Dual-axes differential confocal microscopy with high axial resolution and long working distance

We discover that the slight transverse offset of a point detector results in a shift of the axial intensity response curve in a dual-axes confocal microscopy (DCM). Based on this, we propose a new dual-axes differential confocal microscopy (DDCM) with high axial resolution and long working distance, in which two point detectors are placed symmetrically about the collection axis. And a signal is obtained through the differential subtraction of two signals received simultaneously by the two point detectors. Theoretical analyses and preliminary experiments indicate that DDCM is feasible and suitable for the high precision tracing measurement of microstructures and surface contours.     

Transmission confocal laser scanning microscopy with a virtual pinhole based on nonlinear detection

We present a transmission-mode confocal laser scanning microscope system based on the use of second-harmonic generation (SHG) for signal detection. Our method exploits the quadratic intensity dependence of SHG to preferentially reveal unscattered signal light and reject out-of-focus scattered background. The SHG crystal acts as a virtual pinhole that remains self-aligned without the need for descanning.     

Combined phase-sensitive acoustic microscopy and confocal laser scanning microscopy

Combined phase-sensitive acoustic microscopy (PSAM) at 1.2 GHz and confocal laser scanning microscopy (CLSM) in reflection and fluorescence has been implemented and applied to polymer blend films and fluorescently labeled fibroblasts and neuronal cells in order to explore the prospects and the various contrast mechanisms of this powerful technique. Topographic contrast is available for appropriate samples from CLSM in reflection and, with significantly higher precision, from the acoustic phase images. Material contrast can be gained from acoustic amplitude V(z) graphs. In the case of the biological cells investigated, the optical and acoustic images are very different and exhibit different features of the samples.     

Experimental investigation of fibre-optical confocal scanning microscopy: Including a comparison with pinhole detection

Reported in this paper are the experimental results on fibre-optical confocal scanning microscopy (FOCSM). We have performed various measurements in the FOCSM involving axial responses, edge responses, signal levels and interference axial responses in order to demonstrate the differences in confocal scanning imaging between using optical fibres and using pinholes. Experimentally measured results are compared with our theoretical predictions, revealing good agreements. It is shown convincingly that the FOCSM is a purely coherent imaging system as predicted in our previous theory.     

共聚焦显微镜激光高速扫描控制系统设计及实现

 在传统光学扫描方法基础上,有效地将检流式与共振式光学扫描振镜结合起来,提出一种速度可达4 M/s采样率的高速激光扫描方法,并基于单片机系统设计搭建了系统控制硬件平台,编写了上位机端和下位机端应用软件。实验结果表明:该控制系统扫描速度快,性能稳定可靠,能够应用于共聚焦显微镜,实现实时扫描成像 。     

Spin detection and manipulation with scanning tunneling microscopy

Over the past few decades, spin detection and manipulation at the atomic scale using scanning tunneling microcopy has matured, which has opened the possibility of realizing spin-based functional devices with single atoms and molecules.This article reviews the principle of spin polarized scanning tunneling microscopy and inelastic tunneling spectroscopy,which are used to measure the static spin structure and dynamic spin excitation, respectively. Recent progress will be presented, including complex spin structure, magnetization of single atoms and molecules, as well as spin excitation of single atoms, clusters, and molecules. Finally, progress in the use of spin polarized tunneling current to manipulate an atomic magnet is discussed.     

Near-infrared laser scanning confocal microscopy and its application in bioimaging

Near-infrared (NIR) fluorescence imaging is an important imaging technology in deep-tissue biomedical imaging and related researches, due to the low absorption and scattering of NIR excitation and/or emission in biological tissues. Laser scanning confocal microscopy (LSCM) plays a significant role in the family of fluorescence microscopy. Due to the introduction of pinhole, it can provide images with optical sectioning, high signal-to-noise ratio and better spatial resolution. In this study, in order to combine the advantages of these two techniques, we set up a fluorescence microscopic imaging system, which can be named as NIR-LSCM. The system was based on a commercially available confocal microscope, utilizing a NIR laser for excitation and a NIR sensitive detector for signal collection. In addition, NIR fluorescent nanoparticles (NPs) were prepared, and utilized for fluorescence imaging of the ear and brain of living mice based on the NIR-LSCM system. The structure of blood vessels at certain depth could be visualized clearly, because of the high-resolution and large-depth imaging capability of NIR-LSCM.     

Shrinking of ultrathin polyelectrolyte multilayer capsules upon annealing: A confocal laser scanning microscopy and scanning force microscopy study

Heating-induced morphological changes of micrometer size capsules prepared by step-wise deposition of oppositely charged polyelectrolytes onto melamine formaldehyde (MF) latex particles and biological cells with subsequent dissolution of the core have been investigated by confocal laser scanning microscopy (CLSM) and scanning force microscopy (SFM). For poly(styrenesulfonate-Na salt)/poly(allylamine hydrochloride) polyelectrolyte capsules a remarkable heating-induced shrinking is observed. An increase of the wall thickness corresponding to the capsule diameter decrease is found. The morphology of these microcapsules after temperature treatment is characterized. The thickening of the polyelectrolyte multilayer is interpreted in terms of a configurational entropy increase via polyanion-polycation bond rearrangement. Received 20 January 2000     

New prospects in dermatology: Fiber-based confocal scanning laser microscopy

Confocal scanning laser microscopy (CSLM) constitutes an optical, noninvasive method providing visualization of tissue architecture with resolution similar to that of light microscopy. In dermatology, confocal imaging enables in vivo measurements of surface and subsurface skin microstructures. Skin annexes, as well as cutaneous cells from different epidermal layers, can be easily distinguished; their change in morphology from skin surface to the papillary dermis can be observed. Therefore, CSLM possesses a high potential for diagnostical purposes and dermatological research. The aspect of normal skin in contrast to the pathogenic state can be exposed. In our studies, we used in vivo fluorescence CSLM for morphometric analysis of healthy human skin and for imaging a number of clinically relevant inflammatory, proliferative, and neoplastic skin disorders. We report the ability to produce high-resolution histoimages of normal and pathological epidermis using this nondestructive visualization technique. Changes in keratinocyte size, shape, and morphology, as well as changes in the distribution pattern of the fluorescent emission of the dye, can be detected. Furthermore, novel fiber optic elements support a flexible handling of the rigid microscopic gadgetry. Four clinical examples of implementation were elected and instanced for demonstration.     

Attoliter detection volumes by confocal total-internal-reflection fluorescence microscopy

We report a confocal total-internal-reflection fluorescence (TIRF) microscope that generates a detection volume for analyte molecules of less than 5 al (5 x 10(-18) l) at a water-glass interface. Compared with conventional confocal microscopy, this represents a reduction of almost 2 orders of magnitude, which is important in isolating individual molecules at high analyte concentrations, where many biologically relevant processes occur. Diffraction-limited supercritical focusing and fluorescence collection is accomplished by a parabolic mirror objective. The system delivers TIRF images with excellent spatial resolution and detects single molecules with a high signal-to-background ratio.     

差动共焦显微成像高稳定三维扫描技术与系统

激光差动共焦显微镜具备高空间分辨率特点,但因其逐点扫描成像方式,扫描时间长,易受三维扫描系统不稳定和环境干扰等影响,产生系统漂移,影响仪器的空间分辨率。利用楔块机构高稳定特点,结合刹车机构的自由抱闸特性,设计了一种新型的轴向升降机构,由此构建了结构更具稳定特性的电动三维扫描系统。稳定性实验验证在搭建的激光差动共焦显微镜上进行,经过监测系统在90min内的轴向位置,轴向漂移小于50nm,与原三维扫描系统漂移140nm对比,漂移速度明显减慢,稳定性有显著提升,进而明显改善了差动共焦显微成像效果。     

Geometric phase-shifting for low-coherence interference microscopy

A low-coherence Linnik interference microscope using high numerical aperture optics has been constructed. The system uses a tungsten halogen lamp and Köhler illumination, with separate control over field and aperture stops, so that experiments can be conducted with a range of different operating conditions. The novel feature of the system is the use of an achromatic phase-shifter operating on the principle of the geometric phase, achieved by using a polarising beam splitter, a quarter wave plate and a rotating polariser. Image information is extracted from the visibility of the fringes, the position of the visibility peak along the scanning axis yielding the height of the test surface at the corresponding point.     

Phase retrieval in low-coherence interferometric microscopy

We present compensating methods that address inherent errors in quantitative phase reporting for low-coherence interferometric techniques. A brief theoretical treatment of the problem and experimental validation using spectral domain phase microscopy demonstrate mitigation of the degrading effects of phase leakage on accurate measurement of optical path length in the vicinity of closely spaced reflectors. This result has direct implications for phase-sensitive interferometry techniques, such as Doppler imaging, as well as amplitude-based quantitative reporting. Corrected phase retrieval is demonstrated for conversion of interferometric phase to optical path length in cell surface deflections of beating cardiomyocytes.     

Improvement of confocal microscope performance by shaped annular beam and heterodyne confocal techniques

In order to further improve the performance of a confocal microscope (CM) used for measurement of surface profiles and 3D microstructures, a shaped annular beam heterodyne confocal measurement method based on annular pupil filter technique and reflection confocal microscopy, is proposed to expand the measurement range and to improve the defocused property of CM. The approach proposed uses a confocal dual-receiving light path arrangement and a heterodyne subtraction of two signals received from detectors with axial offset to enable CM to be used for bipolar absolute measurement and to improve the defocused property of CM, and it uses the annular pupil filter technique to produce a binary optical shaped annular beam, which expands the measurement range by expanding the full-width at half-maximum of intensity curve received from two detectors in a heterodyne confocal microscopy system. Theoretical analyses and experimental results indicate that a shaped annular beam heterodyne microscope has a measurement range expanded from 4 to 14 μm, achieved an axial resolution of 2 nm and improved the defocused property, when ε=0.5 and NA=0.65. It can be therefore concluded that the shaped annular beam heterodyne confocal measuring method proposed is a new approach to ultraprecision measurement of surface profiles and 3D microstructures.     

Laser-induced thermal-acoustic velocimetry with heterodyne detection

Laser-induced thermal acoustics (LITA) was used with heterodyne detection to measure simultaneously and in a single laser pulse the sound speed and flow velocity of NO>(2) -seeded air in a low-speed wind tunnel up to Mach number M =0.1 . The uncertainties of the velocity and the sound speed measurements were ~0.2 m/s and 0.5%, respectively. Measurements were obtained through a nonlinear least-squares fit to a general, analytic closed-form solution for heterodyne-detected LITA signals from thermal gratings. Agreement between theory and experiment is exceptionally good.     

Subtractive imaging in confocal scanning microscopy using a CCD camera as a detector

We report a scheme for the detector system of confocal microscopes in which the pinhole and a large-area detector are substituted by a CCD camera. The numerical integration of the intensities acquired by the active pixels emulates the signal passing through the pinhole. We demonstrate the imaging capability and the optical sectioning of the system. Subtractive-imaging confocal microscopy can be implemented in a simple manner, providing superresolution and improving optical sectioning.     

Imaging voltage-dependent cell motions with heterodyne Mach-Zehnder phase microscopy

We describe a heterodyne Mach-Zehnder interferometric microscope capable of quantitative phase imaging of biological samples with subnanometer sensitivity and frame rates up to 10 kHz. We use the microscope to image cultured neurons and measure nanometer-scale voltage-dependent motions in cells expressing the membrane motor protein prestin.     

4Pi confocal microscopy with alternate interference

We introduce 4Pi confocal microscopy with destructive interference of converging waves. Linear lobe deconvolution as well as nonlinear restoration of 4Pi confocal raw data with their point-spread functions (PSF's) leads to almost-identical images, irrespective of whether the 4Pi confocal PSF relies on constructive or destructive interference. Three-dimensional imaging of microtubules of a mouse fibroblast cell yielded an axial resolution near 100-nm in both cases. Moreover, restoration of 4Pi confocal images of the same object with alternate phases is introduced as a powerful test for (nonlinear) image restoration.     

Multiframe full-field heterodyne digital holographic microscopy

Digital holographic microscopy using multiframe full-field heterodyne technology is discussed in which two acousto-optic modulators are applied to generate low-frequency heterodyne interference and a high-speed camera is applied to acquire multiframe full-field holograms. We use a temporal frequency spectrum analysis algorithm to extract the object's information. The twin-image problem can be solved and the random noise can be significantly suppressed. The relationship between the frame number and the reconstruction accuracy is discussed. The typical objects of microlenses and biology cells are reconstructed well with 100-frame holograms for illustration.