This award is for the development of techniques for tracking multiple fluorescing particles simultaneously in a confocal microscope. The project will initially develop algorithms for tracking single fluorescent particles in vitro and inside living cells. Techniques from modern systems and control theory, including limited communication control and optimal estimation, will be built upon to enable tracking of multiple particles simultaneously. The capabilities to be introduced by this project will enable the study of communication between cells, processes inside living cells, and the dynamics of individual molecules and of molecular interactions.
The ability to obtain three-dimensional trajectory information at fine temporal resolution is a vital component for developing a deeper understanding of many processes in molecular biology. The project is multi-disciplinary with both theoretical and experimental components. In addition to support for a female graduate student, modules for a pre-university outreach program will be developed and utilized in a university program targeting academically at-risk students in the 7th and 8th grades. Taking advantage of the large number of biotechnology companies in the greater Boston area, a new freshman level course will be developed for exploring emerging areas of science and technology.
In this project, we created a novel algorithm for position estimation of a sub-diffraction limit sized single fluorescent particle. The algorithm is orders of magnitude faster than previous approaches with only a slight degradation in accuracy. It has been shown through both simulation and experiment to work well, even with only a few measurements and in the presence of large noise and has been analyzed from a theoretical point of view, giving it a firm mathematical foundation. We then built upon this algorithm to create a tracking method to follow single fluorescent particles moving in three dimensions using a confocal microscope. The algorithm has been demonstrated through experiments with quantum dots freely diffusing in three dimensions. The maximum diffusion rate tracked to date is on the order of 0.1 microns squared per second with the speed currently limited by the actuator hardware. The algorithm has also been extended to follow multiple particles simultaneously. These techniques significantly bring the benefits of the confocal modality (good signal-to-noise ratio, high sensitivity, 3-D sectioning, and more) to the ability to follow single particles for long periods of time and over large distances. They will therefore enhance our ability to study single molecules and understand their dynamics and their interactions with other particles and features. These tools should find application in studies in molecular and cellular biology. The biological research in this project also spurred research into the time optimal control of dynamic systems. This work provided a new and efficient method for the time optimal control problem. We have already applied it to scanning problems in atomic force microscopy and expect that the work will find broad application.
