共查询到20条相似文献,搜索用时 31 毫秒
1.
A. Schönle S.W. Hell 《The European Physical Journal D - Atomic, Molecular, Optical and Plasma Physics》1999,6(3):283-290
We introduce the concept of repeatedly exciting an excited state of a photostable fluorescent entity to generate a nonlinear
fluorescence signal which is solely based on the linear susceptibility of the molecule. The excitation cycle between the fluorescent
state and another state prolongs the average lifetime of , with emphasis on those molecules that are in the center of the focus. The photons emitted by the long-lived molecules in
the center are recorded by a temporal filter and constitute fluorescence that depends nonlinearly on the excitation intensity.
Theoretical analysis reveals that this concept can provide three-dimensional imaging and improve the spatial resolution in
far-field fluorescence microscopy. We show that despite the presence of diffraction the effective focal waist can in principle
be narrowed down to the molecular scale at the expense of signal.
Received: 3 December 1998 相似文献
2.
Practical 4Pi microscopy has so far exclusively relied on multiphoton excitation of fluorescence, because the nonlinear suppression of contributions from higher-order sidelobes was mandatory for unambiguous axial superresolution. We show that novel lenses of 74 degrees semiaperture angle enable biological 4Pi microscopy with regular one-photon fluorescence excitation, thus increasing the signal and reducing system complexity and cost. An axial resolution of 95 nm, corresponding to a more than fourfold improvement over confocal microscopy, is verified in the imaging of microtubules in mammalian cells. 相似文献
3.
The use of a large apex-angle axicon for common-path interferometric wavefront sensing is proposed. The approach is a variant of point-diffraction interferometry bearing similarities to pyramidal wavefront sensing. A theoretical basis for wavefront sensing with an axicon is developed, and the outcomes of numerical simulations are compared to experimental results obtained with spherical and cylindrical ophthalmic trial lenses. It is confirmed that the axicon can be used for wavefront sensing, although its refraction may ultimately complicate and limit its operational range. 相似文献
4.
Three-dimensional holographic fluorescence microscopy 总被引:3,自引:0,他引:3
Schilling BW Poon TC Indebetouw G Storrie B Shinoda K Suzuki Y Wu MH 《Optics letters》1997,22(19):1506-1508
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. 相似文献
5.
Two-photon excitation provides efficient optical sectioning in three-dimensional fluorescence microscopy, independently of a confocal detection. In two-photon laser-scanning microscopy, the image resolution is governed by the volume of the excitation light spot, which is obtained by focusing the incident laser beam through the objective lens of the microscope. The light spot being strongly elongated along the optical axis, the axial resolution is much lower than the transverse one. In this Letter we show that it is possible to strongly reduce the axial size of the excitation spot by shaping the incident beam and using a mirror in place of a standard glass slide to support the sample. Provided that the contribution of sidelobes can be removed through deconvolution procedures, this approach should allow us to achieve similar axial and lateral resolution. 相似文献
6.
Quan Xiangyu Nitta Kouichi Matoba Osamu Xia Peng Awatsuji Yasuhiro 《Optical Review》2015,22(2):349-353
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... 相似文献
7.
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. 相似文献
8.
We describe a method of obtaining optical sectioning with a standard wide-field fluorescence microscope. The method involves acquiring two images, one with nonuniform illumination (in our case, speckle) and another with uniform illumination (in our case, randomized speckle). An evaluation of the local contrast in the speckle-illumination image provides an optically sectioned image with low resolution. This is complemented with high-resolution information obtained from the uniform-illumination image. A fusion of both images leads to a full resolution image that is optically sectioned across all spatial frequencies. This hybrid illumination method is fast, robust, and generalizable to a variety of illumination and imaging configurations. 相似文献
9.
Two-photon near- and far-field fluorescence microscopy with continuous-wave excitation 总被引:7,自引:0,他引:7
Hell SW Booth M Wilms S Schnetter CM Kirsch AK Arndt-Jovin DJ Jovin TM 《Optics letters》1998,23(15):1238-1240
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. 相似文献
10.
11.
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。 相似文献
12.
E.J. Botcherby 《Optics Communications》2006,268(2):253-260
We describe the design and implementation of a light efficient binary phase-only diffraction optical element which converts a laser beam into an annular ring with the loss of less than 25% of the overall power. The use of annular illumination permits us to obtain an increase in the axial extent of the point spread function of a microscope objective without compromising the lateral resolution. The light efficiency of the system permitted two photon fluorescence images with extended depth of field to be obtained. The combination of two such extended depth of field images obtained at different parallaxes allowed the generation of high resolution two photon stereo pairs. 相似文献
13.
14.
H. Schneckenburger 《Laser Physics》2006,16(5):799-807
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. 相似文献
15.
Subdiffraction resolution in far-field fluorescence microscopy 总被引:2,自引:0,他引:2
We overcame the resolution limit of scanning far-field fluorescence microscopy by disabling the fluorescence from the outer part of the focal spot. Whereas a near-UV pulse generates a diffraction-limited distribution of excited molecules, a spatially offset pulse quenches the excited molecules from the outer part of the focus through stimulated emission. This results in a subdiffraction-sized effective point-spread function. For a 1.4 aperture and a 388-nm excitation wavelength spatial resolution is increased from 150 +/- 8 nm to 106 +/- 8 nm with a single offset beam. Superior lateral resolution is demonstrated by separation of adjacent Pyridine 2 nanocrystals that are otherwise indiscernible. 相似文献
16.
Sampling in fluorescence microscopy is treated using the concept of the three-dimensional (3D) optical transfer function (OTF). The border of the OTF frequency surface defines the required minimum sampling. The shape of the OTF is derived from simple considerations and valid for far-field high numerical aperture, vector theory. Optimal regular sampling is achieved by a hexagonal grid in 2D, and corresponding hexagonal structures, body-centered cubic (bcc) and hexagonal close-packed (hcp) structures, in 3D. As compared to standard (rectilinear grid) sampling a reduction of 13.4% in 2D and 29.3% in 3D can be achieved with optimized sampling. This reduction in data size is also accompanied by an imaging speed improvement, a reduction of sample bleaching, and can lead to imaging with better signal to noise ratio. 相似文献
17.
Two-color two-photon 4Pi fluorescence microscopy 总被引:1,自引:0,他引:1
In 4Pi fluorescence microscopy the point-spread function is composed of a strong central lobe accompanied by interference sidelobes that produce artifacts in the image. We propose to combine two-color two-photon fluorescence microscopy and 4Pi fluorescence microscopy to overcome this sidelobe problem. Simulation results show that a single sharp fluorescence spot can be produced by use of two excitation wavelengths of 400 and 800 nm and detected at 350-nm emission wavelength. 相似文献
18.
This Letter presents a theoretical and experimental study of an axicon illuminated by an off-axis paraxial point source. The Fresnel diffraction integral is applied to show that a paraxial point source produces a Bessel beam. A simple analytical relationship is demonstrated between the location of the point source and the spatial frequency and the center of the resulting Bessel beam in the image plane of a camera. Finally, experimental verification is given by translating a point source of light along the optical axis of an axicon and comparing the resulting predicted and recorded beam intensity profiles. The resulting images are then analyzed to predict the location of the point source with excellent accuracy. 相似文献
19.
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. 相似文献
20.
We establish the accuracy of the spectrum that is estimated with an inexpensive fluorescence spectral microscope utilizing a small set of spectral filters [Soriano et al, Opt. Exp. 10, 1458–1464 (2002)]. The spectrum at an arbitrary image location of the fluorescent sample is estimated as a linear superposition of basis spectra that are derived by singular value decomposition (SVD) or principal component analysis (PCA) from a spectral library of fluorescence spectra. Estimation performance is analyzed as a function of library statistics, filter selection and sequencing, minimum negativity constraint and signal to noise ratio (SNR) of fluorescence image. We consider image SNR degradations that arise from weakening of image intensity, additive Gaussian noise, intensity-dependent Poisson noise and quantization errors. The recovery of specific spectral features like spectral resolution, general similarity and peak alignments, is assessed using Linfoot’s criteria of fidelity, structural content, and correlation. We found that estimation with SVD basis spectra is more robust against image noise than that with PCA basis spectra. However for high SNR images, accurate estimation is achieved more quickly with PCA basis spectra and with better response to the application of minimum negativity constraint. 相似文献