eSIP: A Novel Solution-Based Sectioned Image Property Approach for Microscope Calibration.

Abstract

Fluorescence confocal microscopy represents one of the central tools in modern sciences. Correspondingly, a growing amount of research relies on the development of novel microscopic methods. During the last decade numerous microscopic approaches were developed for the investigation of various scientific questions. Thereby, the former qualitative imaging methods became replaced by advanced quantitative methods to gain more and more information from a given sample. However, modern microscope systems being as complex as they are, require very precise and appropriate calibration routines, in particular when quantitative measurements should be compared over longer time scales or between different setups. Multispectral beads with sub-resolution size are often used to describe the point spread function and thus the optical properties of the microscope. More recently, a fluorescent layer was utilized to describe the axial profile for each pixel, which allows a spatially resolved characterization. However, fabrication of a thin fluorescent layer with matching refractive index is technically not solved yet. Therefore, we propose a novel type of calibration concept for sectioned image property (SIP) measurements which is based on fluorescent solution and makes the calibration concept available for a broader number of users. Compared to the previous approach, additional information can be obtained by application of this extended SIP chart approach, including penetration depth, detected number of photons, and illumination profile shape. Furthermore, due to the fit of the complete profile, our method is less susceptible to noise. Generally, the extended SIP approach represents a simple and highly reproducible method, allowing setup independent calibration and alignment procedures, which is mandatory for advanced quantitative microscopy.