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.     

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.     

Measurement of steep edges and undercuts in confocal microscopy

Confocal microscopy is widely used to measure the surface topography of specimen with a precision in the micrometer range. The measurement uncertainty and quality of the acquired data of confocal microscopy depends on various effects, such as optical aberrations, vibrations of the measurement setup and variations in the surface reflectivity. In this article, the influence of steep edges and undercuts on measurement results is examined. Steep edges on the specimen's surface lead to a reduced detector signal which influences the measurement accuracy and undercuts cause surface regions, which cannot be captured in a measurement. The article describes a method to overcome the negative effects of steep edges and undercuts by capturing several measurements of the surface with different angles between the surface and the optical axis of the objective. An algorithm is introduced which stitches different angle measurements together without knowledge of the exact position and orientation of the rotation axis. Thus, the measurement uncertainty due to steep edges and undercuts can be avoided without expensive high-precision rotation stages and time consuming adjustment of the measurement setup.     

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.     

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.     

Noiseless amplification of images in a confocal cavity

We study the noiseless amplification of an optical image by means of a confocal cavity containing a parametric medium. We demonstrate, in the ideal situation, the possibility of preserving the signal-to-noise ratio while amplifying uniformly the entire image. Some specific effects, which may degrade the performances of the scheme, are taken into account. Received 23 March 2000     

Enhancement of lateral resolution in confocal self-interference microscopy

We describe confocal self-interference microscopy with enhanced lateral resolution. A uniaxial anisotropic crystal is used to cause interference between two linearly polarized beams that are reflected from the same pointlike object in the focal plane of the objective lens. Theory and the optimal design that maximizes the sensitivity of the interference signal are presented. A numerical experiment shows a 38% decrease in the lateral FWHM for simple confocal self-interference microscopy.     

Contrast of images of small-size objects observed through a scattering medium by the method of transmission confocal microscopy

Using the theory of vision in scattering media, we have developed a model of inhomogeneity observation in biological tissues by the method of transmission confocal microscopy. We introduce general formulas for calculating the contrast of the image of a local inhomogeneity with sinusoidal distribution of the transmission factor, which is located in a homogeneous scattering medium. The influence of observation system parameters on the maximum depth of inhomogeneity visibility is analyzed. Institute of Applied Physics, Russian Academy of Sciences, Russia. Translated from Izvestiya Vysshikh Uchebnykh Zavedenii, Radiofizika, Vol. 40, No. 8, pp. 980–990, August, 1997.     

Characterization of a confocal microscope for time-resolved photon counting fluorescence

A confocal microscope setup is developed for time-resolved fluorescence measurements. It is added to a traditional cuvette time-resolved setup, with a pumped Ti-Sa light source. The temporal resolution of 37 ps (FWHM) is not degraded, in comparison with the cuvette setup also described. These setups allow both decay lifetime and anisotropy relaxation time determination. Fluorescence correlation spectroscopy (FCS) is used to determine the observation point size. When associated with the calcium probe calcium green, calcium concentration in single cells can be determined in 10 ms by simultaneous acquisition of early and late fluorescence photons.     

Improving FRET efficiency measurement in confocal microscopy imaging

Spectral bleedthrough (SBT) ratio is dependent on the level of fluorescence intensity in confocal imaging. Precision Frster resonance energy transfer (FRET) algorithm corrects SBT ratio according to fluorescence intensity and avoids over-or under-estimation of SBT ratio. In this letter, we propose a new method to accurately measure the FRET efficiency of FRET plasmid in single living cells by combining the calculation of SBT in precision FRET algorithm with E-FRET formulae. We also use this method to measure the FRET efficiency of FRET-Bid, and find that in healthy A549 cells it is about 15%, which is verified by FRET acceptor photobleaching method.     

Optical inspection and characterization of microoptics using confocal microscopy

With the growing demand for microoptics in different areas the importance of the characterization increases. Methods for a fast defect detection in microlens arrays are developed. We present a technique where the confocal principle is applied for determining the variation and the absolute value of the focal length. Additionally, using a self–filtering method the deviation of the periodic structure of microlens arrays is investigated theoretically and experimentally. Point-like defects as well as aberrations have been detected. The introduced methods allow the fast, parallel characterization of microlens arrays.     

Image of a straight edge in confocal self-interference microscopy

An image of a straight edge in confocal self-interference microscopy (CSIM) is analyzed. Simulations of edge images based on a two-dimensional imaging equation are presented that show a 103% increase in edge gradient and a 43.1% decrease in the 10-90% width. The first experimental results, to our knowledge, for CSIM are presented and show good agreement with the simulation results and a 23% decrease in the 10-90% width.     

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.     

Visualizing endomembranes with confocal microscopy in protoplasts and cells of tobacco

The distribution and redistribution of the endoplasmic reticulum (ER) was studied with Confocal Laser Scanning Microscopy (CLSM) during the regeneration of tobacco protoplasts. In freshly isolated protoplasts the ER is cisternal in appearance, forming aggregates of plate-like structures clustered around the chloroplasts. During the cell's development, the ER profiles first lose the clustered appearance, then gradually tubular forms develop. Finally mixed population of some cisternal and tubular shapes and a lot of discrete spherical or cylindrical vesicles is spread through the cell's cytoplasm. The probe 3,3'-dihexyloxacarbocyanine iodide (DiOC₆(3)) proved to be a very efficient tool, allowing observations with a resolution of 0.2 μm.     

High-resolution confocal microscopy by saturated excitation of fluorescence

We demonstrate the use of saturated excitation in confocal fluorescence microscopy to improve the spatial resolution. In the proposed technique, we modulate the excitation intensity temporally and detect the harmonic modulation of the fluorescence signal which is caused by the saturated excitation in the center of the laser focus. Theoretical and experimental investigations show that the demodulated fluorescence signal is nonlinearly proportional to the excitation intensity and contributes to improve the spatial resolution in three dimensions beyond the diffraction limit of light.     

Improvements in confocal microscopy imaging using serrated divided apertures

Confocal microscopy with serrated divided apertures is presented. Compared with using two smooth D-shaped apertures in confocal microscopy, the serrated divided apertures can suppress the out-of-focus central bright spot, thus increasing the efficiency of rejection of scattered light. Diffraction analysis shows that the serrated apertures maintain the optical sectioning strength while attenuating the background coming from far from the focal plane. In addition, the signal to background ratio (S/B) is also improved.     

Improved axial resolution in confocal fluorescence microscopy using annular pupils

The axial image of a thick fluorescent layer is studied in a confocal microscope consisting of either circular or annular pupils. The response for circular pupils is sharper than that for annular pupils if a small pinhole detector is used. But when the size of the pinhole is larger than a certain value, the response in the latter case can become sharper than that in the former. This result implies that for a given detector of finite size, axial resolution in confocal fluorescence microscopy can be improved when an annular lens is used. Our experimental results qualitatively demonstrate the theoretical prediction. The strength of optical sectioning and the axial cross-section of the three-dimensional optical transfer function are also derived from the measured data.     

Nanoscale characterization of single Au nanorods by confocal microscopy

We analyzed the scattering patterns of individual Au nanorods detected by means of confocal interference scattering microscopy in combination with a higher order laser mode. We placed the Au nanorods at the interface between two dielectric media and examined the influence of different interfaces on the signal amplitude, the signal-to-noise ratio as well as on the precision of topological measurements. Approaching the index matching regime allows for topological measurements with high accuracy minimizing the acquisition time. We were also able to track the position and the orientation of particles embedded in water even when they were not thoroughly sticking to the glass surface. These results underscore the potential of the presented technique for applications in life sciences.