PubMed Health⌕ Search

Biomedical subjects

Joachim D Müller

Publications and source records attributed to Joachim D Müller.

15 recordsLinked to original sources
PubMed2006

Dual-color photon counting histogram analysis of mRFP1 and EGFP in living cells.

We investigate the potential of dual-color photon counting histogram (PCH) analysis to resolve fluorescent protein mixtures directly inside cells. Because of their small spectral overlap, we have chosen to look at the fluorescent proteins EGFP and mRFP1. We experimentally demonstrate that dual-color PCH quantitatively resolves a mixture of EGFP and mRFP1 in cells from a single measurement. To mimic the effect of protein association, we constructed a fusion protein of EGFP and mRFP1 (denoted EGFP-mRFP1). Fluorescence resonant energy transfer within the fusion protein alters the dual-channel brightness of the fluorophores. We describe a model for fluorescence resonant energy transfer effects on the brightness and incorporate it into dual-color PCH analysis. The model is verified using fluorescence lifetime measurements. Dual-color PCH analysis demonstrated that not all of the expressed EGFP-mRFP1 fusion proteins contained a fluorescent mRFP1 molecule. Fluorescence lifetime and emission spectra measurements confirmed this surprising result. Additional experiments show that the missing fluorescent fraction of mRFP1 is consistent with a dark state population of mRFP1. We successfully resolved this mixture of fusion proteins with a single dual-color PCH measurement. These results highlight the potential of dual-color PCH to directly detect and quantify protein mixtures in living cells.

Animals↗
PubMed2006

Dual-color time-integrated fluorescence cumulant analysis.

We introduce dual-color time-integrated fluorescence cumulant analysis (TIFCA) to analyze fluorescence fluctuation spectroscopy data. Dual-color TIFCA utilizes the bivariate cumulants of the integrated fluorescent intensity from two detection channels to extract the brightness in each channel, the occupation number, and the diffusion time of fluorophores simultaneously. Detecting the fluorescence in two detector channels introduces the possibility of differentiating fluorophores based on their fluorescence spectrum. We derive an analytical expression for the bivariate factorial cumulants of photon counts for arbitrary sampling times. The statistical accuracy of each cumulant is described by its variance, which we calculate by the moments-of-moments technique. A method that takes nonideal detector effects such as dead-time and afterpulsing into account is developed and experimentally verified. We perform dual-color TIFCA analysis on simple dye solutions and a mixture of dyes to characterize the performance and accuracy of our theory. We demonstrate the robustness of dual-color TIFCA by measuring fluorescent proteins over a wide concentration range inside cells. Finally we demonstrate the sensitivity of dual-color TIFCA by resolving EGFP/EYFP binary mixtures in living cells with a single measurement.

Animals↗
PubMed2006

Probing nucleocytoplasmic transport by two-photon activation of PA-GFP.

Two-photon activation of photoactivatable green fluorescent protein (PA-GFP) provides a unique tool for probing cellular transport processes, because activation is strictly limited to the sub-femtoliter optical volume of the two-photon spot. We demonstrate two-photon activation of PA-GFP immobilized in a gel and freely diffusing within cells and recover a quadratic power dependence. Illumination at 820 nm allows simultaneous activation and fluorescence monitoring by two-photon excitation. Alternatively, we activate PA-GFP using two-photon excitation and monitor the fluorescence of the photoconverted product with one-photon excitation. We probe nucleocytoplasmic transport through the nuclear pore complex of COS-1 cells, by observing the time-dependent fluorescence at various locations within the cell after two-photon activation of PA-GFP in the nucleus and in the cytoplasm. Two-photon activation of a tandem construct of two PA-GFPs showed a markedly slower rate of crossing through the nuclear pore. Analysis based on a restricted diffusion model yields a nuclear pore radius of 4.5 nm, which is in good agreement with previously reported values. This application demonstrates the attractive features of two-photon photoactivation over traditional techniques, such as photobleaching, for studying transport processes in cells.

Active Transport, Cell Nucleus↗
PubMed2005

Molecular brightness determined from a generalized form of Mandel's Q-parameter.

Mandel's Q-parameter, which is determined from the first two photon count moments, provides an alternative to PCH analysis for determining the brightness of fluorophores. Here, the definition of the Q-parameter is generalized to include correlations between photon counts that are separated by a time tau. We develop and experimentally verify a theory that takes the effects of dead time, afterpulsing, and the finite sampling time on the generalized parameter Q(tau) into account. Q(0), which corresponds to the original Q-parameter, is severely affected by dead time and afterpulsing. Q(tau) for tau>0, on the other hand, is quite robust with respect to nonideal detector effects. Thus, analysis of Q(tau) provides a robust method for determining the brightness of fluorophores. We extend the theory to a mixture of species, which is characterized by an apparent brightness. The brightness of EGFP in CV-1 cells is measured as a function of protein concentration to demonstrate the feasibility of Q(tau) analysis in cells. In addition, we monitor protein association of the ligand-binding domain of retinoid X receptor in the presence and absence of 9-cis-retinoic acid by Q(tau) analysis.

Alitretinoin↗
PubMed2005

The dual-color photon counting histogram with non-ideal photodetectors.

Dual-color photon counting histogram (PCH) analysis utilizes the photon counts in two detection channels to distinguish species by differences in brightness and color. Here we modify the existing dual-color PCH theory, which assumes ideal detectors, to include the non-ideal nature of the detector. Specifically, we address the effects of deadtime and afterpulsing. Both effects modify the shape of the dual-color PCH and thus potentially lead to incorrect values for the brightness and number of molecules if an ideal model is assumed. We use the modified theory to predict the effects of detector non-idealities on dual-color PCH as a function of concentration and brightness. In addition, we introduce a method based on moment analysis to determine the error in brightness due to non-ideal detector effects. We verify our theory experimentally by measuring a dye solution as a function of concentration and brightness. We determine the deadtime and afterpulse probability of our detectors and show that both effects play an important role in the analysis of dual-color PCH experiments. We demonstrate that resolving a mixture of CFP and YFP requires taking non-ideal detector effects into account. These corrections are also crucial for cellular measurements, as shown for GFP and RFP in mammalian cells.

Algorithms↗
PubMed2005

Time-integrated fluorescence cumulant analysis in fluorescence fluctuation spectroscopy.

We introduce a new analysis technique for fluorescence fluctuation data. Time-integrated fluorescence cumulant analysis (TIFCA) extracts information from the cumulants of the integrated fluorescence intensity. TIFCA builds on our earlier FCA theory, but in contrast to FCA or photon counting histogram (PCH) analysis is valid for arbitrary sampling times. The motivation for long sampling times lies in the improvement of the signal/noise ratio of the data. Because FCA and PCH theory are not valid in this regime, we first derive a theoretical model of cumulant functions for arbitrary sampling times. TIFCA is the first exact theory that describes the effects of sampling time on fluorescence fluctuation experiments. We calculate factorial cumulants of the photon counts for various sampling times by rebinning of the original data. Fits of the data to models determine the brightness, the occupation number, and the diffusion time of each species. To provide the tools for a rigorous error analysis of TIFCA, expressions for the variance of cumulants are developed and tested. We demonstrate that over a limited range rebinning reduces the relative error of higher order cumulants, and therefore improves the signal/noise ratio. The first four cumulant functions are explicitly calculated and are applied to simple dye systems to test the validity of TIFCA and demonstrate its ability to resolve species.

Algorithms↗
PubMed2005

Position-sensitive scanning fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) uses a stationary laser beam to illuminate a small sample volume and analyze the temporal behavior of the fluorescence fluctuations within the stationary observation volume. In contrast, scanning FCS (SFCS) collects the fluorescence signal from a moving observation volume by scanning the laser beam. The fluctuations now contain both temporal and spatial information about the sample. To access the spatial information we synchronize scanning and data acquisition. Synchronization allows us to evaluate correlations for every position along the scanned trajectory. We use a circular scan trajectory in this study. Because the scan radius is constant, the phase angle is sufficient to characterize the position of the beam. We introduce position-sensitive SFCS (PSFCS), where correlations are calculated as a function of lag time and phase. We present the theory of PSFCS and derive expressions for diffusion, diffusion in the presence of flow, and for immobilization. To test PSFCS we compare experimental data with theory. We determine the direction and speed of a flowing dye solution and the position of an immobilized particle. To demonstrate the feasibility of the technique for applications in living cells we present data of enhanced green fluorescent protein measured in the nucleus of COS cells.

Algorithms↗
PubMed2005

Unraveling protein-protein interactions in living cells with fluorescence fluctuation brightness analysis.

Fluorescence correlation spectroscopy is a potentially powerful tool for measuring protein-protein interactions directly in single living cells. We previously reported on the detection of homodimer formation in cells using molecular brightness analysis. Here, we extend the technique to detect binding between different proteins. Proteins are labeled with the fluorescent markers YFP and CFP. We first determine the coexpression ratio of both proteins by measuring the intensity ratio with a dual-color setup. The effect of fluorescence resonance energy transfer on the intensity ratio is explicitly taken into account. The brightness of cells coexpressing both proteins is measured in a single-color setup. Selecting the laser wavelength of the two-photon light source allows us to either coexcite both proteins or to selectively excite YFP-labeled proteins. This approach enables us to distinguish between homodimer and heterodimer formation. We first present the theory and then demonstrate experimental feasibility using the ligand binding domains of retinoic acid receptor (RARLBD) and of retinoid X receptor (RXRLBD). Both proteins form heterodimers, and RXRLBD also forms homodimers in the presence of its agonist. We explore binding between these proteins in the presence and absence of RXR agonist. Our results demonstrate that brightness analysis offers a quantitative method for determining protein interactions in cells.

Animals↗
PubMed2004

Dual-color photon-counting histogram.

We report on the development of dual-color photon-counting histogram (PCH) analysis. Dual-color PCH is an extension of regular PCH and considers the photon counts received in two detection channels instead of one. Because each detection channel records a different color, dual-color PCH distinguishes fluorescent species not only by differences in their brightness, but also according to their color. The additional discrimination by color increases the sensitivity of PCH in resolving a mixture of species considerably. Most dual-color fluorescence fluctuation experiments are performed on fluorophores with overlapping emission spectra. This overlap results in spectral cross talk between the detector channels, which reduces resolvability. Here, we demonstrate that dual-color PCH is able to resolve binary dye mixtures in the presence of cross talk from a single measurement without any additional information about the sample. We discuss the effect of sampling time on the fit parameters of dual-color PCH. Differences between dual-color fluorescence correlation spectroscopy and dual-color PCH will also be addressed. We quantitatively resolve a mixture of the two fluorescent proteins CFP and YFP, which is challenging because of the strong spectral overlap of their emission spectra. Dichroic mirrors are needed to direct the light into the two detection channels. We quantify the influence of these filters on dual-color PCH analysis and determine the optimal transition wavelength of the dichroic mirror for the CFP-YFP pair.

Algorithms↗
PubMed2004

Cumulant analysis in fluorescence fluctuation spectroscopy.

A novel technique for the analysis of fluorescence fluctuation experiments is introduced. Fluorescence cumulant analysis (FCA) exploits the factorial cumulants of the photon counts and resolves heterogeneous samples based on differences in brightness. A simple analytical model connects the cumulants of the photon counts with the brightness epsilon and the number of molecules N in the optical observation volume for each fluorescent species. To provide the tools for a rigorous error analysis of FCA, expressions for the variance of factorial cumulants are developed and tested. We compare theory with experiment by analyzing dye mixtures and simple fluorophore solutions with FCA. A comparison of FCA with photon-counting histogram (PCH) analysis, a related technique, shows that both methods give identical results within experimental uncertainty. Both FCA and PCH are restricted to data sampling times that are short compared to the diffusion time of molecules through the observation volume of the instrument. But FCA theory, in contrast to PCH, can be extended to treat arbitrary sampling times. Here, we derive analytical expressions for the second factorial cumulant as a function of the sampling time and demonstrate that the theory successfully models fluorescence fluctuation data.

Fluorescence↗
PubMed2003

Probing protein oligomerization in living cells with fluorescence fluctuation spectroscopy.

Fluorescence fluctuation spectroscopy provides information about protein interactions in the intercellular environment from naturally occurring equilibrium fluctuations. We determine the molecular brightness of fluorescent proteins from the fluctuations by analyzing the photon counting histogram (PCH) or its moments and demonstrate the use of molecular brightness in probing the oligomerization state of proteins. We report fluorescence fluctuation measurements of enhanced GFP (EGFP) in cells up to concentrations of 10 microM by using an improved PCH theory. The molecular brightness of EGFP is constant in the concentration range studied. The brightness of a tandem EGFP construct, which carries two fluorophores, increases by a factor of two compared with EGFP alone, demonstrating the sensitivity of molecular brightness as a probe for protein complex formation. Oligomerization of nuclear receptors plays a crucial role in the regulation of gene expression. We probe the oligomerization state of the testicular receptor 4 and the ligand-binding domains of retinoid X receptor and retinoic acid receptor by observing molecular brightness changes as a function of protein concentration. The large concentration range accessible by experiment allows us to perform titration experiments on EGFP fusion proteins. An increase in the molecular brightness with protein concentration indicates the formation of homocomplexes. We observe the formation of homodimers of retinoid X receptor ligand binding domain upon addition of ligand. Resolving protein interactions in a cell is an important step in understanding cellular function on a molecular level. Brightness analysis promises to develop into an important tool for determining protein complex formation in cells.

Animals↗
PubMed2003

The photon counting histogram in fluorescence fluctuation spectroscopy with non-ideal photodetectors.

Fluorescence fluctuation spectroscopy utilizes the signal fluctuations of single molecules for studying biological processes. Information about the biological system is extracted from the raw data by statistical methods such as used in fluctuation correlation spectroscopy or photon counting histogram (PCH) analysis. Since detectors are never ideal, it is crucial to understand the influence of photodetectors on signal statistics to correctly interpret the experimental data. Here we focus on the effects of afterpulsing and detector dead-time on PCH statistics. We determine the dead-time and afterpulse probability for our detectors experimentally and show that afterpulsing can be neglected for most experiments. Dead-time effects on the PCH are concentration-dependent and become significant when more than one molecule is present in the excitation volume. We develop a new PCH theory that includes dead-time effects and verify it experimentally. Additionally, we derive a simple analytical expression that accurately predicts the effect of dead-time on the molecular brightness. Corrections for non-ideal detector effects extend the useful concentration range of PCH experiments and are crucial for the interpretation of titration and dilution experiments.

Biophysical Phenomena↗
PubMed2003

High-pressure fluorescence correlation spectroscopy.

We demonstrate that a novel high-pressure cell is suitable for fluorescence correlation spectroscopy (FCS). The pressure cell consists of a single fused silica microcapillary. The cylindrical shape of the capillary leads to refraction of the excitation light, which affects the point spread function of the system. We characterize the influence of these beam distortions by FCS and photon-counting histogram (PCH) analysis and identify the optimal position for fluorescence fluctuation experiments in the capillary. At this position within the capillary, FCS and photon-counting histogram experiments are described by the same equations as used in standard FCS experiments. We report the first experimental realization of fluorescence fluctuation spectroscopy under high pressure. A fluorescent dye was used as a model system for evaluating the properties of the capillary under pressure. The autocorrelation function and the photon count distribution were measured in the pressure range from 0 to 300 MPa. The fluctuation amplitude and the diffusion coefficient show a small pressure dependence. The changes of these parameters, which are on the order of 10%, are due to the pressure changes of the viscosity and the density of the aqueous medium.

Capillary Action↗
PubMed2002

Molecular brightness characterization of EGFP in vivo by fluorescence fluctuation spectroscopy.

We characterize the molecular properties of autofluorescence and transiently expressed EGFP in the nucleus and in the cytoplasm of HeLa cells by fluorescence correlation spectroscopy (FCS) and by photon counting histogram (PCH) analysis. PCH has been characterized and applied in vitro, but its potential for in vivo studies needs to be explored. Thus, this study mainly focuses on the characterization of PCH analysis in vivo. The strength of PCH lies in its ability to distinguish biomolecules by their molecular brightness value. Because the concept of molecular brightness is crucial for PCH analysis, we study the molecular brightness of EGFP and determine the statistical accuracy of its measurement under in vivo conditions. We started by characterizing the influence of autofluorescence on EGFP measurements. We found a molecular brightness of EGFP that is a factor of 10 higher than the brightness of the autofluorescence. Moment analysis demonstrates that the contribution of autofluorescence to fluorescence fluctuation experiments is negligible at EGFP concentrations of one protein per excitation volume. The molecular brightness of EGFP measured in the nucleus, the cytoplasm, and in vitro are identical and our study demonstrates that molecular brightness is a very stable and predictable quantity for cellular measurements. In addition to PCH, we also analyzed the autocorrelation function of EGFP. The diffusion coefficient of EGFP is a factor of 3 lower in vivo than compared to in vitro, and a simple diffusion process describes the autocorrelation function. We found that in the nucleus the fluorescence intensity is stable as a function of time, while measurements in the cytoplasm display fluorescence intensity drifts that complicate the data analysis. We introduce and discuss an analysis method that minimizes the influence of the intensity drifts on PCH analysis. This method allows us to recover the correct molecular brightness of EGFP even in the presence of drifts of the fluorescence intensity signal. We found the molecular brightness of EGFP to be a very robust parameter, and anticipate the use of PCH analysis for the study of oligomerization processes in vivo.

Cell Nucleus↗