ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03512.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

Activated sludge flocs are complex consortia of various micro‐organisms. The community structures of samples taken from municipal sewage treatment plants were characterized using fluorescently labelled, 16S and 23S rRNA‐targeted oligonucleotide probes in combination with confocal scanning laser microscopy (CSLM). In comparison with conventional epifluorescence microscopy, CSLM considerably improved the capability to visualize directly the spatial distribution of defined bacterial populations inside the sludge flocs. Analyses could be performed at high resolution undisturbed by problems such as autofluorescence or limited spatial resolution in thick samples. In addition, CSLM was used to analyse some structural properties of paraformaldehyde‐fixed activated sludge flocs, such as floc size and homogeneity. Typical floc sizes were found to be in the range between 5 and 50 μm. Whereas most of the flocs were completely colonized by bacteria, there were also examples of flocs containing gas bubbles or particles in the in

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03513.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

The form of the interference term image in scanning confocal and scanning conventional interference microscopes is identical in all respects including optical sectioning. This observation is used to obtain confocal images and surface profiles from conventional scanning interference microscope images.

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03514.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

Two‐photon excited fluorescence spectroscopy has been performed at a microscopic scale in combination with normal, white‐light microscopy. This gave simultaneously a spectral resolution of 20 nm and a temporal resolution of 20 ps, from a volume element less than 5 μm in all three dimensions. The sample was excited with the light from a continuously mode‐locked Ti: sapphire laser that was focused on the sample in a fluorescence microscope. A polychromator and a streak‐camera were used for detection. The method has been used on tissue, plant and paper samples. It has also been demonstrated how substances naturally occurring in the samples can be identified from their spectroscopic properties and the spatial distribution of these substances can be

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03515.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

The intracellular distribution of the anthracyclinic antibiotic adriamycin in living cultured cells has been investigated by confocal microscopy.In human melanoma cells (M14), adriamycin was localized inside the nuclei. When adriamycin‐treated M14 cells were allowed to recover in drug‐free medium, a complete efflux of the drug from the nucleus was revealed. In recovered cells, a weakly fluorescent signal was observed in the perinuclear region. When M14 cells were recovered in a medium containing colcemid, a microtubule depolymerizing agent, the drug transport from the nucleus to the cell periphery appeared to be inhibited, suggesting that the microtubule network is strongly involved in drug transport mechanisms. In multidrug‐resistant (MDR) cells the intracellular location of adriamycin was shown to be noticeably different from that of the parental wild‐type cells. In particular, in resistant human breast carcinoma cells (MCF‐7), adriamycin appeared to be exclusively located within the cytoplasm whereas the nuclei were shown to be completely negative. When adriamycin treatment was performed in association with MDR revertants, such as Lonidamine (inhibitor of the energy metabolism) or verapamil (inhibitor of the P‐glycoprotein efflux pump), a marked enhancement of the cytoplasmic signal was observed in resistant cells. Under these conditions, adriamycin appeared concentrated in the perinuclear region, but the nuclei were still negative. Confocal microscopy proved to be a very useful method for the study of the intracellular transport of fluorescent substances, such as anthracyclinic antibiotics, and for the investigation of the multidrug resistance phenomenon in t

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03516.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

A tilting device for biological specimens (rotation angle up to 2π), especially fluorescence‐labelled cell nuclei, was developed. It consists of a quartz glass capillary and a mounting adapter for the microscope stage. The applicability of the device was tested for several epifluorescence and confocal scanning laser fluorescence microscopes. The axis of rotation is perpendicular to the optical axis of the microscope. The capillary can be tilted around its axis at any desired angle or in equiangular steps. This can be done manually or by remote control using a stepping motor.The three‐dimensional (3‐D) image‐forming properties of the capillary system were experimentally examined using an inverse confocal scanning laser microscope. The results were compared with measurements obtained from the same microscope with the standard stage for plane slides with cover glasses. The measured point spread function suggested that in spite of aberration effects, the optical arrangement used allows a gain in the 3‐D resolution by tilting the object.A low‐cost, fully automated 3‐D imaging system was built on the basis of a conventional epifluorescence microscope with a cooled black‐and‐white CCD camera. The system was operated by a personal computer. The online visualization (‘movie’) of rotating objects indicates the fea

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03517.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

Two‐photon excitation fluorescence imaging is feasible with continuous wave lasers. Images of biological specimens are obtained by employing photon counting in conjunction with an increased recording time. The approach allows two‐photon three‐dimensional imaging of fluorescently labelled specimens with inexpensive l

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03518.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

The effect of refractive index mismatch on the image quality in two‐photon confocal fluorescence microscopy is investigated by experiment and numerical calculations. The results show a strong decrease in the image brightness using high‐aperture objectives when the image plane is moved deeper into the sample. When exciting at 740 nm and recording the fluorescence around 460 nm in a glycerol‐mounted sample using a lens of a numerical aperture of 1·4 (oil immersion), a 25% decrease in the intensity is observed at a depth of 9 μm. In an aqueous sample, the same decrease is observed at a depth of 3 μm. By reducing the numerical aperture to 1·0, the intensity decrease can be avoided at the expense of the overall resolution and signal intensity. The experiments are compared with the predictions of a theory that takes into account the vectorial character of light and the refraction of the wavefronts according to Fermat's principle. Advice is given concerning how the effects can be taken into account in

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03519.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

The tetrahedral tip is introduced as a new type of a probe for scanning near‐field optical microscopy (SNOM). Probe fabrication, its integration into a scheme of an inverted photon scanning tunnelling microscope and imaging at 30 nm resolution are shown. A purely optical signal is used for feedback control of the distance of the scanning tip to the sample, thus avoiding a convolution of the SNOM image with other simultaneous imaging modes such as force microscopy. The advantages of this probe seem to be a very high efficiency and its potential for SNOM at high lateral resolution below 30 n

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03520.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

A spectrometer has been developed for use with a confocal scanning laser microscope. With this unit, spectral information from a single point or a user‐defined region within the microscope specimen can be recorded. A glass prism is used to disperse the spectral components of the recorded light over a linear CCD photodiode array with 256 elements. A regulated cooling unit keeps the detector at 277 K, thereby allowing integration times of up to 60 s. The spectral resolving power, λ/Δλ, ranges from 350 at λ = 400 nm to 100 at λ = 700 nm. Since the entrance aperture of the spectrometer has the same size as the detector pinhole used during normal confocal scanning, the three‐dimensional spatial resolution is equivalent to that of normal confocal scanning. Light from the specimen is deflected to the spectrometer by a solenoid controlled mirror, allowing fast and easy switching between normal confocal scanning and spectrometer readings.With this equipment, studies of rodent liver specimens containing porphyrins have been made. The subcellular localization is of interest for the mechanisms of photo‐dynamic therapy (PDT) of malignant tumours. Spectroscopic detection is necessary to distinguish the porphyrin signal from other fluorescent components in the specimen. Two different substances were administered to the tissue, Photofrin, a haematoporphyrin derivative (HPD) and δ‐amino levulinic acid (ALA), a precursor to protoporphyrin IX and haem in the haem cycle. Both are substances under clinical trials for PDT of malignant tumours. Following administration of these compounds to the tissue, the potent photosensitizer and fluorescent compound Photofrin, or protoporphyrin IX, respectively, is accumulated. For our study Wistar/Furth rats were injected either with Photofrin or with ALA 3–5 h before they were killed. The organs were removed directly after, and snap‐frozen in carbon dioxide ice with isopentane. No further staining or fixation proced

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03521.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY