Use Of Fluorophores Research Articles

Fluorophore toxicity in mouse eggs and zygotes.

Ion-sensitive fluorophores are commonly used for quantitative measurements of intracellular ion concentrations. However, both the method of intracellular loading--which for many fluorophores involves endogenous esterase-mediated removal of hydrophobic groups such as acetoxymethyl esters (AM)--and fluorescence excitation of fluorophores in the cell, can produce toxic metabolites and reactive species. Techniques used to measure intracellular ion concentrations in mammalian eggs and embryos are being increasingly employed, yet little information is available about any detrimental effects of the use of fluorophores. We have therefore used in vitro fertilisation (IVF) to assess potential fluorophore toxicity in mouse eggs, and whole cell patch-clamp recordings to detect fluorophore-associated membrane damage in zygotes. Four fluorophores were examined: SNARF-1 and BCECF (pH indicators), Fura-2 (Ca2+) and MQAE (Cl-). Cleavage of AM groups alone had no effect either on the success of IVF or on membrane electrical properties of mouse zygotes. Intracellularly loaded BCECF, SNARF-1 and Fura-2 followed by fluorescence excitation were not cell-toxic under the conditions examined. In contrast, MQAE demonstrated significant toxicity both alone and in combination with fluorescence excitation.

Evaluation of sperm damage and techniques for sperm clean-up.

The cryopreservation of bull semen adversely affects the metabolism, motility and membrane integrity of the spermatozoa, thereby decreasing the fertilizing ability of the processed sample. The present review covers methods available for examining the functionality of bull spermatozoa after thawing, including the use of fluorophores in combination with morphological examination. Procedures for clean-up, separation and selection of morphologically-normal, viable spermatozoa in vitro are also reviewed. Among the reviewed procedures are methods for dilution and washing and the selective fractionation of sperm subpopulations (especially density-gradient centrifugation in silica-bead suspensions), as well as methods based on adherence to glass (differential filtration) and sperm self-migration (particularly swim-up through hyaluronic acid). Emphasis is placed on assessing the value of these techniques for diagnostic purposes and for optimizing the methodology of in vitro fertilization.

Double-Label Immunofluorescence with the Laser Scanning Confocal Microscope Using Cyanine Dyes

The laser scanning confocal microscope, when used with the krypton-argon ion laser, is well suited for the simultaneous detection of pairs of antigens by immunofluorescence. Traditionally, double-label studies have utilized secondary antibodies conjugated to fluorescein isothiocyanate (FITC), excited by the 488-nm line (blue), and to tetramethyl rhodamine isothiocyanate or Texas Red, excited by the 568-nm line (yellow). However, the use of fluorophores excited by the 488 nm line produces unsatisfactory results when tissue contains low wavelength-excitable autofluorescence. In the amphibian cardiac ganglion, for example, autofluorescent granules within parasympathetic neurons obscure cell surface-derived signals and prevent one from analyzing the relative position of acetylcholine receptor clusters and synaptic boutons by double-label immunofluorescence. This problem has been solved by using cyanine 3.18 (Cy3)- and cyanine 5.18 (Cy5)-conjugated secondary antibodies, which are excited efficiently by the 568-nm (yellow) and the 647-nm (red) lines and which emit in the orange/red and in the far-red, respectively, and thus by avoiding the 488-nm line altogether. The resulting images are as good or better than those obtained with FITC and Texas Red, even without consideration of autofluorescence.

Correction of timing errors in photomultiplier tubes used in phase-modulation fluorometry

The measurement of fluorescence lifetimes is known to be hindered by the wavelenght-dependent and photocathode area-dependent time response of photomultiplier tubes. A simple and direct method is described to minimize the effects in photomultiplier tubes for phase-modulation fluorometry. Reference fluorophores of known lifetime were used in place of the usual scattering reference. The emission wavelenghts of the reference and sample were matched by either filters or a monochromator, and the use of a fluorophore rather than a scatter decreases the differences in spatial distribution of light emanating from the reference and sample. Thus photomultiplier tube artifacts are minimized. Five reference fluorophores were selected on the basis of availability, ease of solution preparation, and constancy of lifetime with temperature and emission wavelenght. These compounds are p-terphenyl, PPO, PPD, POPOP and dimethyl POPOP. These compounds are dissolved in ethanol to give standard solutions that can be used over the temperature range from −55 to +55°C. Purging with inert gas is not necessary. The measured phase and modulation of the reference solution is used, in conjunction with the known reference, lifetime, to calculate the actual phase and modulation of the exictation beam. The use of standard fluorophores does not require separate experiments to quantify photomultiplier effects, and does not increase the time required for the measurement of fluorescence lifetimes. Examples are presented which demonstrate the elimination of artifactual photomultiplier effects in measurements of the lifetimes of DADH (0.4 ns) and indole solutions quenched by iodide. In addition, the use of these reference solutions increases the accuracy of fluorescence lifetime measurements ranging ranging to 30 ns. We judge this method to provide more reliable lifetime measurements by the phase and modulation method. The test solutions and procedures we describe may be used by other laboratories to evaluate the performance of their phase fluorometers.