A Two-Stage Dissociation System for Multilayer Imaging of Cancer Biomarker-Synergic Networks in Single Cells

Retinal,cell aggregates develop in culture in a pattern similar to the in ovo retina, forming neurites first and then synapses. In the present study, we continuously exposed chick retinal cell aggregates to a high concentration (1 mM) of carbamylcholine (carbachol), an acetylcholine (ACh) analog that resists hydrolysis by acetylcholinesterase (AChE). This situation is similar to organophosphorus anticholinesterase poisoning in which the ACh level is elevated at synaptic junctions due to inhibition of AChE, Our objective was to determine whether continuous carbachol exposure either damaged cholino- ceptive neurites, cell bodies, and synaptic elements of the aggregates or influenced (hastened or retarded) their development.The retinal tissue was isolated aseptically from 11 day embryonic White Leghorn chicks and then enzymatically (trypsin) and mechanically (trituration) dissociated into single cells. After washing the cells by repeated suspension and low (about 200 x G) centrifugation twice, aggregate cell cultures (about l0 cells/culture) were initiated in 1.5 ml medium (BME, GIBCO) in 35 mm sterile culture dishes and maintained as experimental (containing 10-3 M carbachol) and control specimens.

Download Full-text

Selective Staining of Acid Mucopolysaccharides By Ruthenium Red

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100099933 ◽

1968 ◽

Vol 26 ◽

pp. 38-39

Author(s):

J. H. Luft

Keyword(s):

Electron Microscope ◽

Light Microscopy ◽

Light Microscope ◽

Osmium Tetroxide ◽

Single Cells ◽

Ruthenium Red ◽

Collagen Fibers ◽

Selective Staining ◽

Pectic Substances ◽

Solid Tissues

Ruthenium red is one of the few completely inorganic dyes used to stain tissues for light microscopy. This novelty is enhanced by ignorance regarding its staining mechanism. However, its continued usefulness in botany for demonstrating pectic substances attests to selectivity of some sort. Whether understood or not, histochemists continue to be grateful for small favors.Ruthenium red can also be used with the electron microscope. If single cells are exposed to ruthenium red solution, sufficient mass can be bound to produce observable density in the electron microscope. Generally, this effect is not useful with solid tissues because the contrast is wasted on the damaged cells at the block surface, with little dye diffusing more than 25-50 μ into the interior. Although these traces of ruthenium red which penetrate between and around cells are visible in the light microscope, they produce negligible contrast in the electron microscope. However, its presence can be amplified by a reaction with osmium tetroxide, probably catalytically, to be easily visible by EM. Now the density is clearly seen to be extracellular and closely associated with collagen fibers (Fig. 1).

Download Full-text

Fluorescent potentiometric dyes for membrane-potential imaging

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100168566 ◽

1994 ◽

Vol 52 ◽

pp. 166-167

Author(s):

Leslie M. Loew

Keyword(s):

Membrane Potential ◽

Single Cells ◽

Quantitative Imaging ◽

Optical Recording ◽

Biological Processes ◽

Major Focus ◽

The Past ◽

Excitable Systems ◽

Major Application ◽

The Impact

A major application of potentiometric dyes has been the multisite optical recording of electrical activity in excitable systems. After being championed by L.B. Cohen and his colleagues for the past 20 years, the impact of this technology is rapidly being felt and is spreading to an increasing number of neuroscience laboratories. A second class of experiments involves using dyes to image membrane potential distributions in single cells by digital imaging microscopy - a major focus of this lab. These studies usually do not require the temporal resolution of multisite optical recording, being primarily focussed on slow cell biological processes, and therefore can achieve much higher spatial resolution. We have developed 2 methods for quantitative imaging of membrane potential. One method uses dual wavelength imaging of membrane-staining dyes and the other uses quantitative 3D imaging of a fluorescent lipophilic cation; the dyes used in each case were synthesized for this purpose in this laboratory.

Download Full-text

Two-stage self-seeding method for preparation of single crystals of poly(phenylene sulfide)

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100153816 ◽

1989 ◽

Vol 47 ◽

pp. 368-369

Author(s):

Sengshiu Chung ◽

Peggy Cebe

Keyword(s):

Single Crystals ◽

High Performance ◽

Dilute Solution ◽

Two Stage ◽

Annealing Behavior ◽

High Performance Polymers ◽

Seeding Method ◽

Phenylene Sulfide

We are studying the crystallization and annealing behavior of high performance polymers, like poly(p-pheny1ene sulfide) PPS, and poly-(etheretherketone), PEEK. Our purpose is to determine whether PPS, which is similar in many ways to PEEK, undergoes reorganization during annealing. In an effort to address the issue of reorganization, we are studying solution grown single crystals of PPS as model materials.Observation of solution grown PPS crystals has been reported. Even from dilute solution, embrionic spherulites and aggregates were formed. We observe that these morphologies result when solutions containing uncrystallized polymer are cooled. To obtain samples of uniform single crystals, we have used two-stage self seeding and solution replacement techniques.

Download Full-text