KEYWORDS: Simulation of CCA and DLA aggregates, Statistical analysis, Molecules, Photodetectors, Confocal microscopy, Error analysis, Luminescence, Current controlled current source, Proteins, Quantum efficiency
Typical problems in molecular biology, like oligomerization of proteins, appear on non-resolvable length scales.
Therefore a method which allows counting numbers of fluorescent emitters beyond this barrier can help to
unveil these questions. One approach engaging this task makes use of the photon antibunching (PAB) effect.
Most fluorophores are single photon emitters. Therefore upon a narrow excitation pulse they might only run
through one excitation cycle and emit one photon at a time. This behavior is known as PAB. By analyzing
coincident events of photon detections (Coincidence Analysis, CCA ) over many excitation cycles the number of
fluorophores residing in the confocal volume can be estimated. Simulations have shown that up to 40 fluorophores
can be distinguished with a reasonable error. In follow-up experiments five fluorophores could be distinguished
by CCA. In this work the method is applied to a whole sample set and statistical variance and robustness are
determined. CCA is critical to several parameters like photo stability, background noise, label efficiency and
photopysical properties of the dye, like brightness and blinking. Therefore a reasonable scheme for analysis is introduced and setup parameters are optimized. To proof the superiority of CCA, it has been applied to estimate the number of dyes for a well-defined probe and the results have been compared with bleach step analysis (BS analysis), a method based on the ability to observe single bleach-steps.
Fluorescence lifetime-based glucose sensing does not depend on fluctuations of the intensity of the light source, light
scattering, or changes in the transmission of optical components. Here we demonstrate the sensing of glucose based on
the fluorescence lifetime properties of dihydro nicotinamide adenine dinucleotide (NADH), which is reduced from NAD
in the presence of glucose and glucose dehydrogenase. In particular we use the difference in the fluorescence properties
of free and protein-bound NADH and calculate an average fluorescence lifetime, which arises from the two short
lifetimes τ1=0.28ns and τ2=0.60ns (representing free NADH) and the longer lifetime of τ3=2.9ns (for the protein-bound NADH). While initial results were derived from measurements in aqueous solution, we also demonstrate the suitability of this method for determining the concentration of glucose in blood using test strips. We find that the average
fluorescence lifetime changes linearly by a factor of 0.17 per 100mg/dl change in glucose concentration. As an
alternative the ratio between free and protein-bound components Rs/l may also be used for quantification. Rs/l increases by
a factor of 0.74 per 100mg/dl change in glucose concentration.
The approach of modeling intracellular networks of biochemical reactions in systems biology demands novel methods
suited for acquiring quantitative data about transport and interaction of proteins and metabolites within the
heterogeneous environment of living cells. Single-molecule fluorescence spectroscopy (SMFS) has proven a valuable
tool for investigating complex structures and processes in biochemistry and molecular biology providing a rich set of
methods for in vitro studies of protein/protein and protein/DNA interactions. Although especially designed to reveal
spatial and temporal heterogeneities, few applications of SMFS to living cells were reported. Multi-parameter
approaches like spectrally-resolved fluorescence lifetime imaging microscopy (SFLIM) have the unique advantage of
acquiring individual photons from single molecules carrying along characteristic properties of the fluorescence emission,
such as fluorescence lifetime and fluorescence emission spectrum. Herein we present different possibilities for data
analysis that can be applied to single molecule data recorded on single photon bases. The photons be used to generate
microscopic images with additional contrast based on different fluorescence lifetimes or emission spectra. Information
about dynamic processes influencing the characteristic fluorescence signal, like local quenching or molecular diffusion,
can be investigated by use of auto- or cross-correlation or on the basis of individual events. Additional information about
the number of molecules contributing to the fluorescence emission can be obtained by use of photo-antibunching. Yet
the versatile methods of SMFS need further development in view of acceleration and automation of data acquisition and
analysis to meet the demands of their application in systems biology.
Some fluorescent dyes, like Atto635 or Cy5, exhibit alterations in fluorescence emission in the presence of various metal
ions and metal ion complexes. We used this effect to design dye-ligand conjugates that can be immobilized on glass
surfaces and allow studying metal-ion binding using time-resolved single-molecule fluorescence spectroscopy (SMFS).
Double-stranded DNA served as a rigid scaffold carrying 2,2'-bipyridene-4,4'-dicarboxylic acid as chelating ligand and
a fluorescent dye as reporter, placed in close vicinity to the ligand. In the absence of metal ions, the probes showed high
fluorescence quantum yield, whereas strong fluorescence quenching upon binding of Cu2+-ions was observed. Time-resolved
single-molecule measurements revealed stochastic switching between a highly fluorescent ("on") and a low
fluorescent ("off") state. The coordination of the metal ion to the ligand is thus indicated by intramolecular fluorescence
quenching of the dye. We screened various fluorescent dyes for their sensitivity to Cu2+-coordination, and found that
both Atto620 and MR121 are well-suited for this application. Ensemble studies of the fluorescence lifetimes of metalsensors
with Atto620 showed only small dependence on the metal-ion concentration, while single-molecule studies
reported strong changes in the fluorescence lifetimes which were correlated with the observed on- and off-states. Our
results further indicate that the fluorescence of Atto620 is not completely quenched upon association of the metal-ion
complex; either because a less fluorescent complex is formed or because of intramolecular collisional quenching due to
conformational changes of the C6-linker used for covalent coupling of the fluorescent dye.
The investigation of the structure and dynamics of biomolecules and biomolecular assemblies in living cells is of current interest in molecular biology. Recent developments in single molecule fluorescence spectroscopy (SMFS) have opened ways for investigating the dynamics and stoichiometry of individual biomolecular complexes e.g., by application of single pair fluorescence resonance energy transfer (spFRET) with alternating laser excitation (ALEX), and by improved labels and labeling techniques. In the recent years, we have developed a set of techniques that allow the determination of the spatial distribution of single fluorescent molecules and their identification by spectrally-resolved fluorescence lifetime imaging microscopy (SFLIM) as well as the observation of the dynamics of individual molecules immobilized on surfaces. Based on SFLIM we currently focus on investigating the diffusion kinetics of biomolecules in living cells. By combining high-resolution confocal fluorescence microscopy of single molecules with fluorescence correlation spectroscopy (FCS) we seek to quantitate diffusion coefficients and concentrations of relevant fluorescently labeled biomolecules within living cells thereby visualizing the heterogeneous distribution of local mobilities in the sample. The simultaneously acquired fluorescence intensity and lifetime images can further be used for additional single point measurements for obtaining i.e., information about the stoichiometry of immobilized biomolecular complexes based on photon anti-bunching. In addition the simultaneous acquisition of multiple characteristic properties by SFLIM, like spectral emission bands and fluorescence lifetime, offers the opportunity to discriminate different fluorescent probes and autofluorescence.
Recently, we have shown that single fluorescent dye molecules within the diffraction limited detection volume can be counted by coincidence analysis. In combination with spectrally resolved fluorescence lifetime imaging microscopy (SFLIM), polarization modulation and high-resolution colocalization we suggested to use these techniques for the structural and dynamic investigation of functional protein assemblies and molecular machines in cells. Here we present the application of these techniques within fixed and living cells since quantification and observation of protein assembly in-vivo is of great interest for biological research. We show that appropriately chosen dyes, e.g. ATTO 620, can be discriminated from autofluorescent background within the cells by determination of their spectral emission and their fluorescence lifetimes measured by time correlated single photon counting (TCSPC) under pulsed laser excitation on a confocal microscope. Whereas a lot of autofluorescent signal can be found in the cytoplasm especially in living cells, the nucleus contains almost no fluorescent signal. This offers the opportunity to apply the above methods to protein assemblies, e.g. transcription units, within the cell nucleus. By investigation of fluorescently labeled poly-T40-oligonucleotides hybridized to poly-A-termini of mRNA or tethered within the cell nucleus we demonstrate the feasibility of coincidence analysis for counting single fluorescent molecules within fixed and living cells as a fundamental step for structural investigation below the diffraction limit of optical resolution.
We demonstrate how photoinduced electron transfer (PET) reactions can be successfully applied to monitor conformational dynamics in individual biopolymers. Single-pair fluorescence resonance energy transfer (FRET) experiments are ideally suited to study conformational dynamics occurring on the nanometer scale, e.g. during protein folding or unfolding. In contrast, conformational dynamics with functional significance, for example occurring in enzymes at work, often appear on much smaller spatial scales of up to several Angströms. Our results demonstrate that selective PET-reactions between fluorophores and amino acids or DNA nucleotides represent a versatile tool to measure small-scale conformational dynamics in biopolymers on a wide range of time scales, extending from nanoseconds to seconds, at the single-molecule level under equilibrium conditions. That is, the monitoring of conformational dynamics of biopolymers with temporal resolutions comparable to those within reach using new techniques of molecular dynamic simulations. We present data about structural changes of single biomolecules like DNA hairpins and peptides by using quenching electron transfer reactions between guanosine or tryptophan residues in close proximity to fluorescent dyes. Furthermore, we demonstrate that the strong distance dependence of charge separation reactions on the sub-nanometer scale can be used to develop conformationally flexible PET-biosensors. These sensors enable the detection of specific target molecules in the sub-picomolar range and allow one to follow their molecular binding dynamics with temporal resolution.
We present a new technique for high-resolution colocalization of fluorescent dyes. The technique is based on polarization modulated excitation and spectrally-resolved fluorescence lifetime imaging microscopy (SFLIM) as well as on coincidence analysis of the detected photon counts following pulsed laser excitation. The method takes advantage of single fluorescent dyes that can be efficiently excited by a single pulsed diode laser emitting at 635 nm but differ in their emission maxima, and in their fluorescence lifetime. A combined analysis of the fractional intensities and fluorescence lifetimes recorded on two spectrally-separated detectors enables the classification of the portion of each dye per pixel in a point-spread-function (PSF) image with high accuracy, even though only a limited number (generally a few thousand) photons are detected per single dye. From these portions two separate PSF images are calculated and fitted to two-dimensional (2D) Gaussian functions to localize their centers with a precision of a few nanometers. To reveal the number of absorbing and emitting molecules polarization modulated excitation and coincidence analysis of the detected photon counts is used. We demonstrate that by the use of appropriately selected dyes, the presented technique permits (1) the counting of the number of molecules present in the observation volume, and (2) the determination of the distance between two single molecules down to approximately 30 nm with a precision of approximately 10 nm without any chromatic aberrations. The developed techniques are promising for applications in molecular biology, e.g. to determine the number of polymerase molecules active within a transcription factory and/or to measure their distances to nanscent transcripts.
We developed a simple, tiny setup with only a few optical devices for fast and sensitive identification of DNA with a short-pulse semiconductor laser operating at 20 MHz. In combination with newly synthesized fluorescent dyes (rhodamine derivatives) which exhibit high fluorescence quantum efficiencies and distinct fluorescence lifetimes at semiconductor laser excitation wavelength a sensitive detection and time-resolved identification of DNA can be achieved. At an excitation wavelength of 635 nm the fluorescence background is greatly reduced. We demonstrate the DNA identification of A- and G-terminated DNA fragments labeled at the 5'-end with the rhodamine derivatives MR 200- 1 and JA 169 during capillary gel electrophoresis. The characteristic time-resolved data are acquired by the time- correlated single-photon counting technique. Time-resolved identification analysis is realized by the maximum likelihood estimator. For prediction of the error rate (misclassification) Cramers equation in combination with a pattern recognition technique is applied. These methods deliver high reliabilities at low classification error rates for low fluorescence light level applications.
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