动. 本文的工作表明荧光非共线光参量放大光谱技术有益同时关注荧光强度动力学以及光谱谱型演化的研究领域.     

Fluorescence lifetime tomography of live cells expressing enhanced green fluorescent protein embedded in a scattering medium exhibiting background autofluorescence

We present a novel fluorescence lifetime tomography system applied to a highly scattering autofluorescent phantom containing live cells expressing the fluorophore enhanced green fluorescent protein (EGFP). The fluorescence signal was excited using a fiber-laser-pumped supercontinuum source and detected using wide-field time gating imaging. To facilitate rapid 3D reconstruction of the fluorescence lifetime distribution, the time-resolved data were Fourier-transformed in time to give complex functions that formed a data set for the Fourier domain reconstruction. Initially the presence of an unspecified background autofluorescence signal impeded reconstruction of the lifetime distribution, but we show that this problem can be addressed using a simple iterative technique.     

Amplification of fluorescence using collinear picosecond optical parametric amplification at degeneracy

We demonstrate the output characteristic of broadband parametric amplification of incoherent light pulses in a 355-nm pumped degenerate picosecond optical parametric amplification with either saturated or unsaturated amplification.The optical parametric amplifier is seeded by the fluorescence generated in a solution of pyridine-1 dye in ethanol.With the saturated amplification,we can obtain high energy incoherent light pulses,whose full width at half maximum bandwidth varies from 16 nm to 53 nm for the different phase matching angles near degeneracy.Moreover,the unsaturated bandwidth of the amplified pulses fits well to the calculated result at degeneracy.Selecting s-polarized fluorescence with a Glan-Taylor prism,the maximum bandwidth of the amplified fluorescence is found to be 59 nm for a purely s-polarized seed.The maximum output energy is 0.67 mJ for the optical parametric amplifier.By using an optical filter and compressor,the generated high energy incoherent light has great potential as the incoherent pump,signal or idler wave of a parametric down-conversion process,so that a wave with a high degree of coherence can be generated from an incoherent pump light.     

Near-Field Fluorescence and Topography Characterization of a Single Nanometre Fluorophore by Apertureless Tip-Enhanced Scanning Near-Field Microscopy

Tip-enhanced near-field fluorescence and topography characterization of a single nanometre fluorophore is conducted by using an apertureless scanning near-field microscopy system. A fluorophore with size 80hm is mapped with a spatial resolution of 10hm. The corresponding near-field fluorescence data shows significant signal enhancement due to the apertureless tip-enhanced effect. With the nanometre spatial resolution capability and nanometre local tip-enhanced effect, the apertureless tip-enhanced scanning near-field microscopy may be further used to characterize a single molecule by realizing the local near-field spectrum assignment corresponding to topography at nanometre scale.     

第Ⅰ类BBO晶体中的参量荧光空间特性理论研究

 采用第Ⅰ类相位匹配BBO晶体的放大传递函数方法,分别对光参量放大过程中单波长信号光注入和多波长信号光注入出现的参量荧光的空间特性进行了详细的理论研究。结果表明:一方面单波长信号光注入时,在某一特定的非共线角和相位匹配角下,它是以泵浦光为中心,呈锥形分布的,而在其它相位匹配区域内,则呈环形分布;另一方面,在多波长信号光入射情况下,参量荧光的光谱在某一特定的相位匹配角下集中分布在很宽的范围内,而在其余相位匹配区域内,参量荧光光谱呈分散分布状态。该结论对于光参量放大中三波群速失配的补偿,高增益和窄脉冲宽度的参量光的产生以及对于参量荧光的控制提供了理论依据,并且对于近年来量子图像处理和量子通讯等领域所关注的纠缠光场的产生也具有参考价值。     

New Insights in the Interpretation of Tryptophan Fluorescence

Origin of tryptophan fluorescence is still up to these days a quiz which is not completely solved. Fluorescence emission properties of tryptophan within proteins are in general considered as the result of fluorophore interaction within its environment. For example, a low fluorescence quantum yield is supposed to be the consequence of an important fluorophore–environment interaction. However, are we sure that the fluorophore has been excited upon light absorption? What if fluorophore excitation did not occur as the result of internal conformation specific to the fluorophore environment? Are we sure that all absorbed energy is used for the excitation process? Fluorescence lifetimes of Trp residues are considered to originate from rotamers or conformers resulting from the rotation of the indole ring within the peptide bonds. However, how can we explain the fact that in most of the proteins, the two lifetimes 0.5 and 3 ns, attributed to the conformers, are also observed for free tryptophan in solution? The present work, performed on free tryptophan and tyrosine in solution and on different proteins, shows that absorption and excitation spectra overlap but their intensities at the different excitation wavelengths are not necessarily equal. Also, we found that fluorescence emission intensities recorded at different excitation wavelengths depend on the intensities at these excitation wavelengths and not on the optical densities. Thus, excitation is not equal to absorption. In our interpretation of the data, we consider that absorbed photons are not necessary used only for the excitation, part of them are used to reorganize fluorophore molecules in a new state (excited structure) and another part is used for the excitation process. A new parameter that characterizes the ratio of the number of emitted photons over the real number of photons used to excite the fluorophore can be defined. We call this parameter, the emission to excitation ratio. Since our results were observed for fluorophores free in solution and present within proteins, structural reorganization does not depend on the protein backbone. Thus, fluorescence lifetimes (0.5 and 3 ns) observed for tryptophan molecules result from the new structures obtained in the excited state. Our theory allows opening a new way in the understanding of the origin of protein fluorescence and fluorescence of aromatic amino acids.     

Near-field terahertz imaging with a dynamic aperture

By introduction of an optical gating beam on a semiconductor wafer, near-field terahertz (THz) imaging with a dynamic aperture has been realized. The spatial resolution is determined by the focus size of the optical gating bean and the near-field diffraction effect. THz imaging with subwavelength spatial resolution (better than 50mum) is demonstrated.     

Whole-Object Fluorescence Lifetime Setup for Efficient Non-Imaging Quantitative Intracellular Fluorophore Measurements

In the present study we introduce a Whole-Object Fluorescence Life Time (wo-FLT) measurement approach for ease and a relatively inexpensive method of tracing alterations in intracellular fluorophore distribution and in the physical-chemical features of the microenvironments hosting the fluorophore. Two common fluorophores, Rhodamine 123 and Acridine Orange, were used to stain U937 cells which were incubated, with and without either Carbonyl cyanide 3-chlorphenylhydrazon or the apoptosis inducer H₂O₂. The wo-FLT, which is a non-imaging quantitative measurement, was able to detect several fluorescence decay components and corresponding weights in a single cell resolution. Following cell treatment, both decay time and weight were altered. Results suggest that the prominent factor responsible for these alterations and in some cases to a shift in emission spectrum as well, is the intracellular fluorophore local concentration. In this study it was demonstrated that the proposed wo-FLT method is superior to color fluorescence based imaging in cases where the emission spectrum of a fluorophore remains unchanged during the investigated process. The proposed wo-FLT approach may be of particular importance when direct imaging is impossible.     

Simultaneous optically sectioned fluorescence and optical coherence microscopy with full-field illumination

Full-field optical coherence microscopy (FF-OCM) and optically sectioned fluorescence microscopy are two imaging techniques that are implemented here in a novel dual modality instrument. The two imaging modalities use a broad field illumination to acquire the entire field of view without raster scanning. Optical sectioning is achieved in both imaging modalities owing to the coherence gating property of light for FF-OCM, and a structured illumination setup for fluorescence microscopy. Complementary image data are provided by the dual modality instrument in the context of biological tissue screening. FF-OCM imaging modality shows the tissue microarchitecture, while fluorescence microscopy highlights specific tissue features with cellular-level resolution by using targeting contrast agents. Complementary tissue morphology and biochemical features could potentially improve the understanding of cellular functions and disease diagnosis.     

Time-resolved fluorescence of carbon nanotubes and its implication for radiative lifetimes

The temporal evolution of fluorescence from isolated single-wall carbon nanotubes (SWNTs) has been investigated using optical Kerr gating. The fluorescence emission is found to decay on a time scale of 10 ps. This fast relaxation arises from nonradiative processes, the existence of which explains the relatively low observed fluorescence efficiency in isolated SWNTs. From the measured decay rate and a determination of fluorescence quantum efficiency, we deduce a radiative lifetime of 110 ns.     

Multi-color ultrafast laser platform for nonlinear optical imaging based on independently tunable optical parametric oscillators

We report on design of a multi-color laser set up that allows for high spectral, time and spatial resolution imaging based on second- and third-order optical nonlinearities in soft condensed matter. Two femtosecond optical parametric oscillators (OPOs) are pumped simultaneously to provide intrinsically synchronized pulses at more than a dozen tunable colors across visible and infrared wavelengths. We demonstrate the use of independently tunable OPOs in a variety of imaging modalities. In one useful application, we explore brain tissue in a two-photon absorption fluorescence imaging experiment with near infrared optical pulses (λ ~ 1,070 nm). We also demonstrate second and sum-frequency generation microscopies in different tissues. Results from application of time-resolved, three-color coherent anti-stokes Raman scattering in tissue are presented to demonstrate feasibility of quantitative spectroscopic imaging.     

Direct measurement of the effective input noise power of an optical parametric amplifier

The spontaneous fluorescence background in optical parametric amplifiers is generally attributed to the zero‐point fluctuations of the electromagnetic field. These are amplified in parallel to the seed light and lead to an uncompressible superfluorescence background that deteriorates the contrast in optical parametric chirped pulse amplifiers (OPCPA). The absolute level of the underlying parametric fluorescence has not been reported so far. Comparing the fluorescence to low level cw seed light and quantitatively monitoring the output of a noncollinear optical parametric amplifier for both sources, the level is now determined. In a situation of 50 nm visible output bandwidth and low Gaussian spatial modes about 58 photons are found in the signal direction within the femtosecond time window of the amplifier. The superfluorescence level is observed to be proportional to the pump area for constant signal amplification. The implications for the background in high power OPCPA are discussed.     

Pupil-segmentation-based adaptive optical microscopy with full-pupil illumination

Optical aberrations deteriorate the performance of microscopes. Adaptive optics can be used to improve imaging performance via wavefront shaping. Here, we demonstrate a pupil-segmentation based adaptive optical approach with full-pupil illumination. When implemented in a two-photon fluorescence microscope, it recovers diffraction-limited performance and improves imaging signal and resolution.     

Visible pulse compression to 4 fs by optical parametric amplification and programmable dispersion control

Angular dispersion of pump frequencies is shown to be an efficient mechanism for bandwidth enhancement in a noncollinear optical parametric amplifier. We demonstrate the generation of a continuous, simultaneously phase-matched 250-THz parametrically amplified spectrum. The resultant visible-near-IR signal-wave pulses were compressed to a 4-fs duration by a micromachined flexible mirror. Feedback for an iterative computer-controlled dispersion compensation algorithm is based on pulse characterization by second-harmonic generation frequency-resolved optical gating.     

Electromagnetic interaction of fluorophores with designed two-dimensional silver nanoparticle arrays

We study the fluorescence enhancement of dye molecules adsorbed on regular two-dimensional arrays of designed silver nanoparticles. The silver particles show two orthogonal optical resonances at different wavelengths because of their elongated shape. The short-wavelength resonance was designed to fit to the absorption maximum of the fluorophore. When the excitation light drives the short-wavelength resonance, the measured fluorescence intensity is strongly enhanced compared to that for the orthogonal particle orientation. This shows directly a strong electromagnetic coupling between the nanoparticles and the fluorophore. Additionally enhanced photochemical bleaching is observed due to the interaction of fluorophores with the particles. Using a rate model describing the fluorescence enhancement and the bleaching enhancement, an average value for the particle-induced increase in the radiative fluorescence rate is obtained, together with a lower limit for the averaged particle-induced field intensity enhancement factor. Received: 3 July 2001 / Revised version: 3 September 2001 / Published online: 15 October 2001     

Investigation of the photon statistics of parametric fluorescence in a traveling-wave parametric amplifier by means of self-homodyne tomography

Photon-number distributions for parametric fluorescence from a nondegenerate optical parametric amplifier are measured with a novel self-homodyne technique. These distributions exhibit the thermal-state character predicted by theory. However, a difference between the fluorescence gain and the signal gain of the parametric amplifier is observed. We attribute this difference to a change in the signal-beam profile during the traveling-wave pulsed amplification process.