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.