[6] Preparation of whole mounts and thick sections for confocal microscopy

1999 ◽  
pp. 84-107 ◽  
Author(s):  
Hisashi Hashimoto ◽  
Hiroshi Ishikawa ◽  
Moriaki Kusakabe
Keyword(s):  
Confocal Microscopy ◽  
Whole Mounts

scholarly journals In vivo confocal microscopy features and clinicohistological correlation of limbal nerve corpuscles

2019 ◽  
pp. bjophthalmol-2018-313199 ◽  
Author(s):  
Mouhamed Ali Al-Aqaba ◽  
Fady S Anis ◽  
Imran Mohammed ◽  
Anjali Dias Samarawickrama Yapa ◽  
Winfried M Amoaku ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Digital Pathology ◽  
In Vivo Confocal Microscopy ◽  
Eye Bank ◽  
Histological Features ◽  
Corneal Nerves ◽  
Normal Human ◽  
Epithelial Layers ◽  
Whole Mounts

AimsTo describe the in vivo confocal microscopy (IVCM) features of human limbal nerve corpuscles (LNCs) and correlate these with the histological features.MethodsWe examined 40 eyes of 29 healthy living subjects (17 female, 12 male; mean age=47.6) by IVCM. Four limbal quadrants were scanned through all epithelial layers and stroma to identify the LNCs and associated nerves. Ten fresh normal human corneoscleral discs from five deceased patients with a mean age of 67 years and 17 eye-bank corneoscleral rims with a mean age of 57.6 years were stained as whole mounts by the acetylcholinesterase (AChE) method to demonstrate LNCs and corneal nerves. Stained tissue was scanned in multiple layers with the NanoZoomer digital pathology microscope. The in vivo results were correlated to the histological findings.ResultsOn IVCM, LNCs were identified in 65% of the eyes studied and were mainly (84%) located in the inferior or superior limbal regions. They appeared either as bright (hyper-reflective) round or oval single structures within the hyporeflective, relatively acellular fibrous core of the palisades or were clustered in groups, often located anterior to the palisades of Vogt. They measured 36 µm in largest diameter (range 20–56 µm). The in vivo features were consistent with the histology, which showed LNCs as strongly AChE positive round or oval structures.ConclusionThe strong correlation with histology will enable use of IVCM to study LNCs in normal and disease conditions.

Detection of transcripts via fluorescencein-situ hybridization and confocal microscopy in whole mounts of roots ofArabidopsis thaliana

1997 ◽  
Vol 15 (1) ◽  
pp. 22-37 ◽  
Author(s):  
Serge Bauwens ◽  
Els Verhoeyen ◽  
Janice de Almeida Engler ◽  
Marc Van Montagu ◽  
Gilbert Engler
Keyword(s):  
Confocal Microscopy ◽  
Fluorescencein Situ Hybridization ◽  
Ofarabidopsis Thaliana ◽  
Whole Mounts

Immunocytochemical localization of multiple cholecystokinin-like peptides in the stomatogastric nervous system of the crab Cancer borealis.

1995 ◽  
Vol 198 (1) ◽  
pp. 263-271 ◽  
Author(s):  
A E Christie ◽  
D Baldwin ◽  
G Turrigiano ◽  
K Graubard ◽  
E Marder
Keyword(s):  
Nervous System ◽  
Confocal Microscopy ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Immunocytochemical Localization ◽  
Stomatogastric Nervous System ◽  
Cancer Borealis ◽  
Scanning Confocal Microscopy ◽  
Whole Mounts

Three anti-cholecystokinin antibodies were used to label the stomatogastric nervous system of the crab Cancer borealis. Labeled tissues were examined as whole mounts using laser scanning confocal microscopy. Although each of the anti-cholecystokinin antibodies labeled a variety of structures within the stomatogastric nervous system (including somata, fibers and neuropil), the pattern of labeling produced by each antibody was distinct. These results indicate that there is a family of cholecystokinin-like molecules that are differentially distributed among a subpopulation of the neurons in the stomatogastric nervous system of Cancer borealis.

Mapping CGRP-IR innervation of male mice stomach with Neurolucida 360 v1

2021 ◽  
Author(s):  
Duyen not provided Nguyen ◽  
Jichao Ma
Keyword(s):  
Confocal Microscopy ◽  
Calcitonin Gene Related Peptide ◽  
Male Mice ◽  
Muscular Layer ◽  
Related Peptide ◽  
Topographical Organization ◽  
Calcitonin Gene ◽  
Whole Mounts ◽  
Immunoreactive Axons

This protocol describes the process of using Neurolucida 360 software to map the topographical organization of Calcitonin gene related peptide – immunoreactive axons and terminals in the muscular layer of mice stomach. Stomachs were removed, layers were separated and gone under immunohistochemistry as whole mounts, then scanned using confocal microscopy.

Three-Dimensional Reconstruction Using Stereo Pairs from Serial Thick Sections

1977 ◽  
Vol 35 ◽  
pp. 562-563
Author(s):  
Robert Glaeser ◽  
Thomas Bauer ◽  
David Grano
Keyword(s):  
Three Dimensional ◽  
Dimensional Structure ◽  
Serial Sections ◽  
Thin Sections ◽  
3 Dimensional ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Dimensional Reconstruction ◽  
Stereo Imagery ◽  
Whole Mounts

In transmission electron microscopy, the 3-dimensional structure of an object is usually obtained in one of two ways. For objects which can be included in one specimen, as for example with elements included in freeze- dried whole mounts and examined with a high voltage microscope, stereo pairs can be obtained which exhibit the 3-D structure of the element. For objects which can not be included in one specimen, the 3-D shape is obtained by reconstruction from serial sections. However, without stereo imagery, only detail which remains constant within the thickness of the section can be used in the reconstruction; consequently, the choice is between a low resolution reconstruction using a few thick sections and a better resolution reconstruction using many thin sections, generally a tedious chore. This paper describes an approach to 3-D reconstruction which uses stereo images of serial thick sections to reconstruct an object including detail which changes within the depth of an individual thick section.

Two-photon excitation microscopy in cellular biophysics

1995 ◽  
Vol 53 ◽  
pp. 62-63
Author(s):  
David W. Piston ◽  
Brian D. Bennett ◽  
Robert G. Summers
Keyword(s):  
Confocal Microscopy ◽  
Laser Beam ◽  
Duty Cycle ◽  
Excited Electronic State ◽  
Input Power ◽  
Two Photon ◽  
Simultaneous Absorption ◽  
Photon Excitation ◽  
Very High ◽  
Two Photon Excitation

Two-photon excitation microscopy (TPEM) provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging and photochemistry. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet. In practice, two-photon excitation is made possible by the very high local instantaneous intensity provided by a combination of diffraction-limited focusing of a single laser beam in the microscope and the temporal concentration of 100 femtosecond pulses generated by a mode-locked laser. Resultant peak excitation intensities are 106 times greater than the CW intensities used in confocal microscopy, but the pulse duty cycle of 10-5 maintains the average input power on the order of 10 mW, only slightly greater than the power normally used in confocal microscopy.

Measurement of spontaneous neuronal membrane activity by time lapse confocal microscopy

1995 ◽  
Vol 53 ◽  
pp. 972-973
Author(s):  
R H. Selinfreund ◽  
A. H. Cornell-Bell
Keyword(s):  
Membrane Potential ◽  
Confocal Microscopy ◽  
Patch Clamp ◽  
Electrical Activity ◽  
Time Lapse ◽  
Neuronal Firing ◽  
Neuronal Membrane ◽  
Pituitary Cells ◽  
Patch Clamp Recording ◽  
Spontaneous Electrical Activity

Cellular electrophysiological properties are normally monitored by standard patch clamp techniques . The combination of membrane potential dyes with time-lapse laser confocal microscopy provides a more direct, least destructive rapid method for monitoring changes in neuronal electrical activity. Using membrane potential dyes we found that spontaneous action potential firing can be detected using time-lapse confocal microscopy. Initially, patch clamp recording techniques were used to verify spontaneous electrical activity in GH4\C1 pituitary cells. It was found that serum depleted cells had reduced spontaneous electrical activity. Brief exposure to the serum derived growth factor, IGF-1, reconstituted electrical activity. We have examined the possibility of developing a rapid fluorescent assay to measure neuronal activity using membrane potential dyes. This neuronal regeneration assay has been adapted to run on a confocal microscope. Quantitative fluorescence is then used to measure a compounds ability to regenerate neuronal firing.The membrane potential dye di-8-ANEPPS was selected for these experiments. Di-8- ANEPPS is internalized slowly, has a high signal to noise ratio (40:1), has a linear fluorescent response to change in voltage.

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