scholarly journals Using laser scanning confocal microscopy as a guide for electron microscopic study: a simple method for correlation of light and electron microscopy.

1995 ◽  
Vol 43 (3) ◽  
pp. 329-335 ◽  
Author(s):  
X J Sun ◽  
L P Tolbert ◽  
J G Hildebrand
Keyword(s):  
Electron Microscopy ◽  
Confocal Microscopy ◽  
Microscopic Study ◽  
Electron Microscopic Study ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Synaptic Connections ◽  
Electron Microscopic ◽  
Simple Method ◽  
Scanning Confocal Microscopy

Anatomic study of synaptic connections in the nervous system is laborious and difficult, especially when neurons are large or have fine branches embedded among many other processes. Although electron microscopy provides a powerful tool for such study, the correlation of light microscopic appearance and electron microscopic detail is very time-consuming. We report here a simple method combining laser scanning confocal microscopy and electron microscopy for study of the synaptic relationships of the neurons in the antennal lobe, the first central neuropil in the olfactory pathway, of the moth Manduca sexta. Neurons were labeled intracellularly with neurobiotin or biocytin, two widely used stains. The tissue was then sectioned on a vibratome and processed with both streptavidin-nanogold (for electron microscopic study) and streptavidin-Cy3 (for confocal microscopic study) and embedded in epon/araldite. Interesting areas of the labeled neuron were imaged in the epon/araldite blocks with laser scanning confocal microscopy and then thin-sectioned at the indicated depth for electron microscopic study. This method provides an easy, reliable way to correlate three-dimensional light microscopic information with electron microscopic detail, and can be very useful in studies of synaptic connections.

scholarly journals A Rapid Method for Combined Laser Scanning Confocal Microscopic and Electron Microscopic Visualization of Biocytin or Neurobiotin-labeled Neurons

1998 ◽  
Vol 46 (2) ◽  
pp. 263-273 ◽  
Author(s):  
Xue J. Sun ◽  
Leslie P. Tolbert ◽  
John G. Hildebrand ◽  
Ian A. Meinertzhagen
Keyword(s):  
Confocal Microscopy ◽  
Laser Scanning ◽  
Fluorescent Dyes ◽  
Laser Scanning Confocal Microscopy ◽  
Single Step ◽  
Three Dimensions ◽  
Fluorescent Label ◽  
Electron Microscopic ◽  
Scanning Confocal Microscopy ◽  
Long Term Storage

Intracellular recording and dye filling are widely used to correlate the morphology of a neuron with its physiology. With laser scanning confocal microscopy, the complex shapes of labeled neurons in three dimensions can be reconstructed rapidly, but this requires fluorescent dyes. These dyes are neither permanent nor electron dense and therefore do not allow investigation by electron microscopy. Here we report a technique that quickly and easily converts a fluorescent label into a more stable and electron-dense stain. With this technique, a neuron is filled with Neurobiotin or biocytin, reacted with fluorophore-conjugated avidin, and imaged by confocal microscopy. To permit long-term storage or EM study, the fluorescent label is then converted to a stable electron-dense material by a single-step conversion using a commercially available ABC kit. We find that the method, which apparently relies on recognition of avidin's excess biotin binding sites by the biotin–peroxidase conjugate, is both faster and less labor intensive than photo-oxidation procedures in common use. The technique is readily adaptable to immunocytochemistry with biotinylated probes, as we demonstrate using anti-serotonin as an example.

A new approach to the study of apical meristem development using laser scanning confocal microscopy

1998 ◽  
Vol 76 (5) ◽  
pp. 899-904 ◽  
Author(s):  
Gordon D Lemon ◽  
Usher Posluszny
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Confocal Microscopy ◽  
Light Microscopy ◽  
Laser Scanning ◽  
Apical Meristem ◽  
Laser Scanning Confocal Microscopy ◽  
Shoot Apices ◽  
Scanning Confocal Microscopy ◽  
Scanning Electron

Epi-illumination light microscopy and scanning electron microscopy have been standard techniques for developmental studies of shoot apices. Recently, laser scanning confocal microscopy has gained popularity as a tool for biological imaging. We have adapted laser scanning confocal microscopy to study development in whole shoot apices. It was tested on angiosperm and fern apices using three fluorescent dyes; acriflavine, safranin O, and acid fuchsin, and compared with epi-illumination light microscopy and scanning electron microscopy. In all cases, acid fuchsin proved to be the best fluorochrome for examining shoot apices; having a high affinity for cell walls and nuclear material. The images produced with laser scanning confocal microscopy were sharper and clearer than images generated with epi-illumination light microscopy and scanning electron microscopy. Laser scanning confocal microscopy allows one to map patterns of cell division on the surface of an apical meristem, which is extremely difficult using other techniques such as scanning electron microscopy or epi-illumination light microscopy. Since the laser scanning light microscope records images digitally a method for digital plate production is described. Our methods can easily be applied to study the development of other plant structures on a cellular level such as root apical meristems, floral meristems, stomata, or trichomes, and reproductive organs in lower plants.Key words: confocal microscopy, apical meristem, development, fluorochrome, cytokinesis.

Vesicular system associated with spermiogenesis of bullfrog

1987 ◽  
Vol 45 ◽  
pp. 818-819
Author(s):  
K. C. Liu ◽  
S. F. Tsay
Keyword(s):  
Electron Microscopy ◽  
Microscopic Study ◽  
Electron Microscopic Study ◽  
Reproductive System ◽  
Seminiferous Tubule ◽  
Rana Catesbeiana ◽  
Routine Procedure ◽  
Electron Microscopic ◽  
Twisted Tubules ◽  
Electron Lucent

In the histologic and electron microscopic study of the male reproductive system of bullfrog, Rana catesbeiana, a vesicular system associated with spermiogenesis was observed. It appeared in the lumenal space of the seminiferous tubule (Fig. 1), in the heads of spermatids (Fig. 2), associated with the chromatins of the spermatid (Fig. 4). As deduced from sections, this vesicular system consisted of vesicles of various size or a large group of waving and twisted tubules (Fig. 3), After routine procedure of treatment for electron microscopy, the lumens of both of the vesicles and tubules were electron lucent.In human, vesicles and vesicular system associated with reproductive cell and tissue were reported. In abnormal spermiogenesis, flower-like body, actually vesicles, and giant vesicle associated with the head of spermatid were observed. In both cases the number of vesicle was limited from a single one to a few.

Laser Scanning Confocal Microscopy and Three-Dimesnsional Reconstruction of the Mitotic Spindles of Unequally Dividing Embryonic Cells

1990 ◽  
Vol 48 (3) ◽  
pp. 166-167
Author(s):  
J. Holy ◽  
G. Schatten
Keyword(s):  
Confocal Microscopy ◽  
Mitotic Spindle ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Confocal Microscopy ◽  
Two Dimensions ◽  
Embryonic Cells ◽  
Mitotic Spindles ◽  
Cleavage Division ◽  
Scanning Confocal Microscopy

One of the classic limitations of light microscopy has been the fact that three dimensional biological events could only be visualized in two dimensions. Recently, this shortcoming has been overcome by combining the technologies of laser scanning confocal microscopy (LSCM) and computer processing of microscopical data by volume rendering methods. We have employed these techniques to examine morphogenetic events characterizing early development of sea urchin embryos. Specifically, the fourth cleavage division was examined because it is at this point that the first morphological signs of cell differentiation appear, manifested in the production of macromeres and micromeres by unequally dividing vegetal blastomeres.The mitotic spindle within vegetal blastomeres undergoing unequal cleavage are highly polarized and develop specialized, flattened asters toward the micromere pole. In order to reconstruct the three-dimensional features of these spindles, both isolated spindles and intact, extracted embryos were fluorescently labeled with antibodies directed against either centrosomes or tubulin.

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