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iPALM: PALM in Three Dimensions
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Interferometry is a technique that can measure positions to nanometer accuracy. To gain access to the third axial dimension, we have recently combined interferometry with PALM in a method we call iPALM that can resolve individual fluorescent protein locations in three dimensions. Requirements of self-calibration and tolerance to fluctuating brightness of the labels can be met with simultaneous multiphase interferometry and inspired the invention of a custom three-way beam splitter. The following movie illustrates how light from a single-molecule source interferes into three different beams, depending on the axial location of the molecule. The relative brightness of the three different images of the molecule can be read out to determine its axial position.
The first comprehensive application of iPALM was to tease apart the molecular architecture of a cellular structure called focal adhesions. These ~100 nm thick plaques consist of over 100 different proteins that connect an object outside the cell, in our case the glass slide surface, through the membrane to the actin fibers of the cytoskeleton. With data from the iPALM we were able to see the layering of the different proteins and in certain cases even determine the orientation of longer proteins. The following movie is an example of iPALM imaging of a plasma membrane marker, integrin αv and actin.
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