Antonio Virgilio Failla

Spatially modulated illumination microscopy allows axial distance resolution in the nanometer range

For an improved understanding of the structural basis of cellular mechanisms, it is highly desirable to develop methods for a detailed topological analysis of biological nanostructures and their dynamics in the interior of three-dimensionally conserved cells. We present a method of far-field laser fluorescence microscopy to measure relative axial positions of pointlike fluorescent targets and the distance between each target in the range of a few nanometers. The physical principle behind this approach can be extended to the determination of three-dimensional (3D) positions and 3D distances between any number of objects that can be discriminated owing to their spectral signature, thus allowing topological measurements so far regarded to be beyond the capabilities of light microscopy.

Nanosizing of fluorescent objects by spatially modulated illumination microscopy

A new approach to measuring the sizes of small fluorescent objects by use of spatially modulated illumination (SMI) far-field light microscopy is presented. This method is based on SME measurements combined with a new SMI virtual microscopy (VIM) data analysis calibration algorithm. Here, experimental SMI measurements of fluorescent objects with known diameter (size) were made. From the SMI data obtained, the size was determined in an independent way by use of the SMI VIM algorithm. The results showed that with SMI microscopy in combination with SMI VIM calibration, subwavelength object size measurements as small as 40 nm are experimentally feasible with high accuracy.

Subwavelength size determination by spatially modulated illumination virtual microscopy

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 approximately 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.

Superresolution size determination in fluorescence microscopy: A comparison between spatially modulated illumination and confocal laser scanning microscopy

Recently developed far field light optical methods are a powerful tool to analyze biological nanostructures and their dynamics, in particular including the interior of three-dimensionally conserved cells. In this article, the recently described method of spatially modulated illumination (SMI) microscopy has been further extended to the online determination of the extension of small, subwavelength sized, fluorescent objects (nanosizing). Using fluorescence excitation with 488 nm, the determination of fluorescent labeled object diameters down to 40 nm corresponding to about 1/12th of the wavelength used for one-photon excitation could be shown. The results of the SMI nanosizing procedure for a detailed, systematic variation of the object diameter are presented together with a fast algorithm for online size evaluation. In addition, we show a direct comparison of the diameter of “colocalization volumes” between SMI nanosizing and conventional confocal laser scanning microscopy.

Virtual spatially modulated illumination microscopy predicts nanometer precision of axial distance measurment

Far field optical light microscopy with its unique capability for contactless, non destructive imaging inside thick transparent specimen such as cell nuclei has contributed widely to the present knowledge of the three- dimensional (3D-) architecture of the interphase nucleus. A serious drawback, however, is the limited optical resolution. A recently introduced light microscopical approach, Spectral Precision Distance Microscopy (SPDM) allows the measurement of distances between point-like fluorescent objects of different spectral signature far below the optical resolution criterion as defined by the Full Width at Half Maximum (FWHM) of the point spread function (PSF). Here, an aspect of the theoretical limits of this method was studied by virtual microscopy. The precision of the axial distance measurements was studied, taking into account photon statistics and image analysis. The results indicate that even under low fluorescence intensity conditions typical for biological structure research, a precision of distance measurements in the nanometer range can be determined.