Quantitative immunocytochemistry using an image analyzer. I. Hardware evaluation, image processing, and data analysis

doi:10.1016/0165-0270(88)90125-2

Journal of Neuroscience Methods

Volume 26, Issue 1, November 1988, Pages 1-23

https://doi.org/10.1016/0165-0270(88)90125-2 Get rights and content

Abstract

In this review we describe how video-based image analysis systems are used to measure immunocytochemically labeled tissue. The general principles underlying hardware and software procedures are emphasized. First, the characteristics of image analyzers are described, including the densitometric measure, spatial resolution, gray scale resolution, dynamic range, and acquisition and processing speed. The errors produced by these instruments are described and methods for correcting or reducing the errors are discussed. Methods for evaluating image analyzers are also presented, including spatial resolution, photometric transfer function, short- and long-term temporal variability, and measurement error. The procedures used to measure immunocytochemically labeled cells and fibers are then described. Immunoreactive profiles are imaged and enhanced using an edge sharpening operator and then extracted using segmentation, a procedure which captures all labeled profiles above a threshold gray level. Binary operators, including erosion and dilation, are applied to separate objects and to remove artifacts. The software then automatically measures the geometry and optical density of the extracted profiles. The procedures are rapid and efficient methods for measuring simultaneously the position, geometry, and labeling intensity of immunocytochemically labeled tissue, including cells, fibers, and whole fields. A companion paper describes non-biological standards we have developed to estimate antigen concentration from the optical density produced by antibody labeling (Nabors et al., 1988).

References (57)

L.F. Agnati et al.
Computer-assisted morphometry and microdensitometry of transmitter-identified neurons with special reference to the mesostriatal dopamine pathway. I. Methodological aspects
Acta Physiol. Scand.
(1984)
L.F. Agnati et al.
Computer-assisted morphometry and microdensitometry of transmitter-identified neurons with special reference to the mesostriatal dopamine pathway. II. Further studies on the effects of the GM₁ ganglioside on the degenerative and regenerative features of mesostriatal dopamine neurons
Acta Physiol. Scand.
(1984)
G. Alexander et al.
Quantitative computer analysis of autoradiographs utilizing a charge-coupled device solid-state camera
J. Neurosci. Methods
(1984)
C.A. Altar et al.
Computer assisted video analysis of [³H]spiroperidol binding autoradiographs
J. Neurosci. Methods.
(1984)
H. Baker et al.
Strain differences in pituitary prolactin content: relationship to number of hypothalamic dopamine neurons
Brain Res.
(1985)
D.G. Baskin et al.
An inexpensive microcomputer digital imaging system for densitometry: quantitative autoradiography of insulin receptors with ¹²⁵I and LKB ultrofilm
J. Neurosci. Methods
(1986)
R.H. Benno et al.
Quantitative immunocytochemistry of tyrosine hydroxylase in rat brain. I. Development of a computer assisted method using the peroxidase-antiperoxidase technique
Brain Res.
(1982)
R.H. Benno et al.
Quantitative immunocytochemistry of tyrosine hydroxylase in rat brain. II. Variations in the amount of tyrosine hydroxylase among individual neurons of the locus coeruleus in relationship to neuronal morphology and topography
Brain Res.
(1982)
D.J. Berck et al.
Microcomputer-assisted densitometry for quantitative receptor autoradiography
J. Neurosci. Methods
(1985)
S. Bradbury
Automatic image analyzers and their use in anatomy
Prog. Clin. Biol. Res.
(1981)

K.P. Briski et al.

Effect of ovariectomy on the hypothalamic content of immunoreactive gonadotropin-releasing hormone in the female mouse as revealed by quantitative immunocytochemistry and radioimmunoassay

Am. J. Anat.

(1983)

M.D. Cassell et al.

Computer-assisted image analysis of the distributions of peptidergic terminals in the central nucleus of the amygdala: a preliminary study

Peptides

(1982)

K.R. Castelman

Digital Image Processing

(1979)

E. Feingold et al.

Hardware and software design consideration in engineering and image processing workstation: autoradiographic analysis with DUMAS and the BRAIN autoradiograph analysis software package

C.R. Gallistel et al.

Computer assisted analysis of 2-DG autoradiographs

Neurosci. Biobehav. Rev.

(1982)

C.R. Gallistel et al.

Microcomputer systems for analyzing 2-deoxyglucose autoradiographs

R.C. Gonzalez et al.

Digital Image Processing

(1977)

C. Goochee et al.

Computerized densitometry and color coding of ¹⁴C-deoxyglucose autoradiographs

Ann. Neurol.

(1979)

D.S. Gross et al.

Quantitative immunocytochemistry of hypothalamic and pituitary hormones: validation of an automated, computerized image analysis system

J. Histochem. Cytochem.

(1985)

M.G. Hartman et al.

Glutamate-like antibody staining in the cat superior colliculus is reduced by visual decortication

Invest. Opthalmol. Visual. Sci.

(1988)

L.S. Hibbard et al.

Three-dimensional reconstruction of metabolic data from quantitative autoradiography of rat brain

Am. J. Physiol.

(1984)

R.N. Holdefer et al.

Laminar organization and ultrastructure of GABA-immunoreactive neurons and processes in the dorsal lateral geniculate nucleus of the tree shrew (Tupaia belangeri)

Visual Neurosci.

(1988)

C.R. Houser et al.

Time course of the reduction of GABA terminals in a model of focal epilepsy: a glutamic acid decarboxylase immunocytochemical study

Brain Res.

(1986)

A. Isseroff et al.

An inexpensive microcomputer-based image-analysis system: novel applications to quantitative autoradiography

J. Neurosci. Methods

(1985)

S.C. Jones et al.

The evaluation of quantitative autoradiogram processing systems for cerebrovascular research

J. Neurosci. Methods

(1988)

M.J. Kuhar

Receptor localization with the microscope

J.L. Lear et al.

High-performance digital image analyzer for quantitative autoradiography

J. Cereb. Blood Flow Metabol.

(1986)

T.K. Lewellen et al.

Quantitative autoradiography using a personal computer

J. Nucl. Med.

(1986)

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Research paperQuantitative immunocytochemistry using an image analyzer. I. Hardware evaluation, image processing, and data analysis

Abstract

Computer-assisted morphometry and microdensitometry of transmitter-identified neurons with special reference to the mesostriatal dopamine pathway. I. Methodological aspects

Acta Physiol. Scand.

Computer-assisted morphometry and microdensitometry of transmitter-identified neurons with special reference to the mesostriatal dopamine pathway. II. Further studies on the effects of the GM1 ganglioside on the degenerative and regenerative features of mesostriatal dopamine neurons

Acta Physiol. Scand.

Quantitative computer analysis of autoradiographs utilizing a charge-coupled device solid-state camera

J. Neurosci. Methods

Computer assisted video analysis of [3H]spiroperidol binding autoradiographs

J. Neurosci. Methods.

Strain differences in pituitary prolactin content: relationship to number of hypothalamic dopamine neurons

Brain Res.

An inexpensive microcomputer digital imaging system for densitometry: quantitative autoradiography of insulin receptors with 125I and LKB ultrofilm

J. Neurosci. Methods

Quantitative immunocytochemistry of tyrosine hydroxylase in rat brain. I. Development of a computer assisted method using the peroxidase-antiperoxidase technique

Brain Res.

Quantitative immunocytochemistry of tyrosine hydroxylase in rat brain. II. Variations in the amount of tyrosine hydroxylase among individual neurons of the locus coeruleus in relationship to neuronal morphology and topography

Brain Res.

Microcomputer-assisted densitometry for quantitative receptor autoradiography

J. Neurosci. Methods

Automatic image analyzers and their use in anatomy

Prog. Clin. Biol. Res.

Effect of ovariectomy on the hypothalamic content of immunoreactive gonadotropin-releasing hormone in the female mouse as revealed by quantitative immunocytochemistry and radioimmunoassay

Am. J. Anat.

Computer-assisted image analysis of the distributions of peptidergic terminals in the central nucleus of the amygdala: a preliminary study

Peptides

Digital Image Processing

Hardware and software design consideration in engineering and image processing workstation: autoradiographic analysis with DUMAS and the BRAIN autoradiograph analysis software package

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Neurosci. Biobehav. Rev.

Microcomputer systems for analyzing 2-deoxyglucose autoradiographs

Digital Image Processing

Computerized densitometry and color coding of 14C-deoxyglucose autoradiographs

Ann. Neurol.

Quantitative immunocytochemistry of hypothalamic and pituitary hormones: validation of an automated, computerized image analysis system

J. Histochem. Cytochem.

Glutamate-like antibody staining in the cat superior colliculus is reduced by visual decortication

Invest. Opthalmol. Visual. Sci.

Three-dimensional reconstruction of metabolic data from quantitative autoradiography of rat brain

Am. J. Physiol.

Laminar organization and ultrastructure of GABA-immunoreactive neurons and processes in the dorsal lateral geniculate nucleus of the tree shrew (Tupaia belangeri)

Visual Neurosci.

Time course of the reduction of GABA terminals in a model of focal epilepsy: a glutamic acid decarboxylase immunocytochemical study

Brain Res.

An inexpensive microcomputer-based image-analysis system: novel applications to quantitative autoradiography

J. Neurosci. Methods

The evaluation of quantitative autoradiogram processing systems for cerebrovascular research

J. Neurosci. Methods

Receptor localization with the microscope

High-performance digital image analyzer for quantitative autoradiography

J. Cereb. Blood Flow Metabol.

Quantitative autoradiography using a personal computer

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Research paper
Quantitative immunocytochemistry using an image analyzer. I. Hardware evaluation, image processing, and data analysis

Computer-assisted morphometry and microdensitometry of transmitter-identified neurons with special reference to the mesostriatal dopamine pathway. II. Further studies on the effects of the GM₁ ganglioside on the degenerative and regenerative features of mesostriatal dopamine neurons

Computer assisted video analysis of [³H]spiroperidol binding autoradiographs

An inexpensive microcomputer digital imaging system for densitometry: quantitative autoradiography of insulin receptors with ¹²⁵I and LKB ultrofilm

Computerized densitometry and color coding of ¹⁴C-deoxyglucose autoradiographs