RESUMEN
Two-photon fluorescence microscopy is one of the most important recent inventions in biological imaging. This technology enables noninvasive study of biological specimens in three dimensions with submicrometer resolution. Two-photon excitation of fluorophores results from the simultaneous absorption of two photons. This excitation process has a number of unique advantages, such as reduced specimen photodamage and enhanced penetration depth. It also produces higher-contrast images and is a novel method to trigger localized photochemical reactions. Two-photon microscopy continues to find an increasing number of applications in biology and medicine.
Asunto(s)
Microscopía Fluorescente/métodos , Animales , Ingeniería Biomédica , Colorantes Fluorescentes , Historia del Siglo XX , Humanos , Procesamiento de Imagen Asistido por Computador , Microscopía Confocal , Microscopía Fluorescente/historia , Microscopía Fluorescente/instrumentación , Microscopía por Video , Óptica y Fotónica/instrumentación , FotonesRESUMEN
The analysis of the intensity fluctuation of a fluorescence signal from a relatively small volume and from a few molecules contains information about the distribution of different species present in the solution and about kinetic parameters of the system. The same information is generally averaged out when the fluorescence experiment is performed in a much larger volume, typically a cuvette experiment. The fundamental reason for this difference is that the fluctuations of the fluorescence signal from a few molecules directly reflect the molecular nature of the matter. Only recently, with the advent of confocal microscopy and two-photon excitation, it has become practical to achieve small excitation volumes in which only a few fluorescent molecules are present. We introduce the concept of fluctuation spectroscopy and highlight some of the technical aspects. We discuss different analysis methods used in fluctuation spectroscopy and evaluate their use for studying protein-protein interactions.
Asunto(s)
Microscopía Confocal/métodos , Proteínas/química , Espectrometría de Fluorescencia/métodos , Calibración , Dimerización , Diseño de Equipo , Rayos Láser , Microscopía Confocal/instrumentación , Fotones , Unión Proteica , Proteínas/metabolismo , Espectrometría de Fluorescencia/instrumentaciónRESUMEN
Scanning fluctuation correlation spectroscopy (FCS) is an experimental technique capable of measuring particle number concentrations by monitoring spontaneous equilibrium fluctuations in the local concentration of a fluorescent species in a small (femtoliter) subvolume of a sample. The method can be used to detect molecular aggregation for dilute, submicromolar samples by directly "counting particles". We introduce the application of two-photon excitation to scanning FCS and discuss its important advantages for this technique. We demonstrate the capability of measuring particle number concentrations in solution, first with dilute samples of monodisperse 7-nm and 15-nm radius latex spheres, and then with B phycoerythrin. The detection of multiple species in a single sample is shown, using mixtures containing both sphere sizes. The method is then applied to study protein aggregation in solution. We monitor the concentration-dependent association/ dissociation equilibrium for glycogen phosphorylase A and malate dehydrogenase. The measured dissociation constants, 430 nM and 144 nM respectively, are in good agreement with previously published values. In addition, oligomer dissociation induced by pH titration from pH 8 to pH 5.0 is detectable for the enyme phosphofructokinase. The possibility of measuring dissociation kinetics by scanning two-photon FCS is also demonstrated using phosphofructokinase.
Asunto(s)
Análisis Espectral/métodos , Fenómenos Biofísicos , Biofisica , Estudios de Evaluación como Asunto , Concentración de Iones de Hidrógeno , Cinética , Látex , Sustancias Macromoleculares , Malato Deshidrogenasa/química , Microesferas , Modelos Químicos , Tamaño de la Partícula , Fosfofructoquinasa-1/química , Fosforilasas/química , Fotones , SolucionesRESUMEN
We report on the application of two photon molecular excitation to fluorescence correlation spectroscopy. We demonstrate the first fluorescence correlation spectroscopy measurements of translational mobility in the cytoplasm of living cells. Two-photon excitation inherently excites small sample volumes in three dimensions, providing depth discrimination similar to confocal microscopy, without emission pinholes. We demonstrated accurate measurements of the diffusion constant, D, for particles of several different known sizes, in bulk solutions of different viscosity. We then showed measurements of translational diffusion for 7- and 15-nm radius latex beads in the cytoplasm of mouse fibroblast cells. We measured time-dependent diffusion coefficients. When first injected in the cells, the spheres moved from two to five times slower than in water, with average rates of 18 x 10(-8) cm2/s for the 7 nm and 5 x 10(-8) cm2/s for the 15 nm radius spheres. After a few hours, spheres stick to the cells, and the motion slows down 10 to 100 times.