In optical microscopy fluorescent molecules are used to label target structures like proteins or DNA.With confocal microscopy, a complex of multiple fluorescent molecules is detected as a point spread function due to the diffraction limit. A particular challenge is to determine the number of molecules hiding behind the point spread function. In this work an extended method for determining the number of fluorescent molecules is presented. The method is based on photon antibunching, which is the phenomenon that a single fluorescent molecule can emit only one photon at a time. A statistical analysis of coincidently detected photons can be used to determine the number of photon emitters. In previous works the maximal number of molecules that can be distinguished was about 3. This has now been extended by doubling the number of detectors from 2 to 4, so that up to 4 simultaneously emitted photons can be detected. A new data analysis procedure was established according to the changes of the scheme. Simulations have shown that in theory up to 50 molecules can be resolved under realistic conditions. These predictions were experimentally validated with immobilized dsDNA labeled with 5 fluorophores. The consideration of photobleaching in the data analysis and the use of a photo-stabilizer enable up to 15 molecules to be determined. Thus, this method provides a promising tool for determining the stoichiometry of various biomolecular complex, which can not be achieved by normal microscopic methods.
In optical microscopy fluorescent molecules are used to label target structures like proteins or DNA.With confocal microscopy, a complex of multiple fluorescent molecules is detected as a point spread function due to the diffraction limit. A particular challenge is to determine the number of molecules hiding behind the point spread function. In this work an extended method for determining the number of fluorescent molecules is presented. The method is based on photon antibunching, which is the phenomenon that a single fluorescent molecule can emit only one photon at a time. A statistical analysis of coincidently detected photons can be used to determine the number of photon emitters. In previous works the maximal number of molecules that can be distinguished was about 3. This has now been extended by doubling the number of detectors from 2 to 4, so that up to 4 simultaneously emitted photons can be detected. A new data analysis procedure was established according to the changes of the scheme. Simulations have shown that in theory up to 50 molecules can be resolved under realistic conditions. These predictions were experimentally validated with immobilized dsDNA labeled with 5 fluorophores. The consideration of photobleaching in the data analysis and the use of a photo-stabilizer enable up to 15 molecules to be determined. Thus, this method provides a promising tool for determining the stoichiometry of various biomolecular complex, which can not be achieved by normal microscopic methods.
In single-molecule fluorescence spectroscopy photon-antibunching is frequently used to prove the occurrence of single fluorophores. Furthermore, the relative frequency of coincident photon pairs was also used to determine the number of fluorophores in the diffraction limited observation volume of a confocal microscope. However, the ability to count fluorophores is so far limited to approximately 3 molecules due to saturation of the calibration curve with increasing number of fluorophores. Recently, we introduced a novel theoretical framework for counting the number of emitting molecules by analyzing photon-distributions acquired with a confocal microscope using four single-photon detectors. Here, we present the experimental realization of the proposed scheme in a confocal setup using novel multi-channel photon-counting electronics and DNA constructs that were labelled with five fluorophores. Our experimental results give a clear correlation between the number of estimated fluorophores and the number of bleaching steps for DNA probes conjugated with five ATTO647N labels with an error of approximately 20%. Moreover, we could acquire experimental data for up to 15 fluorophores indicating the simultaneous occurrence of three DNA probes. Our experiments put into perspective that the analysis of photon-distributions acquired with four detection channels is suited to count the number of fluorescently labelled molecules in larger aggregates or clusters with potential for applications in molecular and cell biology and for time-resolved analysis of multi-chromophoric compounds in material sciences.
Acquisition of quantitative information from microscopic biological samples is highly desirable in the context of the emerging field of systems biology. We derive a statistical approach to estimate the number of fluorescent molecules in the observation volume based on a confocal microscope for single-molecule detection. The method employs ps-pulsed laser sources for excitation and time-correlated single-photon counting with 4 avalanche photon diodes (APDs) for detection of individual photons. The feasibility for estimating the number of molecules is shown based on simultaneous emission and detection of multiple photons (photon-antibunching) under realistic experimental conditions. In theory, it should be possible to estimate the number of molecules with errors of less than 1% by using novel photo-stabilizing agents. The proposed method puts into perspective its application for high-resolution microscopy without the need for photo-switching or photo-activation of fluorescence dyes.
Single-molecule fluorescence microscopy has been established in life science as an essential tool for studying the characteristics and dynamics of individual fluorescent emitters both in vitro as well as in vivo. Acquiring quantitative information from the confocal observation volume is still a challenging task and knowing the absolute number or concentration of proteins in, e.g., cellular structures can significantly improve our understanding of cell biology, which is an important step towards quantitative microscopy. In this talk, a new quantitative analytical tool called Counting by Photon Statistics (CoPS) will be presented. The approach relies on a statistical analysis of detected photon coincidences to estimate the number of independent fluorescent labels in the observation volume [1]. CoPS does exploit the photon antibunching effect: a single photon emitter can only generate one photon at a time. Originally developed for point measurements, CoPS has recently been extended to an imaging scheme [2]. Using a confocal fluorescence microscopy setup with pulsed excitation, four single-photon, detectors and parallellized time-correlated single photon counting electronics (MicroTime 200, PicoQuant), we prove the applicability of the method with artificial model systems (immobilized DNA origami) and present first steps towards biological samples. [1] Ta, H., Wolfrum, J., Herten, D.-P., An extended scheme for counting fluorescent molecules by photon-antibunching Laser Phys. 20:119 (2010). [2] Ta, H. et al., Mapping molecules in scanning far-field fluorescence nanoscopy. Nat. Commun. 6:7977 (2015).
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.
The lack of reliable quantitative methods in fluorescence microscopy prevents the detailed investigation of basic molecular processes. Single-molecule fluorescence spectroscopy offers several techniques to determine the stoichiometry of fluorescent probes. Among them, an approach using the single-photon emission of an individual quantum system, also known as photon antibunching, yields access to both the emitter number and their molecular brightness. Previous work demonstrated the feasibility of the method to estimate up to five dyes without further physical characterization. In this work, I investigated and improved the reliability and robustness of this method. I developed different fluorescent probes with a defined label stoichiometry to explore the performance of our approach \nin experiments. I could demonstrate, in combination with simulations, that it can provide reliable estimates of up to 18 fluorescent emitters at a time resolution of approximately 200 ms. Furthermore, I inspected the dependency of the estimation bias and precision on the laser intensity, the analysis time and the number of expected molecules in detail. My results designate the scope for future stoichiometry determinations. In initial experiments, I analyzed the labeling distributions of different fluorescent probes. In addition, I could combine our counting approach with super-resolution STED-microscopy and identify critical parameters for possible applications
The formation of protein complexes with other proteins and nucleic acids is critical to biological function. Although it is relatively easy to identify the components present in these complexes, it is often difficult to determine their exact stoichiometry and obtain information about the homogeneity of the sample from bulk measurements. We demonstrate the use of single molecule photon-pair correlation spectroscopy to distinguish between discrete numbers of molecules in biological complexes. Fluorescence photon antibunching is observed from a single molecule by employing time-correlated single photon counting in combination with a Hanbury-Brown and Twiss coincidence setup. In addition, pulsed laser excitation and time-tagged time-resolved data collection allow for the measurement of photon arrival times with nanosecond time resolution. The interphoton time distribution between consecutively arriving photons can be calculated and provides a measure of the second-order temporal correlation function. Analysis of this function yields an absolute measure of the number of molecules, N, present in a given complex. It is this ability to measure N that renders this technique powerful for determining stoichiometries in complex biological systems at the single molecule level. We investigate the counting efficiency and statistics of photon antibunching of specifically designed biological samples labeled with multiple copies of the same fluorescent dye and derive conclusions about its use in the analytical evaluation of complex biological samples.