PubMed · 6607402
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F Daniels, S R Rothstein, S Blum. A quantitative skin touch-retouch test: a fluorescence method.. https://pubmed.ncbi.nlm.nih.gov/6607402/
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Submicroscopic gold particle suspensions scatter colored light when illuminated with white light, and we have observed that a light-scattering gold particle suspension has the same appearance as a fluorescing solution. Thus, when illuminated by a narrow beam of white light, a 40-nm gold sol displays a clear (not cloudy), green scattered light (Tyndall) beam and has the same appearance as a fluorescing fluorescein solution. These, as well as other, observations have suggested to us that, in general, light-scattering particles can be treated as fluorescent analogs and used as fluorescent analog tracers in immuno- and DNA probe assays as well as in cell and molecular biology studies. Light-scattering particles are advantageous in these applications because particles such as gold and silver have very high light-scattering powers, which allows these particles to be easily detected, by light-scattering, at particle concentrations as low as 10(-16) M. The scattered light can be detected by the unaided eye for qualitative measurements or with a simple light-sensitive detector for quantitative measurements. Moreover, individual particles can be easily detected by eye or a video camera using a simple light microscope with a proper illuminating system. In addition, submicroscopic particles which scatter blue, green, yellow, orange, or red light can be readily synthesized. Antibodies, DNA probes, and other tracer substances can be readily attached to gold and other particles without altering their light-scattering properties. In this article we present the theory which allows one to predict the light-scattering properties of particles of different sizes and compositions and identify those particle sizes and compositions which appear most adequate for particular applications. Furthermore, we calculate molar extinction coefficients and emission efficiencies for particles of different sizes and compositions which allows us to compare the light-producing powers of these particles with those of well known fluorescent tracers. A 60-nm gold particle, for example, is equivalent to about 3 x 10(5) fluorescein molecules. Very simple, easy to use, low-cost, ultrasensitive immuno- and DNA probe assays can be developed using light-scattering particles as fluorescent analog tracers. Single particles can be detected on cell surfaces and inside cells using light microscopy techniques with proper illumination as described in the article. At high particle densities, particle-labeled cells have the same appearance as fluorescent cells.
We demonstrate the first use of Cerenkov radiation for the measurement of fluorescence lifetimes. Relativistic beta particles from the nuclear decay of 90 Sr and 90Y generate a spectral continuum in a quartz waveguide. Light flashes of < 100-ps duration are delivered simultaneously to a sample cell and a reference photomultiplier. A simple, digitally based cross-correlation signal processor allows extraction of the sample fluorescence decay kinetics without distortions which can result from the random excitation pulse sequence. We characterize both the pulse duration and the pulse intensity of the light that is emitted from the waveguide. Although the excitation intensity is very weak, we demonstrate that accurate lifetime measurements are possible with only a few hundred seconds of integration time. Tests on a variety of compounds illustrate the utility of the light source throughout the UV and blue regions of the spectrum. We also discuss future design improvements and potential applications of this new approach to time-resolved fluorescence.
The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a versatile reporter protein for monitoring gene expression and protein localization in a variety of systems. Applications using GFP reporters have expanded greatly due to the availability of mutants with altered spectral properties, including several blue emission variants, all of which contain the single point mutation Tyr-66 to His in the chromophore region of the protein. However, previously described "BFP" reporters have limited utility, primarily due to relatively dim fluorescence and low expression levels attained in higher eukaryotes with such variants. To improve upon these qualities, we have combined a blue emission mutant of GFP containing four point mutations (Phe-64 to Leu, Ser-65 to Thr, Tyr-66 to His, and Tyr-145 to Phe) with a synthetic gene sequence containing codons preferentially found in highly expressed human proteins. These mutations were chosen to optimize expression of properly folded fluorescent protein in mammalian cells cultured at 37 degreesC and to maximize signal intensity. The combination of improved fluorescence and higher expression levels yield an enhanced blue fluorescent protein that provides greater sensitivity and is suitable for dual color detection with green-emitting fluorophores.