High-resolution 3D imaging of single semiconductor nanocrystals

A method of imaging a microscopic object comprising:

• arranging a microscopic object associated with at least a first and a second semiconductor nanocrystal in the optical field of an optical microscope, wherein at least one of the semiconductor nanocrystals has a coating that has an affinity for a biological structure;
• recording a first fluorescence image of the first and second semiconductor nanocrystals at a first focal depth;
• recording a second fluorescence image of the first and second semiconductor nanocrystals at a second focal depth different from the first focal depth;
• and
• calculating the relative depths of the first and second semiconductor nanocrystals based at least in part on the first and second fluorescence images,
• wherein calculating the relative depths of the first and second semiconductor nanocrystals includes measuring a radius of the first semiconductor nanocrystal as it appears in each of the first and second fluorescence images, wherein measuring the radius includes determining a radial intensity profile, and wherein determining the radial intensity profile includes calculating an integrated radial intensity profile for a plurality of different angles.