Single-Molecule Measurements in Solution by FCS/FCCS

Fluorescence from a single molecule can be distinguished from the small background associated with a femtoliter of solvent. At a solution concentration of about 1 nM, the probability that there is an analyte molecule in the confocal probe volume is less than one. Although fluorescence from individual molecules is collected, the data are analyzed by an auto-correlation or two-color cross-correlation function that is the average of thousands of molecules. Properties of single molecules are not obtained by FCS and two-color FCCS. We want to open the door for biophysical testing in an environment, ideal for cell biologists and clinicians, by enabling these measurements to take place directly in solution without immobilization or hydrodynamic focusing in order to measure the dynamics of single particles undergoing reactions in solution on a timescale not previously available. Probing a biological process in live cells with good spatial and temporal resolution in a selective way offers a significant potential for the understanding of many biological problems. The work presented in this project proposal involves the development of a new analysis technique for singlemolecule spectroscopy. For the first time, it is turned into a potential opportunity. The new concept of analysis of just one single molecule in solution over extended periods of time will make it possible to observe the single molecules without bias to the millisecond range in solution of freely diffusing molecules or to artificial immobilization on surfaces or hydrodynamic focusing. The following main idea is put forward: is it possible to statistically recognize the same molecule multiple times as it diffuses through a small confocal probe volume? If so, the detection time of an individual molecule freely diffusing in solution could be extended. For the new analysis approach, it is crucial to understand the balance between the time scale and the frequency that a single molecule reenters the confocal probe volume and the overall time necessary for the measurement. The proposed research will provide with a better understanding of some of the theoretical and experimental backgrounds of these types of analyses and biological or medical applications. The originality and innovative nature of the proposal work reside in the strict coupling of latest microscopic and spectroscopic developments with physiological relevance. The new ideas are comprehensively presented and this relationship is a new concept at the time. Possible users for this concept are those working in biotechnological applications dealing with gene technology. Furthermore, the concept is of interest for a great number of medical, pharmaceutical and bio-chemical laboratories. It may serve as a foundation for further work in single-cell biology. The groups involved in this application have been internationally competitive in their respective research areas. We propose a joint, coordinated research program, with studies to be performed at the molecular to the systemic level, covering theoretical as well as experimental approaches, spanning different competence fields, from laser spectroscopy and biophysics to mathematics and biochemistry. Our ambition mounts a broad approach, capable of providing and interpreting data from a range of viewpoints, to cover as many degrees of freedom as possible, needed to sufficiently describe and understand the DNA and protein interactions under study. The combination of all efforts and resources will provide a unique opportunity to develop a leading center for these studies. Without this grant, the integration between the ISS and the MUG partners will not happen, and the efforts will be significantly delayed. If not awarded, the theoretical methods will mainly be developed independently, and the comparison with experimental results will have to wait for other groups to produce the necessary data.

Fluorescence from a single molecule can be distinguished from the small background associated with a femtoliter of solvent. At a solution concentration of about 1 nM, the probability that there is an analyte molecule in the confocal probe volume is less than one. Although fluorescence from individual molecules is collected, the data are analyzed by an auto-correlation or two-color cross-correlation function that is the average of thousands of molecules. Properties of single molecules are not obtained by FCS and two-color FCCS. We want to open the door for biophysical testing in an environment, "ideal" for cell biologists and clinicians, by enabling these measurements to take place directly in solution without immobilization or hydrodynamic focusing in order to measure the dynamics of single particles undergoing reactions in solution on a timescale not previously available. Probing a biological process in live cells with good spatial and temporal resolution in a selective way offers a significant potential for the understanding of many biological problems. The work presented in this project proposal involves the development of a new analysis technique for singlemolecule spectroscopy. For the first time, it is turned into a potential opportunity. The new concept of analysis of just one single molecule in solution over extended periods of time will make it possible to observe the single molecules without bias to the millisecond range in solution of freely diffusing molecules or to artificial immobilization on surfaces or hydrodynamic focusing. The following main idea is put forward: is it possible to statistically recognize the same molecule multiple times as it diffuses through a small confocal probe volume? If so, the detection time of an individual molecule freely diffusing in solution could be extended. For the new analysis approach, it is crucial to understand the balance between the time scale and the frequency that a single molecule reenters the confocal probe volume and the overall time necessary for the measurement. The proposed research will provide with a better understanding of some of the theoretical and experimental backgrounds of these types of analyses and biological or medical applications. The originality and innovative nature of the proposal work reside in the strict coupling of latest microscopic and spectroscopic developments with physiological relevance. The new ideas are comprehensively presented and this relationship is a new concept at the time. Possible users for this concept are those working in biotechnological applications dealing with gene technology. Furthermore, the concept is of interest for a great number of medical, pharmaceutical and bio-chemical laboratories. It may serve as a foundation for further work in single-cell biology. The groups involved in this application have been internationally competitive in their respective research areas. We propose a joint, coordinated research program, with studies to be performed at the molecular to the "systemic" level, covering theoretical as well as experimental approaches, spanning different competence fields, from laser spectroscopy and biophysics to mathematics and biochemistry. Our ambition mounts a broad approach, capable of providing and interpreting data from a range of viewpoints, to cover as many degrees of freedom as possible, needed to sufficiently describe and understand the DNA and protein interactions under study. The combination of all efforts and resources will provide a unique opportunity to develop a leading center for these studies. Without this grant, the integration between the ISS and the MUG partners will not happen, and the efforts will be significantly delayed. If not awarded, the theoretical methods will mainly be developed independently, and the comparison with experimental results will have to wait for other groups to produce the necessary data.