Total internal reflection with fluorescence correlation spectroscopy: nonfluorescent competitors

Total internal reflection with fluorescence correlation spectroscopy is a method for measuring the surface association/dissociation rate constants and absolute densities of fluorescent molecules at the interface of a planar substrate and solution. This method can also report the apparent diffusion coefficient and absolute concentration of fluorescent molecules very close to the surface. Theoretical expressions for the fluorescence fluctuation autocorrelation function when both surface association/dissociation kinetics and diffusion through the evanescent wave, in solution, contribute to the fluorescence fluctuations have been published previously. In the work described here, the nature of the autocorrelation function when both surface association/dissociation kinetics and diffusion through the evanescent wave contribute to the fluorescence fluctuations, and when fluorescent and nonfluorescent molecules compete for surface binding sites, is described. The autocorrelation function depends in general on the kinetic association and dissociation rate constants of the fluorescent and nonfluorescent molecules, the surface site density, the concentrations of fluorescent and nonfluorescent molecules in solution, the solution diffusion coefficients of the two chemical species, the depth of the evanescent field, and the size of the observed area on the surface. Both general and approximate expressions are presented.     

Building Fluorescent Sensors by Template Polymerization: The Preparation of a Fluorescent Sensor for -Tryptophan

The development of fluorescent sensors for organic molecules is of great practical importance in chemical, biological, and pharmaceutical sciences. Using -tryptophan as an example, we have studied a new way of making polymeric fluorescent sensors using template polymerization or molecular imprinting techniques. The fluorescent polymers were prepared using functional monomers with a fluorescent probe attached to it. The fluorescence of this polymer could be quenched by 4-nitrobenzaldehyde. Addition of the template molecules, -tryptophan, increased the fluorescence intensity of the imprinted polymer/quencher mixture in a concentration-dependent fashion, presumably through the displacement of the quencher from the binding sites by -tryptophan. This fluorescence intensity change upon mixing with -tryptophan allows the binding event to be detected easily. The sensor also exhibited enantioselectivity for the template molecules. For example, the effect of -tryptophan on the fluorescence intensity of the polymer is about 70% that of its -enantiomer. Furthermore, the effect of -phenylalanine and -alanine on the fluorescence intensity change is much smaller than that of -tryptophan. Because the approach used does not require the de novo design of the complementary binding site and does not rely on any specific structural features of the template molecule or prior knowledge of its three-dimensional structure, the same principle could potentially be useful for the future construction of practical fluorescent sensors for many other compounds.     

Fluorescence photobleaching recovery using total internal reflection interference fringes

Lateral diffusion measurements on cell membrane molecules, most commonly accomplished through fluorescence photobleaching recovery (FPR or FRAP), provide information on such molecules' size, environment, and participation in intermolecular interactions. However, difficulties arise in FPR measurements of lateral dynamics of materials, such as visible fluorescent protein (VFP) fusion proteins, where fluorescent intracellular species contribute to the fluorescence recovery signal and thus distort measurements intended to reflect surface molecules only. A new method helps eliminate these difficulties. In total internal reflection interference fringe FPR, interfering laser beams enter a 1.65-numercial aperture (NA) Olympus objective at the periphery of the back focal plane where the NA exceeds 1.38. This creates an extended interference pattern totally internally reflected at the coverslip-medium interface which excites fluorescence only from fluorescent molecules located where the cell contacts the coverslip. The large illuminated area interrogates many more membrane receptors than spot methods and hence obtains more diffusion information per measurement while rejecting virtually all interfering intracellular fluorescence. We report successful measurements of membrane dynamics of both VFP-containing and conventionally labeled molecules by this technique and compare them with results of other FPR methods.     

Total internal reflection with fluorescence correlation spectroscopy: combined surface reaction and solution diffusion

Total internal reflection with fluorescence correlation spectroscopy (TIR-FCS) is a method for measuring the surface association/dissociation rates and absolute densities of fluorescent molecules at the interface of solution and a planar substrate. This method can also report the apparent diffusion coefficient and absolute concentration of fluorescent molecules very close to the surface. An expression for the fluorescence fluctuation autocorrelation function in the absence of contributions from diffusion through the evanescent wave, in solution, has been published previously (N. L. Thompson, T. P. Burghardt, and D. Axelrod. 1981, Biophys. J. 33:435-454). This work describes the nature of the TIR-FCS autocorrelation function when both surface association/dissociation kinetics and diffusion through the evanescent wave contribute to the fluorescence fluctuations. The fluorescence fluctuation autocorrelation function depends in general on the kinetic association and dissociation rate constants, the surface site density, the concentration of fluorescent molecules in solution, the solution diffusion coefficient, and the depth of the evanescent field. Both general and approximate expressions are presented.     

Single-molecule fluorescence observed with mercury lamp illumination

We demonstrate that it is possible to observe single fluorescent molecules using a standard fluorescence microscope with mercury lamp excitation and an inexpensive cooled charge-coupled device (CCD) camera. With this equipment, we have been able to observe single molecules of tetramethyl-rhodamine, rhodamine 6G, fluorescein isothiocyanate and green fluorescent protein. Immobilized molecules were observed both in air and in aqueous solution.     

Fluorescence correlation spectroscopy and quantitative cell biology

Fluorescence correlation spectroscopy (FCS) analyzes fluctuations in fluorescence within a small observation volume. Autocorrelation analysis of FCS fluctuation data can be used to measure concentrations, diffusion properties, and kinetic constants for individual fluorescent molecules. Photon count histogram analysis of fluorescence fluctuation data can be used to study oligomerization of individual fluorescent molecules. If the FCS observation volume is positioned inside a living cell, these parameters can be measured in vivo. FCS can provide the requisite quantitative data for analysis of molecular interaction networks underlying complex cell biological processes.     

Ultra-high resolution imaging by fluorescence photoactivation localization microscopy

Biological structures span many orders of magnitude in size, but far-field visible light microscopy suffers from limited resolution. A new method for fluorescence imaging has been developed that can obtain spatial distributions of large numbers of fluorescent molecules on length scales shorter than the classical diffraction limit. Fluorescence photoactivation localization microscopy (FPALM) analyzes thousands of single fluorophores per acquisition, localizing small numbers of them at a time, at low excitation intensity. To control the number of visible fluorophores in the field of view and ensure that optically active molecules are separated by much more than the width of the point spread function, photoactivatable fluorescent molecules are used, in this case the photoactivatable green fluorescent protein (PA-GFP). For these photoactivatable molecules, the activation rate is controlled by the activation illumination intensity; nonfluorescent inactive molecules are activated by a high-frequency (405-nm) laser and are then fluorescent when excited at a lower frequency. The fluorescence is imaged by a CCD camera, and then the molecules are either reversibly inactivated or irreversibly photobleached to remove them from the field of view. The rate of photobleaching is controlled by the intensity of the laser used to excite the fluorescence, in this case an Ar+ ion laser. Because only a small number of molecules are visible at a given time, their positions can be determined precisely; with only approximately 100 detected photons per molecule, the localization precision can be as much as 10-fold better than the resolution, depending on background levels. Heterogeneities on length scales of the order of tens of nanometers are observed by FPALM of PA-GFP on glass. FPALM images are compared with images of the same molecules by widefield fluorescence. FPALM images of PA-GFP on a terraced sapphire crystal surface were compared with atomic force microscopy and show that the full width at half-maximum of features approximately 86 +/- 4 nm is significantly better than the expected diffraction-limited optical resolution. The number of fluorescent molecules and their brightness distribution have also been determined using FPALM. This new method suggests a means to address a significant number of biological questions that had previously been limited by microscope resolution.     

Fluorescent proteins for single‐molecule fluorescence applications

We present single‐molecule fluorescence data of fluorescent proteins GFP, YFP, DsRed, and mCherry, a new derivative of DsRed. Ensemble and single‐molecule fluorescence experiments proved mCherry as an ideally suited fluorophore for single‐molecule applications, demonstrated by high photostability and rare fluorescence‐intensity fluctuations. Although mCherry exhibits the lowest fluorescence quantum yield among the fluorescent proteins investigated, its superior photophysical characteristics suggest mCherry as an ideal alternative in single‐molecule fluorescence experiments. Due to its spectral characteristics and short fluorescence lifetime of 1.46 ns, mCherry complements other existing fluorescent proteins and is recommended for tracking and localization of target molecules with high accuracy, fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM), or multicolor applications. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Expanding the scope of quantitative FRAP analysis

In this study, new mathematical models were developed for analysis of fluorescence recovery after photobleaching (FRAP) data to account for features not represented in previous analysis: conical photobleaching geometry, spatial variations in binding of fluorescent molecules, and directed transport of fluorescent molecules. To facilitate computations in conical geometry, a fast computational method for calculation of fluorescence recovery is presented. Two approximations are presented to aid in FRAP analysis when binding varies spatially, one applying to cases of relatively fast diffusion and slow binding and the other to binding of molecules to small cellular structures. Numerical results show that using a model that represents the influential physical processes and that is formulated in the appropriate geometry can substantially improve the accuracy of FRAP calculations.     

Fluorescent rotors and their applications to the study of G-F transformation of actin.

New fluorescent rotor molecules having hydrophilic functional groups, which are derivatives of p-(N,N-dialkylamino)benzylidenemalononitrile, were synthesized. Their properties as fluorescent rotors were confirmed by an observation of solvent viscosity-dependent fluorescence. Incorporation of hydrophilic groups into the molecules increased the solubility of fluorescent rotors in aqueous media; the application of the compounds to biochemical systems became feasible as a consequence. To demonstrate this applicability, we attempted to monitor the G-F transformation of rabbit skeletal muscle actin with these newly synthesized compounds. All the compounds carrying a malononitrile moiety showed greater fluorescence in F-actin. Among them, 1-(2-hydroxyethyl)-6-[(2,2-dicyano)vinyl]-2,3,4-trihydroquinoli ne gave the best result by the criteria of the difference in fluorescence quantum yield for G- and F-actin, solubility, and stability of the compound. The method has the major advantage of not requiring covalent modification of actin.     

A theory of fluorescence polarization decay in membranes.

Decay of fluorescence polarization after an impulsive excitation is correlated with wobbling motion of fluorescent molecules in membranes. The motion is characterized by two parameters, a "wobbling diffusion constant" and a "degree of orientational constraint" both of which can be determined directly from experimentally obtained decay. Detailed discussion, including theoretically calculated time-courses of polarization decay, is given for several types of molecules embedded in lipid bilayers; these types cover a large part of fluorescent probes available at present. The theory is useful for the analysis of fluorescence polarization decay in any system where the orientation of fluorophore is restricted by the surrounding structure.     

Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery

Multiphoton fluorescence photobleaching recovery (MP-FPR) is a technique for measuring the three-dimensional (3D) mobility of fluorescent molecules with 3D spatial resolution of a few microns. A brief, intense flash of mode-locked laser light pulses excites fluorescent molecules via multiphoton excitation in an ellipsoidal focal volume and photobleaches a fraction. Because multiphoton excitation of fluorophores is intrinsically confined to the high-intensity focal volume of the illuminating beam, the bleached region is restricted to a known, three-dimensionally defined volume. Fluorescence in this focal volume is measured with multiphoton excitation, using the attenuated laser beam to measure fluorescence recovery as fresh unbleached dye diffuses in. The time course of the fluorescence recovery signal after photobleaching can be analyzed to determine the diffusion coefficient of the fluorescent species. The mathematical formulas used to fit MP-FPR recovery curves and the techniques needed to properly utilize them to acquire the diffusion coefficients of fluorescently labeled molecules within cells are presented here. MP-FPR is demonstrated on calcein in RBL-2H3 cells, using an anomalous subdiffusion model, as well as in aqueous solutions of wild-type green fluorescent protein, yielding a diffusion coefficient of 8.7 x 10(-7) cm(2)s(-1) in excellent agreement with the results of other techniques.     

Visualization of a specific sequence on a single large DNA molecule using fluorescence microscopy based on a new DNA-stretching method.

A method for analyzing large DNA which makes it possible to obtain spatial information on the positions of specific sequences along a DNA molecule has been developed. Making use of the fact that large DNA molecules are stably elongated under an alternating-current field in a concentrated linear polymer solution, the direct observation of elongated individual lambda DNA molecules with fluorescence probes was carried out using fluorescence microscopy. The spatial positions of the fluorescent spots of the probe (fluorescence-labeled restriction endonuclease EcoRI) on DNA molecules were determined by image analysis. As expected, fluorescent spots of EcoRI were observed at certain positions on lambda DNA, where sequences to which EcoRI binds are located. Finally, the potential application of single large DNA molecule analysis using this DNA-stretching method is discussed.     

Liquid chromatography method for detecting native fluorescent bioamines in urine using post-column derivatization and intramolecular FRET detection

Liquid chromatography (LC) with fluorescence detection is described for simultaneous determination of native fluorescent bioamines (indoleamines and catecholamines). This is based on intramolecular fluorescence resonance energy transfer (FRET) in an LC system following post-column derivatization of native fluorescent bioamines' amino groups with o-phthalaldehyde (OPA). OPA fluorescence was achieved through an intramolecular FRET process when the molecules were excited at maximum excitation wavelength of the native fluorescent bioamines. Bioamines separated by reversed-phase LC on ODS column were derivatized with OPA and 2-mercaptoethanol. This method provides sufficient selectivity and sensitivity for the determination of normetanephrine, dopamine, tyrosine, 5-hydroxytryptamine, tryptamine, and tryptophan in healthy human urine without prior sample purification.     

Fluorescence recovery after photobleaching of suspensions of vacuoles

In this work we derive theoretical expressions for the FRAP measured on a liquid suspension of vacuoles labelled by a fluorescent probe bound to the surface membrane of these vacuoles. The bleaching laser beam creates an inhomogeneity in the surface concentration of the probe molecules. We consider the case in which the randomization of these probe molecules on the vacuole surface occurs much faster than the fluorescent recovery due to the vacuole diffusion. For a given value of the bleaching parameter K, we found that the bleaching fraction of the fluorescent molecules and the fluorescence recovery rate are decreasing functions of the square ratio of the vacuole to the laser beam radius of the FRAP instrument.     

Measurement of rotational motion in membranes using fluorescence recovery after photobleaching.

A method has been developed for the measurement of the rotational motion of membrane components. In this method fluorescent molecules whose transition dipole moments lie in a given direction are preferentially destroyed with a short intense burst of polarized laser radiation. The fluorescence intensity, excited with a low intensity observation beam of polarized laser radiation, changes with time as the remaining fluorescent molecules rotate. The feasibility of the method has been demonstrated in a study of the rotation of the fluorescent lipid probe, dil ([bis,-2-(N-octadecyl-3,3-dimethyl-1-benzo[b]pyrrole]-trimethincyanine iodide) incorporated into membranes composed of distearoylphosphatidylcholine (DSPC) or dipalmitoylphosphatidylcholine (DPPC) and 0.20 mol% cholesterol, below the main chain-melting transition temperatures of the phosphatidylcholines. Rotation times in the 0.6-800 s range were observed. The fluorescence recovery (or decay) curves are in satisfactory agreement with theoretical calculations.     

Building fluorescent sensors for carbohydrates using template-directed polymerizations

The ability to custom-make fluorescent sensors for different analytes could have a tremendous impact in a variety of areas. Template-directed polymerization or molecular imprinting seems to be a promising approach for the preparation of high-affinity and specific binding sites for different template molecules. However, the application of molecular imprinting in the preparation of fluorescent sensors has been hampered by the lack of suitable fluorescent tags, which would respond to the binding event with significant fluorescence intensity changes. We have designed and synthesized a fluorescent monomer (1) that allows for the preparation of fluorescent sensors of cis diols using molecular imprinting methods. This monomer has been used for the preparation of imprinted polymers as sensitive fluorescent sensors for D-fructose. The imprinted polymers prepared showed significant fluorescence intensity enhancement upon binding with the template carbohydrate.     

FRAP法对内源性GFP在活细胞中动态分布的共焦显微镜成像

各种分子在核质问的动态分布与它们的跨膜转运密切相关。离子、r证矾A和多数小分子量蛋白可以通过核孔复合物（NPG,nuclear pore complexes）在核质问自由扩散,而分子量大于70kDa的分子需要ATP和核定位序列才能实现跨膜转运。本实验利用荧光漂白后恢复（FRAP,fluorescence recovery after photobleaching）法观测人肺腺癌肿瘤细胞（ASTC-a-1）中表达的27 kDa EGFP在核质问的被动扩散,并以激光共焦显微镜进行实时成像。转染EGFP外源基因的肿瘤细胞系在经过半年的传代培养后仍能稳定而高效的表达其荧光标记。实验表明,EGFP分子可以通过核孔在核质间被动扩散,但扩散速度远低于在核内或质内的速度,没有证据表明EGFP可以在细胞问扩散。     

A Rapid and Sensitive Fluorimetric Protocol for the Quantification of Green Fluorescent Protein in Soluble Protein Extracts from Transgenic Arabidopsis

Green fluorescent protein (GFP) is a popular qualitative reporter protein used to study different aspects of plant biology. However, to be used as a reliable quantitative reporter in expression studies using fluorescence based assays, methods to eliminate interfering endogenous molecules must be considered. Therefore, a standard curve based solid phase fluorescent immunoassay that eliminates the effects of interfering endogenous molecules was developed to quantify the GFP levels in soluble green extracts prepared from plants. Microtiter plates coated with anti-GFP were used to capture GFP from soluble plant extracts, interfering endogenous molecules was eliminated by washing without disturbing the anti-GFP binding of GFP, and then the fluorescence intensity of bound GFP was measured using a spectrofluorometer. We report in this study the use of this method to quantify the expression levels of soluble modified GFP in transgenic Arabidopsis thaliana.