Depth of Field Measurements Relevant to Single Photon Detection Using Image-intensified Microscopy

The problems of defining a depth of field d p when individual photons emitted in a low-level luminescent process are recorded via an image-intensified microscope are discussed. Simulation studies of a self-luminous cylindrical volume source whose axis lies along the optical axis of the microscope were carried out by moving a uniformly-illuminated pinhole along the optical axis, and arranging for its in-focus image to fill exactly a circular light detector. The detector output plotted against pinhole position is approximately Gaussian in form for the objectives studied (from 10 2 /0·25 to 74 2 /0·65), and d p is defined as the full width at half maximum. These values of d p adequately fit the theoretical relation d p = 2·45 R/tan sin -1 (NA/n), where NA is the numerical aperture of the objective and n is the refractive index of the immersion medium. With spherical, or near-spherical, volume sources d p is usually significantly greater than the volume of the source. The problems of defining a depth of field p when individual photons emitted in a low-level luminescent process are recorded via an image-intensified microscope are discussed. Simulation studies of a self-luminous cylindrical volume source whose axis lies along the optical axis of the microscope were carried out by moving a uniformly-illuminated pinhole along the optical axis, and arranging for its in-focus image to fill exactly a circular light detector. The detector output plotted against pinhole position is approximately Gaussian in form for the objectives studied (from 10 2 /0·25 to 74 2 /0·65), and d p is defined as the full width at half maximum. These values of d p adequately fit the theoretical relation d p = 2·45 R/tan sin -1 (NA/n), where NA is the numerical aperture of the objective and n is the refractive index of the immersion medium. With spherical, or near-spherical, volume sources d p is usually significantly greater than the volume of the source.