Method for correcting motion artifacts of in vivo fluorescence measurements

A method for correcting motion artifacts of in vivo fluorescence measurements of a live specimen using a 3D laser scanning microscope having an optical axis (Z) and X, Y axis that are perpendicular to the optical axis and to each other and having a first pair of acousto-optic deflectors deflecting a laser beam in an x-z plane defined by the X and Z axis and a second pair of acousto-optic deflectors deflecting the laser beam in an y-z plane defined by the Y and Z axis,

• selecting a fluorescent object on a dark background within the sample,
• providing three perpendicular imaging planes that are centered on the fluorescent object,
• scanning each imaging plane by providing chirp signals for the acoustic frequencies in the deflectors for focusing the laser beam to a focus spot and continuously moving the focus spot along parallel lines lying within the given imaging plane, so as to scan the entire imaging plane,
• determining a 3-dimensional trajectory of the fluorescent object during the predetermined period of time
• determining an average motion direction of the fluorescent object from the 3-dimensional trajectory,
• selecting within the specimen a plurality of points of interest,
• extending each point of interest to a scanning line traversing the given point of interest and being parallel to the average motion direction of the fluorescent object,
• scanning each scanning line by focusing the laser beam at one end of the given scanning line and providing chirp signals for the acoustic frequencies in the deflectors for continuously moving the focus spot along the given scanning line, thereby obtaining scanning data of each scanning line corresponding to one of the plurality of points of interest,
• projecting the scanning data of each scanning line to 1-dimensional frames,
• repeating the measurement to obtain a 1-dimensional time series from the successive frames for each scanning line corresponding to one of the plurality of points of interest,
• correcting motion artifacts by shifting the data of the successive frames with respect to each other so as to maximize fluorescence cross correlation between the data of the frames.