Nanometer-scale optical imaging by the modulation tracking (MT) method

摘要

An optical imaging method based on a feedback principle in which the specific scan pattern is adapted according to the shape of the sample. The feedback approach produces nanometer-resolved three dimensional images of very small and moving features in live cells and in a matter of seconds. Images of microvilli in live cultured opossum kidney cells expressing NaPi co-transporter proteins with different GFP constructs and images of cell protrusions in a collagen matrix are produced with a resolution of about 20 nm. Along cell protrusions in three dimensional cellular adhesions could be identified to the extracellular matrix.

主权项

1. A method of optical imaging for producing a nanometer-resolved three dimensional image of a feature of an object comprising:
moving a laser spot around a surface of the feature in a circular orbit; oscillating a radial position of the laser spot perpendicularly with respect to the surface of the feature as the laser spot is being moved in the circular orbit around the surface of the feature, wherein the laser spot has a cross section characterized by a point spread function with a width, the radial position of the laser spot within the circular orbit further being oscillated within the width of the point spread function of the laser spot using feedback control of the perpendicular distance; detecting a returned fluorescence signal from the feature of the object; measuring the perpendicular distance of the laser spot from the surface in real time upon detection of the returned fluorescence signal using a modulation function; scanning the feature of the object at a plurality of selective feedback controlled scan positions and repeating oscillating, detecting and measuring in repeated three dimensional scans to acquire an ensemble of corresponding measured perpendicular distances; and constructing a three dimensional image of the shape of the feature of the object from the ensemble of corresponding measured perpendicular distances and scan positions of the oscillating laser spot during scanning.