Optical sectioning in reciprocal fiber-optic based chromatic confocal microscope

A theoretical analysis for studying the optical sectioning in a chromatic confocal microscope employing the same single mode fiber for illumination and detection is described. The confocality achieved by different wavelengths due to the fixed size of the fiber core is investigated using diffraction theory. The detected signal level for the different wavelengths is also calculated. The ratio of the numerical aperture of the lens and the fiber is found to play a strong role in the performance of the system.     

Micromachined transmissive scanning confocal microscope

We have developed a miniature scanning confocal microscope that uses electrostatically actuated microlenses for focusing and scanning. Objective lenses, scanners, a pupil, and a pinhole of the confocal microscope are microfabricated and integrated into a volume smaller than 2 mm3 by stacking these components. Objective lenses are composed of two vertically cascaded polymer microlenses integrated into micromachined comb actuators. Raster scanning is implemented by electrostatically actuating each microlens in orthogonal directions. We have demonstrated reflection confocal imaging with 3-microm transverse resolution over a 100-microm field of view and a 0.38-mm working distance at lambda = 633 nm.     

Dual-wedge scanning confocal reflectance microscope

A confocal reflectance microscope has been developed that incorporates a dual-wedge scanner to reduce the size of the device relative to current raster scanning instruments. The scanner is implemented with two prisms that are rotated about the optical axis. Spiral and rosette scans are performed by rotating the prisms in the same or opposite directions, respectively. Experimental measurements show an on-axis lateral resolution of 1.6 microm and optical sectioning of 4.7 microm, which compares with a diffraction-limited resolution of 0.8 and 1.9 microm, respectively.     

Three-dimensional surface measurement using the confocal scanning microscope

The use of the depth discriminination property of the confocal scanning microscope for surface profiling has been adapted to provide a method of high-resolution three-dimensional surface profilometry. Measurements on a semiconductor specimen demonstrate the technique; depth variations of the order of 0.1 m are clearly resolved.     

共焦扫描光学显微镜的高分辨率

讨论了共焦扫描光学显微镜的高分辨率性质,指出共焦扫描显微镜由于采用点探测器,因而视场大大减小,信噪比大大提高,同时每幅图像逐点扫描形成,在光学系统信息能力不变的前提下,系统的空间域通带宽度增加和时域通带宽度减小。因而可成高分辨率的像,特别是其独特的深度分辨率特性使得可以实现光学断层扫描成像。给出了所研制的共焦扫描荧光显微镜所获得光学断层扫描图像     

Demonstration of a no-moving-parts axial scanning confocal microscope using liquid crystal optics

For the first time, to the best of authors’ knowledge, a no-moving-parts axial scanning confocal microscope (ASCM) system is designed and demonstrated using a combination of a large diameter liquid crystal (LC) lens and a classical microscope objective lens. By electrically controlling the 5 mm diameter LC lens, the 633 nm wavelength focal spot is moved continuously over a 48 μm range with a measured 3-dB axial resolution of 3.1 μm using a 0.65 numerical aperture (NA) micro-objective lens. The ASCM is successfully used to image an Indium Phosphide (InP) twin square optical waveguide sample with a 10.2 μm waveguide pitch and 2.3 μm height and width. Using fine analog electrical control of the LC lens, a super-fine sub-wavelength axial resolution of 270 nm is demonstrated. The proposed ASCM can be useful in various precision three-dimensional (3D) imaging and profiling applications.     

Investigation of the structure of water using a laser confocal scanning microscope

Measurements of water as the most available and vitally important element were performed using the laser confocal scanning microscope with the purpose of extending the range of its application. In this work, the measured Raman spectra of water obtained for different water samples—unprocessed, purified, and mineral, in different phase states, and after filtration and processing using different methods—are presented.     

共焦扫描荧光显微镜的信噪比与测量精度

激光扫描共焦显微术和多光子显微术等新的显微成像技术可以对厚的生物样品实现光学断层成像 ,因而在生物医学诊断领域具有重要的应用前境。在Fried的一维分辨度理论的基础上 ,系统地讨论了运用共焦扫描荧光显微术在进行光学断层成像时 ,其光学断层平面分辨度与信噪比之间的定量关系 ,建立了实际显微成像系统平面测量精度的定量计算方法。所得出的结果对于选择共焦扫描显微成像系统的最佳参数及评价所设计的显微成像系统的性能具有重要的意义。     

Imaging biological specimens with the confocal scanning laser microscope/macroscope

A new confocal scanning laser microscope/macroscope (cslm/M) has recently been developed. It combines in one instrument the high resolution capability of a confocal scanning beam microscope for imaging small specimens, with good resolution confocal imaging of macroscopic specimens. Some of its main features include: (a) 0.25 μm lateral resolution in the microscope mode and 5 μm lateral resolution in the macroscope mode; (b) a field of view that can vary from 25 μm × 25 μm to 75,000 μm × 75,000 μm; (c) capability for acquiring large data sets from 512 × 512 pixels to 2048 × 2048 pixels; (d) 0.5 μm depth resolution in the microscope mode and 200 μm depth resolution in the macroscope mode.

In this work the cslm/M was used to image whole biological specimens (> 5 m diameter), including insects which are ideal specimens for the macroscope. Specimens require no preparation, unlike scanning electron microscope (SEM) specimens which require a conductive coating. The specimens described in this paper are too large to be imaged in their entirety by a scanning beam laser microscope, however they can be imaged by slower scanning stage microscopes. In the macroscope mode the cslm/M was used to acquire a large number (e.g. 20–40) of confocal image slices which were then used to reconstruct a three-dimensional image of the specimen. High resolution images were collected by the cslm/M by switching to the microscope mode where high numerical aperture (NA) objectives were used to image a small area of interest. Reflected-light and fluorescence images of plant and insect specimens are presented which demonstrate the morphological details obtained in various imaging modes. A process for three-dimensional visualization of the data is described and images are shown.     

Spectrally encoded slit confocal microscopy

A simple and cost-effective method for real-time imaging in confocal microscopy is proposed. Spectrally encoded slit confocal microscopy (SESCoM) uses a spectral encoding technique together with a confocal slit aperture to achieve two-dimensional images. Simulation and experimental results of the SESCoM's axial and lateral performances are presented. The measured FWHM of the axial response is 1.15 mum when an objective with a NA of 0.95 is used. FWHMs of the lateral line spread functions are measured to be 236 and 244 nm along the x and y directions, respectively. Both the axial and the lateral experimental results agree well with the simulation results.     

Large field of view MEMS-based confocal laser scanning microscope for fluorescence imaging

Although confocal fluorescence microscopes are widely used in biology and have been proven to be promising diagnostic tools in dermatologic diagnostics, they are at present uncommon in medical practice. This is mainly due to high costs of acquisition and their large and complex outline. With the integration of a MEMS scanner we present a demonstration system of a confocal fluorescence laser scanning microscope which is affordable and portable. It has a field of view of 500 μm × 500 μm and is mainly composed of off-the-shelf components.     

Optical sectioning by two-pinhole confocal fluorescence microscopy

A two-pinhole axially superresolving confocal fluorescence imaging system is presented. Based on the concept of subtractive incoherent imaging, the system described here is equipped with a zero-focus complex-transmittance pupil filter in one of the collector paths. The optical sectioning capacity of the system is 25% superior to that of a free-pupil one-pinhole instrument.     

Fiber-coupled multiplexed confocal microscope

We describe a new parallel scanning mechanism for confocal microscopy that is inherently fiber-optic compatible and that retains the simplicity of the line scanning confocal microscope. The method works by employing an incoherent fiber-optic bundle that maps a line illumination pattern back on itself on double passing, while separating the fibers that carry photons from out-of-focus sample planes. The transformation permits efficient rejection of out-of-focus photons by a slit aperture.     

数字共焦显微技术

数字共焦显微技术是一种采用数学算法来实现共焦成像的技术 ,其功能与激光共焦显微技术相同。介绍了数字共焦显微技术的原理和结构组成。与激光共焦显微技术相比 ,数字共焦显微技术具有不产生光漂白、照明激发光谱宽、易于操作使用、工作方式灵活和价格便宜的特点 ,且其成像质量好于激光共焦显微技术。因此 ,数字共焦显微技术已广泛应用于许多领域且具有广阔的应用前景。     

Tracking a single fluorescent molecule with a confocal microscope

We consider the problem of tracking a single fluorescent molecule in both two and three dimensions using a confocal laser scanning microscope. An estimate of the position of the molecule is generated from the measured fluorescence signal through the use of parameter estimation theory. This estimate is used in a nonlinear controller designed both to track the position of the molecule and to provide good measurements for use in the estimation algorithm. The performance of the approach is investigated through numerical simulation for molecules undergoing diffusion and directed transport and the capabilities of the controller relative to experimental limitations are discussed.     

Guiding a confocal microscope by single fluorescent nanoparticles

Confocal optical microscopes offer unparalleled high sensitivity and three-dimensional (3D) imaging capability but require slow point-by-point scanning; they are inefficient for imaging moving objects. We propose a more efficient solution. Instead of indiscriminate scanning, we let the focus of the microscope pursue the object of interest such that no time is wasted on uninformative background, allowing us to visualize 3D trajectories of fluorescent nanoparticles in solution with millisecond temporal and ~200 nm spatial resolution.     

Method of obtaining optical sectioning by using structured light in a conventional microscope

We describe a simple method of obtaining optical sectioning in a conventional wide-field microscope by projecting a single-spatial-frequency grid pattern onto the object. Images taken at three spatial positions of the grid are processed in real time to produce optically sectioned images that are substantially similar to those obtained with confocal microscopes.     

Surface-state Stark shift in a scanning tunneling microscope

We report a quantitative low-temperature scanning tunneling spectroscopy (STS) study on the Ag(111) surface state over an unprecedented range of currents (50 pA to 6 microA) through which we can tune the electric field in the tunnel junction of the microscope. We show that in STS a sizable Stark effect causes a shift of the surface-state binding energy E0. Data taken are reproduced by a one-dimensional potential model calculation, and are found to yield a Stark-free energy E0 in agreement with recent state-of-the-art photoemission spectroscopy measurements.     

Active focus locking in an optically sectioning microscope utilizing a deformable membrane mirror

A significant challenge for in vivo imaging is to remove movement artifacts. These movements (typically due to either respiration and cardiac-related movement or surface chemical response) are normally limited to the axial direction, and hence features move in and out of the focal plane. This presents a real problem for high-resolution optically sectioned imaging techniques such as confocal and multiphoton microscopy. To overcome this we have developed an actively locked focus-tracking system based around a deformable membrane mirror. This has a significant advantage over more conventional focus-tracking techniques where the microscope objective is dithered, since the active element is not in direct, or indirect, contact with the sample. To examine the operational limits and to demonstrate possible applications for this form of focus locking, sample oscillation and movement are simulated for two different biological applications. We were able to track focus over a 400 microm range (limited by the range of the piezomounted objective) with a rms precision on the focal depth of 0.31 microm +/- 0.05 microm.