General analysis and optimization strategy to suppress autofluorescence in microscope lenses.

Abstract

With the development of fluorescence microcopy, the autofluorescence effect of optical glass in microscope lenses has become an important source of stray light which decreases the contrast of the image. However, the autofluorescence effect of various types of microscope lenses has not been thoroughly discussed and the dominant factors of this effect are not clear. Currently, the most commonly used analysis method of the autofluorescence effect is based on the volume scattering model and Monte Carlo raytracing, which is extremely time-consuming due to the large number of rays that need to be traced. In our previous work, we have presented an efficient phase-space-based simulation method, which significantly accelerates the simulation of the autofluorescence effect [Appl. Opt.58, 3589 (2019)APOPAI0003-693510.1364/AO.58.003589]. Here we apply this new method on different types of microscope lenses and perform a systematic analysis of the autofluorescence effect based on the simulation results. In order to obtain an overview of the autofluorescence effect of different types of microscope lenses, more than 100 microscope lenses with different numerical aperture (NA), magnification, working distance, and immersion medium are selected and the corresponding autofluorescence contribution from each element in the lenses is calculated. Following a systematic analysis of the simulation result, we find that the autofluorescence effect of a lens is dependent on the etendue and complexity of the system, while the most critical elements are usually found in the front and rear groups. After the origins of the autofluorescence contribution from these critical lens groups have been found, possibilities to reduce the autofluorescence intensity have been investigated. Finally, effective methods to reduce the autofluorescence effect are presented and compared. The major contribution of this work is a detailed analysis of the impact of different factors on the autofluorescence effect, based on which a guideline is provided for the optical designers as well as the users to design or to select an appropriate microscope lens with low autofluorescence intensity.