Two-Photon Excitation Fluorescence Microscopy Motion Tracking to Study in-vivo Subcellular Structures

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Collaborators

Laboratory of Cardiac Energetics, NHLBI
Laboratory of Cardiac Energetics, NHLBI
Laboratory of Cardiac Energetics, NHLBI
Laboratory of Cardiac Energetics, NHLBI (formerly with IDEAS)
Laboratory of Cardiac Energetics, NHLBI
Laboratory of Cardiac Energetics, NHLBI
Laboratory of Cardiac Energetics, NHLBI
Laboratory of Cardiac Energetics, NHLBI
Laboratory of Cardiac Energetics, NHLBI

Project Brief

IDEAS collaborated with the NHLBI Laboratory of Cardiac Energetics to develop a methodology enabling the study in vivo sub-cellular structures and signaling processes in real-time.  Multi-photon fluorescence imaging provides improved tissue penetration, sensitivity, and information content when investigating dynamic intercellular events within living tissue, but physiological motion degrades the quality of these images and makes temporal observations challenging.  Our system implementation, designed to be functional in tandem with the commercial two-photon microscope system, adjusts the field-of-view and focal plane of the microscope in real-time to compensate for the natural physiological motion of living tissue.  The function and performance of the commercial system is unaffected.  The motion tracking system intercepts the images as they are generated by the microscope, creating a multi-slice volume.  The system utilizes a high-performance GPU platform to correlate each real-time volume to an initial reference volume to quantify the amount and direction of tissue motion.  This motion information is used to adjust the microscope XY stage and objective to dynamically correct for the tissue motion.  IDEAS integrated the custom hardware and GPU, and developed the custom software for this system. 

Slice T racking using an Optical Navigator Desired plane Rapidly run a reduced FOV stack before each image, or fraction of image to find the appropriate plane Motion Control Solution

Two-photon excitation fluorescence microscopy motion tracking concept

Data Processing Computer Position Out T esla c1060 SYNC In Image In Focus Controller Stage Controller X- Y Stage Standard Microscope Computer TC P ethernet USB Interface Laser Scanning Microscope Obj. Z Stepper Motor Z Piezo

Block diagram of two-photon excitation fluorescence microscopy motion tracking system

 

Time-lapse volume of with and without motion tracking correction

Time-lapse volume of with and without motion tracking correction