A procedure to determine the correct thickness of an object with confocal microscopy in case of refractive index mismatch

A difference in refractive index (n) between immersion medium and specimen results in increasing loss of intensity and resolution with increasing focal depth and in an incorrect axial scaling in images of a confocal microscope. Axial thickness measurements of an object on such images are therefore not exact. The present paper describes a simple procedure to determine the correct axial thickness of an object with confocal fluorescence microscopy. We study this procedure for a specimen that has a higher refractive index than the immersion medium and with a thickness up to 100 µm. The measuring method was experimentally tested by comparing the thickness of polymer layers measured on axial images of a confocal microscope in case of a water–polymer mismatch to reference values obtained from an independent technique, i.e. scanning electron microscopy. The case when the specimen has a lower refractive index than the immersion medium is also shown by way of illustration. Measured thickness data of a water layer and an oil layer with the same actual thickness were obtained using an oil-immersion objective lens with confocal microscopy. Good agreement between theory and experiment was found in both cases, consolidating our method.