Polarized confocal theta microscopy

We propose a comprehensive treatment of theta microscopy based on dipole emission, which better describes fluorescence emission than the isotropic emission model, as fluorescence emission is often polarized. Formulas describing the point spread function for polarized confocal fluorescence theta microscopy are given. Examples are given and some advantages of polarized theta fluorescence microscopy are presented. To cite this article: O. Haeberlé et al., C. R. Physique 3 (2002) 1445–1450.     

Spectrally encoded confocal microscopy

An endoscope-compatible, submicrometer-resolution scanning confocal microscopy imaging system is presented. This approach, spectrally encoded confocal microscopy (SECM), uses a quasi-monochromatic light source and a transmission diffraction grating to detect the reflectivity simultaneously at multiple points along a transverse line within the sample. Since this method does not require fast spatial scanning within the probe, the equipment can be miniaturized and incorporated into a catheter or endoscope. Confocal images of an electron microscope grid were acquired with SECM to demonstrate the feasibility of this technique.     

Fibre-optic double-pass confocal microscopy

This paper reports on the construction and characterisation of a novel double-pass confocal scanning microscope that uses a four-port fibre coupler. The new imaging system is self-aligned, compact, vibration-free, and purely coherent. Compared with fibre-optic transmission- and reflection-mode confocal systems, the fibre-optic double-pass confocal imaging system exhibits higher resolution.     

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.     

Heterogeneous photobleaching in confocal microscopy

Photobleaching was studied during recording of confocal scanning laser microscopy. Studies on fluorescent gels of FITC-labeled dextran were used to evaluate differential bleaching along thez-axis. Differential bleaching along the z-axis was observed and it was seen that this was related to the numerical aperture of the objective in use. This points to the conclusion that photon energy flux density is an important parameter in photobleaching. To check if photon energy flux density heterogeneity is affected by local variation in the refractive index of the sample, photobleaching rates were calculated for different fluorescent objects (sections of seeds, animal cells stained with nuclear stains, immunocytochemistry preparations) and a pronounced similarity was found between photobleaching rates and DIC images.     

Optical tweezers for confocal microscopy

In confocal laser scanning microscopes (CLSMs), lasers can be used for image formation as well as tools for the manipulation of microscopic objects. In the latter case, in addition to the imaging lasers, the light of an extra laser has to be focused into the object plane of the CLSM, for example as optical tweezers. Imaging as well as trapping by optical tweezers can be done using the same objective lens. In this case, z-sectioning for 3D imaging shifts the optical tweezers with the focal plane of the objective along the optical axis, so that a trapped object remains positioned in the focal plane. Consequently, 3D imaging of trapped objects is impossible without further measures. We present an experimental set-up keeping the axial trapping position of the optical tweezers at its intended position whilst the focal plane can be axially shifted over a distance of about 15 μm. It is based on fast-moving correctional optics synchronized with the objective movement. First examples of application are the 3D imaging of chloroplasts of Elodea densa (Canadian waterweed) in a vigorous cytoplasmic streaming and the displacement of zymogen granules in pancreatic cancer cells (AR42 J). Received: 24 March 2000 / Revised version: 23 June 2000 / Published online: 11 October 2000     

Microlens arrays for confocal microscopy

A new; high resolution measuring system based on confocal microscopy has been developed for the evaluation of microlens arrays; in particular for applications in confocal microscopy itself. Lenslet arrays for parallel scanning and processing in confocal microscopy were designed as phase-matched Fresnel lenslets and fabricated by direct laser writing. Replica arrays were produced by ultraviolet embossing and hot embossing techniques. Fabricated arrays with a numerical aperture of 0.28 exhibited near diffraction limited performance and a focal length standard deviation of 120 nm in a nominal value of 250 μm. The technique developed represents a convenient and powerful technique for the characterization of lenslet arrays in general.     

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.     

Combined confocal and magnetic resonance microscopy

Confocal fluorescence optical microscopy and magnetic resonance microscopy are each used to study live cells in a minimally invasive way. Both techniques provide complementary information. Therefore, by examining cells simultaneously with both methodologies, more detailed information is obtained than is possible with each microscope individually. In this paper two configurations of a combined confocal and magnetic resonance microscope are described. The first configuration is capable of studying large single cells or three-dimensional cell agglomerates, whereas the second configuration is designed for the investigation of monolayers of mammalian cells. In both cases the sample compartment is part of a temperature regulated perfusion system. Images obtained with the combined system are shown forXenopus laevis oocytes, model JB6 tumor spheroids, and a single layer of Chinese hamster ovary cells. Finally, potential applications of the combined microscope are discussed.     

Antifading embedding media in confocal immunoflourescence microscopy

Fading or bleaching of fluorescence intensity during continuous illumination of stained objects is a serious problem in fluorescence microscopy. Fluorescence intensity as well as bleaching characteristics of dyes are dependent primarily upon physical parameters such as molecular constants (absorption rate and quantum efficiency), excitation energy and brightness (causes photon saturation), and environmental parameters (pH, ions, binding to proteins, etc.) that can strongly influence the properties of fluorochrome molecules. We have studied the effect of various antifading reagents on the behavior of the common dyes fluorescein isothiocyanate (FITC) and phycoerythrin (PE) using immunofluorescent-stained living cells in suspension or membrane-permeabilized dried cells as test systems. As expected, fading cannot be completely eliminated but may be reduced to varying degrees. In our hands, the most efficient antifading reagent for FITC isn-propyl gallate (NPG) dissolved in glycerol. No additive was found to retard fading, but complete dehydration of the cell suspension reduces this effect.     

Laser confocal microscopy for investigating small structures

The applicability of Raman scanning confocal microscopy for investigations in the field of condensed matter physics is demonstrated.     

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.     

Wavelength division multiplexed confocal microscopy using supercontinuum

We investigate both theoretically and experimentally wavelength division multiplexed confocal imaging by using white light supercontinuum. We show that with the optimized pinhole diameter an axial resolution of 0.75 μm and detection efficiency of 80% can be achieved. In addition, we applied the axial WDM confocal system to 3D surface measurement and the result agreed well with that measured by commercially available surface profilometer. The measured sensitivity of the system is 3.25 nm. Finally, we demonstrated lateral confocal imaging by using supercontinuum. An effective lateral scanning range of 130 μm was obtained.     

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.     

Upconversion fiber-optic confocal microscopy under near-infrared pumping

We present a simple upconversion fiber-optic confocal microscope design using a near-infrared laser for pumping of a rare-earth-doped glass powder. The nonlinear optical frequency conversion process is highly efficient with more than 2% upconversion fluorescence efficiency at a near-infrared pumping wavelength of 1.55 microm. The upconversion confocal design allows the use of conventional Si detectors and 1.55 microm near-infrared pump light. The lateral and axial resolutions of the system were equal to or better than 1.10 and 13.11 microm, respectively.     

共焦显微扫描探测技术的发展

传统的共焦显微探测多是采用单点机械扫描方式完成的,存在着扫描效率低的缺点,针对于此,一种多光束共焦显微系统成为当前的研究热点,通过不同方式对光束进行分割,使单点检测变为多路并行检测,提高了测量速度,减少了扫描过程中光源和振动噪声的影响,实现多光束的同步测量。介绍了几种典型的单光束和多光束共焦显微测量系统的原理、研究进展、发展方向及作者在该方面的研究。     

Zeeman laser-scanning confocal microscopy in turbid media

A novel Zeeman laser-scanning confocal microscope (ZLSCM) is proposed. It has the same configuration as the conventional laser-scanning confocal microscope (LSCM) in which a Zeeman laser in conjunction with a Glan-Thompson analyzer is used. In our system, the analyzer with the bandpass filter, which act simultaneously as a polarization gate and a coherence gate, enhance the collection efficiency of the weak-scattering photons and simultaneously suppress the multiple-scattering photons. The improvement in depth resolution of a ZLSCM in a scattering medium compared with that of a conventional LSCM is discussed and demonstrated experimentally.     

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 。     

Chromatic confocal microscopy with a finite pinhole size

Chromatic confocal microscopy has the advantage of short measurement times because of its parallel depth scan. As most white-light sources have limited optical output power, light-efficient setups are necessary. Using an extended detection pinhole is one way to improve light efficiency. We have calculated the effect of extended pinholes in chromatic confocal setups. We found that, for certain pinhole sizes, the FWHM of the confocal signal is nearly constant over a large wavelength interval.     

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.