摘要:

SUMMARYThe effects of various system parameters on the imaging performance of confocal microscopy are discussed, including the effects of pinhole size, type of detector and imaging algorithms on signal‐to‐noise ratio. Careful consideration of these parameters allows the microscope user to obtain the best performance for particular applicati

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb03264.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYA new method of comparing the relative merits of different fluorophores that undergo relatively rapid irreversible photo‐inactivation is described. This method showed that the levels of fluorescent emission seen with both fluorescein isothiocyanate (FITC) and bodipy fl conjugated to streptavidin were similar when examined under conditions where they exhibited equal rates of irreversible photo‐inactivation. Bodipy fl and FITC give lower levels of cross‐talk into images of cells immunofluorescently stained with either rhodamine isothiocyanate (RITC) or tetramethyl rhodamine isothiocyanate (TRITC) than into images of cells stained with Texas red, under conditions where the three red fluorophores exhibited an equal level of sensitivity. Furthermore, bodipy fl gave much lower levels of cross‐talk into images of RITC‐stained cells than either FITC or Lucifer yellow. TRITC, but not RITC or Texas red, gave significant levels of cross‐talk into the green band‐pass filters used to visualize FITC and bodipy fl. From these results it seems that a combination of bodipy fl and RITC provides the best contrast when visualizing dual immunofluorescence with a confocal scanning laser microscope if the 488‐nm line of an argon ion laser is used as the ex

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb03265.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYAn atomic force microscope (AFM) was combined with a conventional optical microscope. The optical microscope proved to be very convenient for locating objects of interest. In addition, the high‐resolution AFM image can be compared directly with the traditional optical image. The instrument was used to study chromosome structures. High‐resolution chromosome images revealed details of the 30‐nm chromatide structure, confirming earlier electron microscopic observations. Chromosomes treated with trypsin revealed a banding pattern in height which is very similar to the optical image observed after staining with Giemsa. Furthermore, it is shown that the AFM can be used to locate DNA probes onin situhybridized chromosomes. Images of the synaptonemal complex isolated from rat spermatocytes revealed details that improve the understanding of the three‐dimensional structure of this

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb03266.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYLow‐energy electron microscopy (LEEM) and mirror electron microscopy (MEM) utilize a parallel beam of slow‐moving electrons backscattered from the specimen surface to form an image. If the electrons strike the surface an LEEM image is produced and if they are turned back just before reaching the surface an MEM image results. The applications thus far have been in surface physics. In the present study, applications of LEEM and MEM in the biological sciences are discussed. The preliminary results demonstrate the feasibility of forming images of uncoated cultured cells and cellular components using electrons in the threshold region (i.e. 0–10 V). The results also constitute a successful test of a novel beam‐separating system for LEEM

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb03267.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYA new method has been developed which allows quick‐freezingin situof primary, cardiac cell cultures grown to confluence on gas‐permeable membranes (Petriperm dishes). Small pieces of the growth substratum, with rhythmically beating myocardial cells, were slam‐frozen, without cryoprotectants, against the surface of a helium‐cooled copper block at approximately 16 K. The quality of the cellular cryopreservation, as judged by ultrastructural criteria, was studied in freeze‐substituted specimens processed for transmission electron microscopy. The ultrastructure of cryofixed cardiac cells was compared with that of unfrozen, chemically fixed samples. The severity of cryodistortions increased progressively with increasing distance from the point of first impact. Of particular interest were the dramatic alterations of the mitochondrial ultrastructure. The concept that the reticular and the outer mitochondrial membranes are intimately and strongly associated was clearly demonstrated. Optimally frozen material revealed cryopreserved ultrastructure of high quality. The method described appears to offer an ideal model system for correlating the information gained by phase‐contrast microscopy of living cell cultures with the ultrastructure of the same samples fixedin situby chemical or physical techniques. Cryofixation would be particularly useful for studying dynamic cellular processes associated with physiological and pathophysiological conditions, e.g. metabolic inhibition, anoxia and substrate

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb03268.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYAnaxial illumination has been used so far for either enhancement of visual acuity, or three‐dimensional (3‐D) measurements of static objects. Here we report the use of anaxial illumination for 3‐D tracking of fast‐moving objects, with only slight modifications to the standard microscope. To achieve this, we have investigated highspeed recording of stereoscopic images in two different

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb03269.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYModern collimator design for energy‐dispersive X‐ray detectors requires very accurate positioning of the crystal/collimator assembly in order to achieve the maximum solid angle of collection for the irradiated volume on the specimen. Thus it is important to have a method of checking the alignment of the detector when mounted on the microscope and under vacuum. This paper describes a number of techniques, principally X‐ray mapping, for performing such an alignment check. These techniques are applicable to windowless detectors as well as to those with integral windows which will support atmospheric pressure. Methods of obtaining the non‐standard modes of microscope operation suitable for this task are described, and some suggestions are made for ways of moving the crystal/collimator assembly and monitoring this movement while it is in p

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb03270.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb03263.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY