Antonio Virgilio Failla,
Antonio Cavallo,
and Christoph Cremer
The authors are with Applied Optics & Information Processing, Kirchhoff Institute for Physics, University of Heidelberg, Albert-Ueberle-Strasse 3-5, 69120 Heidelberg, Germany.
Antonio Virgilio Failla, Antonio Cavallo, and Christoph Cremer, "Subwavelength size determination by spatially modulated illumination virtual microscopy," Appl. Opt. 41, 6651-6659 (2002)
A new approach for determining the sizes of individual, small fluorescent objects with diameters considerably below the optical resolution limit is described in which spatially modulated illumination (SMI) microscopy and 360–647-nm excitation wavelengths are used. The results of SMI virtual microscopy computer simulations indicate that, in this wavelength range, reliable measurements of sizes as small as ∼20 nm are feasible if the low numbers of fluorescence photons that are usually detected from such small objects are taken into account. This method is based on the well-known fact that the modulation of the diffraction image in a SMI microscope is disturbed by the size of the object. Using appropriately calculated calibration functions, one can use this disturbance of the modulation to determine the size of the original object.
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Three VIM approaches determining object size, starting from the evaluation of modulation contrast R, were used. The evaluations were performed for the effective excitation wavelengths shown: t, true size values, i.e., FWHMTrue of the objects fixed at the start of the VIM simulation process (see Fig.
3); , object size, determined by use of the graphic illustration of modulation contrast R (for more details see Figs.
4 and 5 and the text); L, object size determined after inversion of the linear approximation of the calibration function (for more details see Fig.
7 and the text).
Two VIM approaches to determining the object size, starting from the evaluation of modulation contrast R, were used. The evaluations were performed for the effective excitation wavelengths shown: t, true size values, i.e., FWHMTrue of the objects fixed at the start of the VIM simulation process (see Fig.
3); , object size, determined by use of the graphic illustration of modulation contrast R (for more details see Figs.
4 and 5 and the text); L, object size determined after inversion of the linear approximation of the calibration function (for more details see Fig.
8 and the text).
Three VIM approaches determining object size, starting from the evaluation of modulation contrast R, were used. The evaluations were performed for the effective excitation wavelengths shown: t, true size values, i.e., FWHMTrue of the objects fixed at the start of the VIM simulation process (see Fig.
3); , object size, determined by use of the graphic illustration of modulation contrast R (for more details see Figs.
4 and 5 and the text); L, object size determined after inversion of the linear approximation of the calibration function (for more details see Fig.
7 and the text).
Two VIM approaches to determining the object size, starting from the evaluation of modulation contrast R, were used. The evaluations were performed for the effective excitation wavelengths shown: t, true size values, i.e., FWHMTrue of the objects fixed at the start of the VIM simulation process (see Fig.
3); , object size, determined by use of the graphic illustration of modulation contrast R (for more details see Figs.
4 and 5 and the text); L, object size determined after inversion of the linear approximation of the calibration function (for more details see Fig.
8 and the text).