Far-field fluorescence microscopy with repetitive excitation

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     

4Pi microscopy with linear fluorescence excitation

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.     

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.     

Two-photon fluorescence isotropic-single-objective microscopy

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.     

Wavefront sensing with an axicon

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.     

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...     

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.     

Wide-field fluorescence sectioning with hybrid speckle and uniform-illumination microscopy

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.     

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.     

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。     

Scanning two photon fluorescence microscopy with extended depth of field

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.     

结合虚拟单像素成像解卷积的双边照明光片荧光显微技术

光片荧光显微术(light-sheet fluorescence microscopy,LSFM)采用薄片光束从侧面激发样品,在垂直于光片方向上进行成像,具有成像速度快、光学层析能力强以及光漂白和光毒性低等优点,适用于对较大活体生物样品进行高质量、长时间三维动态观测.然而,传统高斯光束LSFM存在分辨率低和成像视场小的...     

轴锥镜成像系统的点扩散函数分析

为了使大景深光学镜头在工业视觉检测中的测量结果更准确,必须对中间图像进行复原,点扩散函数是图像复原的关键。根据轴锥镜最大无衍射距离公式设计了无衍射成像系统,以标量衍射理论为基础,在频域范围内利用稳定相法推导出系统的点扩散函数公式。利用准单色光理论,分析非相干光照明下的点扩散函数与锥镜夹角和离焦量的关系。仿真和实验结果表明:轴锥镜夹角越大,点扩散函数中心光强越强,衍射条纹越密;离焦像差对点扩散函数的影响与之相反。     

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.     

Subdiffraction resolution in far-field fluorescence microscopy

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.     

The sampling limit in fluorescence microscopy

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.     

Two-color two-photon 4Pi fluorescence microscopy

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.     

Three-dimensional locating of paraxial point source with axicon

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.     

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.     

Accuracy of spectrum estimate in fluorescence spectral microscopy with spectral filters

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.