Localization and quantification of epidermal growth factor receptors on single cells by confocal laser scanning microscopy.

We have established a method for quantifying binding of fluorescence-labeled growth factors to their receptors on single cells in situ with the confocal laser scanning microscope (CLSM). Biotinylated epidermal growth factor (EGF) coupled to phycoerythrin-labeled anti-biotin was used to compare the levels of fluorescence on three different cell types for which the number of EGF factors was known from Scatchard analysis of [125I]-EGF binding. The results showed that as few as 10,000 receptors/cell were detectable above back-ground. This method will provide a rapid and quantifiable alternative to autoradiography for ligand binding to single cells in situ.

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Epidermal growth factor binding sites in the mouse exocrine and endocrine pancreas shown by in vivo quantitative microautoradiography and confocal laser scanning microscopy

Acta Histochemica ◽

10.1016/s0065-1281(97)80008-6 ◽

1997 ◽

Vol 99 (1) ◽

pp. 55-62 ◽

Cited By ~ 3

Author(s):

Masahito Watanabe ◽

Nobuo Jo ◽

Yasuichiro Nishimura ◽

Masahiro Takafuchi ◽

Masato Kuno ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Confocal Laser Scanning Microscopy ◽

Binding Sites ◽

Laser Scanning ◽

Endocrine Pancreas ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Epidermal Growth

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3-D imaging of cells using a confocal laser scanning microscope and digital image processing

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102560 ◽

1988 ◽

Vol 46 ◽

pp. 96-97

Author(s):

Thomas M. Jovin ◽

Michel Robert-Nicoud ◽

Donna J. Arndt-Jovin ◽

Thorsten Schormann

Keyword(s):

Laser Scanning ◽

Confocal Laser Scanning Microscope ◽

Laser Scanning Microscopy ◽

Confocal Laser Scanning ◽

Confocal Laser ◽

Scanning Microscope ◽

Laser Scanning Microscope ◽

Biochemical Processes ◽

Adjacent Structures

Light microscopic techniques for visualizing biomolecules and biochemical processes in situ have become indispensable in studies concerning the structural organization of supramolecular assemblies in cells and of processes during the cell cycle, transformation, differentiation, and development. Confocal laser scanning microscopy offers a number of advantages for the in situ localization and quantitation of fluorescence labeled targets and probes: (i) rejection of interfering signals emanating from out-of-focus and adjacent structures, allowing the “optical sectioning” of the specimen and 3-D reconstruction without time consuming deconvolution; (ii) increased spatial resolution; (iii) electronic control of contrast and magnification; (iv) simultanous imaging of the specimen by optical phenomena based on incident, scattered, emitted, and transmitted light; and (v) simultanous use of different fluorescent probes and types of detectors.We currently use a confocal laser scanning microscope CLSM (Zeiss, Oberkochen) equipped with 3-laser excitation (u.v - visible) and confocal optics in the fluorescence mode, as well as a computer-controlled X-Y-Z scanning stage with 0.1 μ resolution.

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), confocal laser scanning microscopy (Eberl et al. 1997) or enumerated by flow cytometry (Tombolini et al. 1997). A further advantage of GFP, over other biomarkers, is the fact that no other energy source or substrate addition is required, other than oxygen during initial formation of the chromophore. Therefore, the GFP biomarker holds tremendous promise for elucidation of specific bacterial numbers and their behaviour, colonization, distribution, interaction, and movement in situ in a diversity of environmental sample types. Additionally, mutations have been introduced into the GFP gene in order to produce fluorescence signals with altered properties (Heim et al. 1994, Delagrave et al. 1995, Crameri et 1996, Heim and Tsien 1996). For example, one of the mutants (P4) is a

Recent Advances in Marine Biotechnology, Vol. 8 ◽

10.1201/9781482279986-29 ◽

2003 ◽

pp. 237-237

Keyword(s):

Energy Source ◽

Flow Cytometry ◽

Confocal Laser Scanning Microscopy ◽

Environmental Sample ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Confocal Laser Scanning ◽

Confocal Laser ◽

Initial Formation

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Monitoring the development of anaerobic biofilms using fluorescent in situ hybridization and confocal laser scanning microscopy

Water Science & Technology ◽

10.2166/wst.2000.0243 ◽

2000 ◽

Vol 41 (12) ◽

pp. 69-77 ◽

Cited By ~ 18

Author(s):

J. C. Araujo ◽

G. Brucha ◽

J. R. Campos ◽

R. F. Vazoller

Keyword(s):

In Situ Hybridization ◽

Confocal Laser Scanning Microscopy ◽

Fluorescent In Situ Hybridization ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Confocal Laser Scanning ◽

Scanning Microscopy ◽

Confocal Laser ◽

Anaerobic Consortium

In this study we investigated the development of anaerobic biofilm using a laboratory reactor. We were especially interested in comparing the organization of anaerobic cells (particularly those that are very common in domestic sewage sludge) in a hydrophilic (glass) versus a hydrophobic (polypropylene) surface. Fluorescent in situ hybridization (FISH) with domain and group specific probes directed against 16S ribosomal RNA were used to quantify microbial composition in the biofilm. FISH and confocal laser scanning microscopy (CLSM) were used to elucidate spatial distribution of microbes in the biofilms. Two experiments were carried out, one with pure methanogenic organisms and the other with a microbial anaerobic consortium. The pure methanogen cultures, Methanobacterium formicicum (DSM 1535); Methanosaeta concilli (DSM 3671) and Methanosarcina barkeri (DSM 800) were used to seed the modified Robbins Device (MRD) to allow the development of biofilms on polypropylene and glass surfaces during the 9-days experiment. The results showed that all the three species were colonizing both surfaces after two and nine days of experimental period. In another experiment, with polypropylene coupons only, MRD was seeded with a microbial anaerobic consortium and biofilm formation was studied during 11 days. At the end of this period, the biofilms generated were of uneven thickness with areas of minimal or no surface coverage and areas where the biofilm attained a thickness of 7.0 to 9.0 μm as revealed by CLSM. The results showed that the modified Robbins Device together with the fluorescent in situ hybridization and confocal laser scanning microscopy are suitable tools to study anaerobic biofilm development in different kinds of support materials.

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