摘要:

AbstractNear‐field scanning optical microscopy (NSOM) has been used to generate high resolution fluorescence images of cytoskeletal actin within fixed mouse fibroblast cells. Comparison with other microscopic methods indicates a transverse resolution well beyond that of confocal microscopy, and contrast far more revealing than in force microscopy. Effects unique to the near field are shown to be involved in the excitation of fluorescence, yet the resulting images remain readily interpretable. As an initial demonstration of its utility, the technique is used to analyse the actin‐based cytoskeletal structure between stress fibers and in cellular protrusions formed in the process of wound heal

ISSN:0966-9051    

DOI:10.1002/1361-6374(199309)1:3<129::AID-BIO1>3.0.CO;2-8

出版商:IOP Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

AbstractStress fibers in serum‐deprived fibroblasts provide an excellent system for studying the assembly dynamics of the actin‐based cytoskeleton. Previous studies suggested that fibers containing actin and myosin II are restructured via two alternative pathways: coupled solation‐contraction and disassembly without contraction. The former process appears to be regulated by Ca++, but regulation of the latter process has not been extensively explored. To assess the possible role of protein phosphorylation, we examined the dynamic behavior of stress fibers in cells treated with inhibitors of protein kinases. Swiss 3T3 fibroblasts were microinjected with fluorescent analogs of actin and myosin II and fiber dynamics monitored using light‐microscope imaging. Staurosporine and KT5926 caused reversible dispersal of stress fibers without contraction, along with dephosphorylation of the regulatory light chain of myosin II, LC20. In order to make direct comparisons between the dose responses of these biochemical and morphological effects, fiber disruption was quantitated using two independent measures: total edge strength in fluorescent images of actin and myosin II, and fiber length determined by automated object‐identification. Loss of stress fibers was shown to parallel LC20dephosphorylation. Quantitation of cytoskeletal organization provides a framework for testing relationships between structural events and potential biochemical regulator

ISSN:0966-9051    

DOI:10.1002/1361-6374(199309)1:3<136::AID-BIO2>3.0.CO;2-D

出版商:IOP Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

AbstractA method for analysing the localization of the Hox‐4 gene complex by three‐dimensional reconstruction of serial cross‐sections is presented. It allows the simultaneous visualization of three Hox expression domains on the same reconstructed embryo and the study of the effect of a graft of chicken polarizing region on thede novoexpression of the genes of the HOX‐4 complex. The method consists of registering the sections, segmenting the biological patterns and finally displaying the reconstructed embryo. The precise relationships between the transcript domains of the three genes can thus be assessed in either the normal wing bud or upon pattern modif

ISSN:0966-9051    

DOI:10.1002/1361-6374(199309)1:3<151::AID-BIO3>3.0.CO;2-N

出版商:IOP Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

AbstractIn the preparation of fluorescencein situhybridization (FISH) specimens, multiple probes, each tagged with a different fluorophore, are often used in combination. This allows several different molecular components to be visualized simultaneously. Usually their relative positions within the specimen are of scientific interest. Multi‐band fluorescence filter sets permit one to view multi‐labelled specimens in color.Given the emission spectra of available fluorophores, and the sensitivity spectra of available cameras and filter sets, it is impossible to obtain complete isolation of each fluorophore to a separate color channel. Even with a monochrome camera and carefully selected fluorophores and filters, spectral overlap still spreads each fluorophore across the color channels.This paper presents a processing method that effectively removes the spreading of fluorophore brightness among the color channels, thereby isolating each fluorophore to a single monochrome image. This simplifies subsequent image analysis, since each molecular component of the specimen can be analysed independently, but in register with the other components. Displayed images also are easier to interpret, since the colors appear more satura

ISSN:0966-9051    

DOI:10.1002/1361-6374(199309)1:3<159::AID-BIO4>3.0.CO;2-X

出版商:IOP Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

AbstractQuantitative fluorescence microscopy may present significant advantages compared to quantitative bright field microscopy, primarily due to the availability of a wide variety of dyes and probes which can specifically label cellular components. Fluorescence signals have excellent contrast with respect to the dark background, but the absolute intensity of the signal is relatively weak and therefore very sensitive light transducers must be employed. In general such devices compromise spatial and photometric resolution, limiting the usefulness of fluorescent dyes. Using a DNA stain (acriflavin) which permits imaging in both fluorescence and absorption microscopy modes, cell‐by‐cell comparisons of nuclear feature measurements were performed on cultured cells. We report that although there is more variance in fluoresence, comparable data can be achieved with either fluorescence or absorption microscopy mode using a digital camera which employs a scientific

ISSN:0966-9051    

DOI:10.1002/1361-6374(199309)1:3<166::AID-BIO5>3.0.CO;2-1

出版商:IOP Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

AbstractWe discuss the consequences of implementing image reconstruction techniques which essentially aim to normalize the three dimensional transfer function to unity. We consider the fluorescence case in detail for confocal and conventional systems and show that a good deal of care needs to be taken in interpreting the result when images from conventional microscopes are processed. Brightfield confocal imaging is also considered.

ISSN:0966-9051    

DOI:10.1002/1361-6374(199309)1:3<176::AID-BIO6>3.0.CO;2-Y

出版商:IOP Publishing Ltd    

年代:1993

数据来源: WILEY

ISSN:0966-9051    

DOI:10.1002/1361-6374(199309)1:3<185::AID-BIO7>3.0.CO;2-X

出版商:IOP Publishing Ltd    

年代:1993

数据来源: WILEY

ISSN:0966-9051    

DOI:10.1002/1361-6374(199309)1:3<186::AID-BIO8>3.0.CO;2-R

出版商:IOP Publishing Ltd    

年代:1993

数据来源: WILEY