Millisecond fading and recovery phenomena in fluorescent biological objects

Summary Cytofluorometric signals derived from some frequently used fluorophores were studied during illumination times in the millisecond range. These rapid signals were recorded on a storage oscilloscope. The objects studied included (1) Berberine sulphate stained mast cell heparin, (2) Acriflavine-Feulgen stained DNA, (3) Acridine orange stained mast cell heparin, (4) Acridine orange stained DNA and (5) Fluorescein isothiocyanate-conjugated IgG in an antinuclear factor test. A new rapid fading phenomenon, appearing as an initial peak upon the familiar slowly declining fluorescence signal, is reported. This fading, which had a duration of about 10 ms, also showed a very rapid recovery. The influence of this phenomenon on fluorometric measurement techniques is discussed. The millisecond fading phenomenon occurred in all the fluorophores studied except Fluorescein isothiocyanate-conjugated IgG. In the case of acridine organge the phenomenon was present when the dye was bound to nuclear DNA but absent when the dye was bound to mast cell heparin. This suggests that the millisecond fading and recovery phenomenon may be used in fluorescent microprobe studies.Supported by grants from the Swedish Medical Research Council (Project 12X-2235) and from the Medical Faculty of Linköping University     

Fluorescence fading in quantitative fluorescence microscopy: a cytofluorometer for the automatic recording of fluorescence peaks of very short duration

Synopsis Rates of photodecomposition were studied in some fluorophores during short (milliseconds) and longer (minutes) illumination periods. A xenon burner served as light source, and care was taken to obtain optimum conditions for activation. The fluorophores studied included (i) the formaldehyde-induced fluorescent product from 5-hydroxytryptamine in mast cells, (ii) Berberine sulphate bound to mast cell polyanions, (iii) Feulgen-Pararosaniline-stained DNA, and (iv) Fluorescein isothiocyanate-conjugated IgG in an antinuclear factor test. All fluorophores showed a significant fading during 3 min illumination. The Fluorescein isothiocyanate-conjugate faded the most rapidly; its fluorescence intensity was reduced to 50% of the initial value after 2 sec continuous illumination. No fluorophore faded significantly during the initial few milliseconds of illumination. On the basis of these findings, an inexpensive measuring device was constructed. It contained a peak-reader and memory circuit triggered by the flash synchronization tap of a camera shutter positioned in the activation beam. The peak-reader has a response time of about 2 msec. Repeated measurements on the various fluorophores indicate that this peak-reading device may be used to measure fluorescence intensity without fading.     

Histochemical studies on mast cells in cattle skin

Synopsis Cells in the skin of cattle which gave a green fluorescence after formaldehyde treatment could be stained orange with Acridine Orange and blue with Astrablau. It is concluded that these cells are mast cells containing heparin and a catecholamine, probably dopamine.     

Quantitation of fluorescence fading phenomena for identifying intracellular biopolymers.

The fading behavior of the 670 nm fluorescence emission band produced by unfixed rat mast cells stained with acridine orange (AO) has been found to be in excellent agreement with the behavior predicted by second order chemical kinetics. The reciprocal of fluorescence intensity plotted against time yields a straight line. When due account is taken of dye/cell ratio and the intensity of fluorescence-exciting radiation, Io (measured with the standard phosphor particle), the slope of this straight line is a constant, k', which is independent of dye/cell ratio and Io. k' differs from the second order photochemical rate constant by a constant factor. The fading of a given AO-biopolymer complex is described by a particular value of k'. Two values of k' have been found for rat mast cell granules, indicating the presence of two different AO-biopolymer complexes. Fading of fluorescence may serve to identify particular intracellular biopolymers in individual cells even when present in a heterogeneous population.     

Fluorochromierung peripherer motorischer Nervenfasern am lebenden Frosch

Summary Normally excitable single motor fibers of the frog's sciatic nerve were treated with Acridine orange of low concentration (0.2 Gamma per ml). The storage of the dye begins in the Schwann nuclei. Then the myelin sheath becomes fluorescent in its full extent showing nodal gaps. The staining process could not be influenced by stimulating the fiber. The excitability is not influenced if a stained fiber is kept in darkness but disappears during exposure to the blue light of the fluorescence microscope.Transsection of a fiber is followed by rapid disintegration of the injured internode, beginning with the appearance of Schmitd-Lanterman's incisures and then leading to splitting of the myelin sheath into several lamellas which protrude into the axon. Application of Acridine orange in this stage fails to stain the cut internode, although the proximal internodes show normal fluorescence. The possibility of active incorporation of the dye into the myelin sheath is discussed.     

Bacterial Growth on Surfaces: Automated Image Analysis for Quantification of Growth Rate-Related Parameters

A fast routine method for estimating bacterial cell growth rates by using the metachromatic dye acridine orange is described. The method allows simultaneous estimates of cellular RNA and DNA contents of single cells. Acridine orange staining can be used as a nonspecific supplement to quantitative species-specific hybridizations with fluorescence-labelled ribosomal probes to estimate the single-cell concentration of RNA. By automated analysis of digitized images of stained cells, we determined four independent growth rate-related parameters: cellular RNA and DNA contents, cell volume, and the frequency of dividing cells in a cell population. These parameters were used to compare physiological states of liquid-suspended and surface-growing Pseudomonas putida KT2442 in chemostat cultures. The major finding is that the correlation between substrate availability and cellular growth rate found for the free-living cells was not observed for the surface-bound cells; in contrast, the data indicate an almost constant growth rate for attached cells which was independent of the dilution rate in the chemostat.     

Chromatin condensation during spermiogenesis in the golden hamster (Mesocricetus aureus): a flow cytometric study

DNA-staining of hamster testis cell suspensions followed by flow cytometry demonstrated appearance of the first haploid cells at 23 days post partum (dpp) and of condensed chromatin (in elongated spermatids and spermatozoa) at 33-34 dpp. Mature spermatozoa were first observed in the caput epididymis at 36-37 dpp, thus completing the first spermatogenic wave. Testicular cell suspensions from animals from 23 to 38 dpp were stained with acridine orange, and flow cytometer gating was adjusted to include only the haploid cells. Acridine orange intercalated into double-stranded DNA to produce green fluorescence. The decrease in green fluorescence intensity from 23 until 37 dpp was caused by changes in the binding of DNA to basic proteins in such a fashion as to impede the access of the dye to the DNA double helix. When the green fluorescence values (of the most advanced spermatids) were plotted against the age of the hamsters (in dpp) or the corresponding steps of spermiogenesis, the decrease in fluorescence could be seen to occur in three phases. The inflection point between the first and second phases was observed at about spermiogenesis step 7, consistent with the hypothesis that this represents removal of histone from the chromatin. The second phase presumably represents the period in which transition proteins are bound to the DNA. At approximately steps 15 or 16 a further inflection point was seen where protamines replaced the transition proteins. The red fluorescence produced when acridine orange bound to RNA in spermatids, increased early in spermiogenesis and decreased dramatically at 34 dpp, consistent with the fact that elongating spermatids discard the bulk of their cytoplasm during the maturation process.     

Na+/H+ exchange is present in basolateral membranes from rabbit small intestine

Fluorescence quenching of the pH gradient sensitive dye acridine orange and that of the membrane potential sensitive dye Di-S-C3(5) have been studied in purified basolateral membrane vesicles obtained from rabbit small intestine. Basolateral membranes contain an electroneutral, carrier mediated, Na+/H+ exchange activity. They also appear to contain an electrogenic pathway for H+ movement. Based on the comparison of acridine orange fluorescence quenching in the presence of an outwardly directed Na+ gradient and in the presence of known K+ diffusion gradients it can be estimated that at least 50% of the observed proton fluxes are due to the activity of the exchanger. Acridine orange fluorescence recovery measurements have been used to assess the kinetic properties of the exchanger.     

Photodynamic activity of acridine orange: peroxide radical induction in DNA and synthetic polynucleotides

As shown by electron paramagnetic resonance, acridine orange induces the formation of peroxide radicals in DNA when dye-DNA mixtures frozen at 77 K are irradiated with visible light. The reaction is oxygen dependent and strongly reduced by the addition of an electron scavenger. Factors of the medium can modulate the reaction: an ionic strength increased up to 0.3 greatly enhances the dye efficiency whereas the presence of phosphate ions has an inhibiting influence. Acridine orange, which is slightly less efficient than proflavine on native DNA, induces an important peroxide radical formation in poly(dG).poly(dC) but has no action on the poly(dA).poly(dT)polymer.     

Fluorescence microscopy of viable mast cells stained with different concentrations of acridine orange.

Freshly harvested rat peritoneal mast cells were stained with different concentrations of acridine orange, a metachromatic fluorochrome known to form complexes with chromatin and muscopolysaccharides. Fluorescence metachromasia was observed in cytoplasmic granules in cell populations with intracelluar dye contents as low as 5 X 10(-16) mole per cell, one-half decade lower than required to produce metachromatic staining of the nucleus. Cytoplasmic granules did not stain uniformly throughout the cell; some granules exhibited red fluorescence and others green. As the amount of acridine orange uptake per cell was increased, cytoplasmic fluorescence became uniformly red and nuclear fluorescence gradually changed from green to yellow.     

Flow cytometric evaluation of boar semen by the sperm chromatin structure assay as related to cryopreservation and fertility

Boar semen from a heterospermic mating trial and semen cryopreserved by various methods were evaluated by the flow cytometric sperm chromatin structure assay (SCSA), which measures the susceptibility of sperm nuclear DNA to acid-induced denaturation in situ. Spermatozoa were treated with a pH 1.4 buffer and then stained with the metachromatic dye acridine orange. Acridine orange intercalated into double-stranded DNA (native) fluoresces green while single-stranded DNA (denatured) fluoresces red when excited with 488 nm light. The ratio of red to total fluorescence provides an index of normality/abnormality. The SCSA data on neat boar semen or semen in either Kiev-Merck or Pursel-Johnson extender and frozen directly on dry ice blocks or plunged into LN(2) did not differ within individual boars. Therefore, chromatin structure, as measured by the SCSA, was not influenced differently by these 2 methods of semen cryopreservation. When semen from 6 boars was mixed in equal sperm numbers in six 3-way combinations and inseminated into at least 3 Duroc gilts per combination, 4 of the 6 combinations yielded 2 litters, while the remaining 2 combinations yielded 3 litters. The SCSA correctly predicted both the high and low fertility boars based on a ratio of offspring as deviated from the theoretical percentage. Thus, the SCSA was found to be a valuable adjunct method for evaluating boar cemen quality.     

Development of a photoreactive probe-based system for detecting heparin

We previously identified a peptide heparin-associated peptide Y (HappY) that binds specifically to heparin. In this article, we report a novel heparin detection system using chemically modified HappY as a probe. The photoreactive HappY probe was serially diluted and dispensed into a 96-well plate coated with biotinylated heparin. After ultraviolet irradiation, the HappY probe crosslinked to the heparin on the plate was detected with fluorescein isothiocyanate-conjugated streptavidin. Furthermore, the photoreactive HappY probe was used to stain cutaneous tissue sections obtained from dermatitis-affected or mastocytoma-affected cats and dogs. The photoreactive HappY probe stained limited resident mast cells in the connective tissue of skin compared with the anti-heparan sulfate monoclonal antibody 10E4, suggesting that the probe can be used to distinguish the structure of heparin in tissues. The interactions between glycosaminoglycans and proteins in vivo tend to be weak. Therefore, our method for enhancing such weak interactions may be a promising tool for intermolecular interaction studies in glycobiology research.     

Cytofluorometric and cytochemical comparisons of normal and abnormal human cells from the female genital tract.

Acridine orange staining of exfoliated cells from epithelial tissues facilitates discrimination between normal and abnormal cells: abnormal cells develop highly elevated nuclear fluorescence. Comparisons of acridine orange (AO) staining with propidium iodide (PI) or Feulgen staining have shown that: (a) PI staining also provides highly elevated nuclear fluorescence from abnormal cells; (b) the distributions of nuclear fluorescence following AO or PI staining were usually not significantly different as judged by the Kolmogorov-Smirnov test; (c) fluorescence emission spectra from AO and PI stained cells are consistent with the hypothesis that both fluorochromes bind to DNA within cell nuclei; (d) DNAse treatment of AO stained normal cells eliminates the nuclear fluorescence peak from slit-scan contours; RNAse treatment has no effect on nuclear fluorescence; (e) the distribution of abnormal cell nuclear fluorescence after AO staining is usually, but not always, significantly different from the distribution of abnormal cell nuclear absorbance after Feulgen staining, with relative nuclear fluorescence being greater than relative nuclear absorbance. The hypothesis currently most consistent with these results is that elevated Feulgen DNA content can account for only part of the discrimination provided by AO staining, and that the chromatin within abnormal cells is altered so as to increase accessibility of DNA to intercalating dyes.     

Reduction in rat mesentery mast cell staining after degranulation by protamine. A competitive antagonism.

Degranulation of rat mesentery mast cells by increasing concentrations of protamine causes a parallel decrease in the numbers of mast cells stained with toluidine blue or with berberine sulfate. No decrease in mast cell numbers occurs when degranulation is inhibited. Since protamine does not enter into non stimulated mast cells, these results suggest that this reduction in mast cell numbers is caused by the binding of protamine to the anionic sites of heparin of exocytosed granules thereby preventing their staining. There seems to be a competitive antagonism between protamine and toluidine blue at the anionic sites of heparin for increasing concentrations of toluidine blue progressively reverse the reduction in mast cell numbers.     

Thymosin alpha(1)--an endogenous modulator of alpha-thrombin recognition site]

Thymosin alpha 1-inhibited fibrinogen clotting activity of alpha-thrombin, but not amidolysis of H-D-Phe-Pip-Arg-pNA. Modulation of thrombin interaction with rat peritoneal mast cells (RPMC) by suppressors of additional recognition binding site (thymosin and heparin) was studied. Thrombin-induced pHi changes of RPMC were controlled with pH-sensitive fluorescent dye, BCECF. Thrombin caused a biphasic changes in pHi: rapid cell acidification (0.02) followed by slow alkalinization (0.06 above baseline for 18 min). Thymosin suppressed thrombin-induced pHi increase above resting level. Similar changes in pHi were observed after modification of additional recognition binding site by heparin. Beta/gamma-thrombin with disrupted additional binding site was shown to induce only a decrease of pHi. It is concluded that thymosin alpha 1 is endogenous modulator of alpha-thrombin activity.     

Iron and Aluminum Lakes of Gallo Blue E As Nuclear and Metachromatic Mucin Stains

Gallo blue E, C. I. No. 51040, Mordant Violet 54, furnishes a blue black nuclear stain when applied to tissue sections in the form of its moderately stable iron lakes. This adoring combined well with such counterstains as orange G and eosin B. The Van Gieson stain tends to decolorize mucins, cartilage, and mast ells previously stained with this dye. Its aluminum lake solutions tend to gel in a few minutes to 24 hours depending on the solvent wed and the amount of Al²⁺ present. Aluminum lake solutions give a moderately good blue to dark blue nuclear stain and a brilliant purplish red to dark purple stain to a variety of epithelial and connective tissue mucins. Acid dye counterstains are poorly tolerated. With either lake, nuclear staining is abolished by deoxyribonuclease digestion or relatively short mineral acid extraction of DNA.     

Iron and aluminum lakes of Gallo blue E as nuclear and metachromatic mucin stains.

Gallo blue E, C. I. No. 51040, Mordant Violet 54, furnishes a blue black nuclear stain when applied to tissue sections in the form of its moderately stable iron lakes. This coloring combined well with such counterstains as orange G and eosin B. The Van Gieson stain tends to decolorize mucins, cartilage, and mast cells previously stained with this dye. Its aluminum lake solutions tend to gel in a few minutes to 24 hours depending on the solvent used and the amount of Al3+ present. Aluminum lake solutions give a moderately good blue to dark blue nuclear stain and a brilliant purplish red to dark purple stain to a variety of epithelial and connective tissue mucins. Acid dye counterstains are poorly tolerated. With either lake, nuclear staining is abolished by deoxyribonuclease digestion or relatively short mineral acid extraction of DNA.     

Flow cytometric analysis of DNA binding and cleavage by cell surface-displayed homing endonucleases

LAGLIDADG homing endonucleases (LHEs) cleave 18–24bp DNA sequences and are promising enzymes for applications requiring sequence-specific DNA cleavage amongst genome-sized DNA backgrounds. Here, we report a method for cell surface display of LHEs, which facilitates analysis of their DNA binding and cleavage properties by flow cytometry. Cells expressing surface LHEs can be stained with fluorescently conjugated double-stranded oligonucleotides (dsOligos) containing their respective target sequences. The signal is absolutely sequence specific and undetectable with dsOligos carrying single base-pair substitutions. LHE–dsOligo interactions facilitate rapid enrichment and viable recovery of rare LHE expressing cells by both fluorescence-activated cell sorting (FACS) and magnetic cell sorting (MACS). Additionally, dsOligos conjugated with unique fluorophores at opposite termini can be tethered to the cell surface and used to detect DNA cleavage. Recapitulation of DNA binding and cleavage by surface-displayed LHEs provides a high-throughput approach to library screening that should facilitate rapid identification and analysis of enzymes with novel sequence specificities.     

Fluorescence microscopy of viable mast cells stained with different concentrations of acridine orange

Summary Freshly harvested rat peritoneal mast cells were stained with different concentrations of acridine orange, a metachromatic fluorochrome known to form complexes with chromatin and mucopolysaccharides. Fluorescence metachromasia was observed in cytoplasmic granules in cell populations with intracellular dye contents as low as 5×10^–16 mole per cell, one-half decade lower than required to produce metachromatic staining of the nucleus. Cytoplasmic granules did not stain uniformly throughout the cell; some granules exhibited red fluorescence and others green. As the amount of acridine orange uptake per cell was increased, cytoplasmic fluorescence became uniformly red and nuclear fluorescence gradually changed from green to yellow.