Foundations of Confocal Scanned Imaging in Light Microscopy -- Fundamental Limits in Confocal Microscopy -- Special Optical Elements -- Points, Pixels, and Gray Levels: Digitizing Image Data -- Laser Sources for Confocal Microscopy -- Non-Laser Light Sources for Three-Dimensional Microscopy -- Objective Lenses for Confocal Microscopy -- The Contrast Formation in Optical Microscopy -- The Intermediate Optical System of Laser-Scanning Confocal Microscopes -- Disk-Scanning Confocal Microscopy -- Measuring the Real Point Spread Function of High Numerical Aperture Microscope Objective Lenses -- Photon Detectors for Confocal Microscopy -- Structured Illumination Methods -- Visualization Systems for Multi-Dimensional Microscopy Images -- Automated Three-Dimensional Image Analysis Methods for Confocal Microscopy -- Fluorophores for Confocal Microscopy: Photophysics and Photochemistry -- Practical Considerations in the Selection and Application of Fluorescent Probes -- Guiding Principles of Specimen Preservation for Confocal Fluorescence Microscopy -- Confocal Microscopy of Living Cells -- Aberrations in Confocal and Multi-Photon Fluorescence Microscopy Induced by Refractive Index Mismatch -- Interaction of Light with Botanical Specimens -- Signal-to-Noise Ratio in Confocal Microscopes -- Comparison of Widefield/Deconvolution and Confocal Microscopy for Three-Dimensional Imaging -- Blind Deconvolution -- Image Enhancement by Deconvolution -- Fiber-Optics in Scanning Optical Microscopy -- Fluorescence Lifetime Imaging in Scanning Microscopy -- Multi-Photon Molecular Excitation in Laser-Scanning Microscopy -- Multifocal Multi-Photon Microscopy -- 4Pi Microscopy -- Nanoscale Resolution with Focused Light: Stimulated Emission Depletion and Other Reversible Saturable Optical Fluorescence Transitions Microscopy Concepts -- Mass Storage, Display, and Hard Copy -- Coherent Anti-Stokes Raman Scattering Microscopy -- Related Methods for Three-Dimensional Imaging -- Tutorial on Practical Confocal Microscopy and Use of the Confocal Test Specimen -- Practical Confocal Microscopy -- Selective Plane Illumination Microscopy -- Cell Damage During Multi-Photon Microscopy -- Photobleaching -- Nonlinear (Harmonic Generation) Optical Microscopy -- Imaging Brain Slices -- Fluorescent Ion Measurement -- Confocal and Multi-Photon Imaging of Living Embryos -- Imaging Plant Cells -- Practical Fluorescence Resonance Energy Transfer or Molecular Nanobioscopy of Living Cells -- Automated Confocal Imaging and High-Content Screening for Cytomics -- Automated Interpretation of Subcellular Location Patterns from Three-Dimensional Confocal Microscopy -- Display and Presentation Software -- When Light Microscope Resolution Is Not Enough:Correlational Light Microscopy and Electron Microscopy -- Databases for Two- and Three-Dimensional Microscopical Images in Biology -- Confocal Microscopy of Biofilms — Spatiotemporal Approaches -- Bibliography of Confocal Microscopy.

Handbook of biological confocal microscopy

A new fixative which primarily stabilizes carbohydrate moieties was developed for immunoelectron microscopy. It contains periodate, lysine and paraformaldehyde. Theoretically, the carbohydrates are oxidized by periodate and cross-linked by lysine. The fixative can preserve antigenicity as well as paraformaldehyde and ultrastructure as well as glutaraldehyde. Using this fixative and the peroxidase-labeled antibody technique, basement membrane antigen was localized within the cisternae of endoplasmic reticulum of parietal yolk sac cells and in extracellular basement membranes with adequate tissue successfully accomplished by conventional

Periodate-lysine-paraformaldehyde fixative a new fixative for immunoelectron microscopy

A battery of monoclonal antibodies (mAbs) against brain cell nuclei has been generated by repeated immunizations. One of these, mAb A60, recognizes a vertebrate nervous system- and neuron-specific nuclear protein that we have named NeuN (Neuronal Nuclei). The expression of NeuN is observed in most neuronal cell types throughout the nervous system of adult mice. However, some major cell types appear devoid of immunoreactivity including cerebellar Purkinje cells, olfactory bulb mitral cells, and retinal photoreceptor cells. NeuN can also be detected in neurons in primary cerebellar cultures and in retinoic acid-stimulated P19 embryonal carcinoma cells. Immunohistochemically detectable NeuN protein first appears at developmental timepoints which correspond with the withdrawal of the neuron from the cell cycle and/or with the initiation of terminal differentiation of the neuron. NeuN is a soluble nuclear protein, appears as 3 bands (46-48 × 10(3) M(r)) on immunoblots, and binds to DNA in vitro. The mAb crossreacts immunohistochemically with nervous tissue from rats, chicks, humans, and salamanders. This mAb and the protein recognized by it serve as an excellent marker for neurons in the central and peripheral nervous systems in both the embryo and adult, and the protein may be important in the determination of neuronal phenotype.

NeuN, a neuronal specific nuclear protein in vertebrates.

Prolactin is a protein hormone of the anterior pituitary gland that was originally named for its ability to promote lactation in response to the suckling stimulus of hungry young mammals. We now know that prolactin is not as simple as originally described. Indeed, chemically, prolactin appears in a multiplicity of posttranslational forms ranging from size variants to chemical modifications such as phosphorylation or glycosylation. It is not only synthesized in the pituitary gland, as originally described, but also within the central nervous system, the immune system, the uterus and its associated tissues of conception, and even the mammary gland itself. Moreover, its biological actions are not limited solely to reproduction because it has been shown to control a variety of behaviors and even play a role in homeostasis. Prolactin-releasing stimuli not only include the nursing stimulus, but light, audition, olfaction, and stress can serve a stimulatory role. Finally, although it is well known that dopamine of hypothalamic origin provides inhibitory control over the secretion of prolactin, other factors within the brain, pituitary gland, and peripheral organs have been shown to inhibit or stimulate prolactin secretion as well. It is the purpose of this review to provide a comprehensive survey of our current understanding of prolactin's function and its regulation and to expose some of the controversies still existing.

/pdf/prolactin-structure-function-and-regulation-of-secretion-3w4x6fqbg3.pdf

Prolactin: Structure, Function, and Regulation of Secretion

Heterogeneity in the distribution and morphology of microglia in the normal adult mouse brain

A new method of conjugating horseradish peroxidase with proteins was developed. The carbohydrate moiety of fluorodinitrobenzene-blocked peroxidase was oxidized with so- dium periodate to form aldehyde groups. The peroxidase-aldehyde was then bound to free amino groups of proteins unidirectionally at high efficiencies. Peroxidase-labeled immune- globulin retained its immunologic as well as enzymatic activities. Horseradish peroxidase (HRPO), when cou- pled to immunoglobulin G (IgG), has proven to be a useful marker for immunohistochemistry (13). In contrast to other immunohistochemical markers, such as fluorescein (7) and ferritin (20), HRPO may be used for both light and electron microscopy. It is especially suitable for the intracellular localization of antigens at the ultrastructural level since HRPO (40,000 molec- ular weight) is considerably smaller than fer- ritin (650,000 molecular weight); thus, HRPO- labeled IgG (HRPO-IgG) has superior penetra- tion properties. HRPO is usually coupled to IgG using bifunc- tional reagents. With earlier methods, the con- jugation reactions were carried out in the pres- ence of HRPO, IgG, and p,p'-difluoro-m,m - din tro-diphenyl sulfone (FNPS) (13), (1-cyclohexyl- 3-(2-morpholinoethyl)) carbodiimide metho-p- toluenesulfonate (4), cyanuric chloride (1), bis-

Peroxidase-labeled antibody. A new method of conjugation

The peroxidase-labeled antibody method was modified to localize multiple tissue antigens in a single histologic section. The first antigen was localized by the indirect method and the antisera were removed from the section by elution, leaving the colored reaction products identifying the antigenic sites. The second and subsequent antigens were localized similarly, using substrates that develop reaction products of different colors. For the demonstration of the method, anti-human growth hormone (GH), anti-human thyrotropic hormone (TSH) and anti-human chorionic gonadotropin, which cross-reacts with rat luteinizing hormone (LH), were used with sections of pituitary gland of the rat. AntiGH reacted with small acidophilic cells. Anti-LH was localized in oval shaped cells, and anti-TSH was localized in angulated basophilic cells. Some cells were devoid of these three antigens and no cell contained more than one of them. A convenient method which would identify more than one amitigen in the same section of tissue would be of considerable value to cellular biologists. In the past, two tissue antigens have been localized simultaneously with the immunofluorescent method using antibodies labeled with fluorescent coml)ounds of different color (4, 5, 1012). This technique has obvious limitations, including the tendency of the fluorescence of one label to mask that of the other, and the tendency for one color to fade more rapidly than the other

Simultaneous localization of multiple tissue antigens using the peroxidase-labeled antibody method: a study on pituitary glands of the rat.

Peroxidase-labeled antibody method was used to localize the six hormones of the anterior pituitary gland of male rats both at the light and electron microscopic levels. Growth hormone (GH), adenoco...

Classifications of anterior pituitary cell types with immunoenzyme histochemistry

Peroxidase-labeled antibody method was used to localize tissue antigens directly on ultrathin sections. For the demonstration of the method, luteinizing hormone, growth hormone and prolactin were localized on ultrathin sections of methacrylate-embedded anterior pituitary gland of the rat. The hormones were localized in secretion granules and in endoplasmic reticulum, but not in nucleus or mitochondria. This approach totally eliminates the problem of penetration of antisera through tissues and permits better immunologically controlled experiments by using serially sectioned materials.

Paul K. Nakane

Papers

Periodate-lysine-paraformaldehyde fixative a new fixative for immunoelectron microscopy

Peroxidase-labeled antibody. A new method of conjugation

Simultaneous localization of multiple tissue antigens using the peroxidase-labeled antibody method: a study on pituitary glands of the rat.

Classifications of anterior pituitary cell types with immunoenzyme histochemistry

Localization of tissue antigens on the ultrathin sections with peroxidase-labeled antibody method.