Refocusing and locating effect of fluorescence scattering field*

Abstract

Optical imaging deep inside scattering medium has always been one of the challenges in the field of bioimaging, which significantly drawbacks the employment of con-focal microscopy system. Although a variety of feedback techniques, such as acoustic or nonlinear fluorescence-based schemes have realized the refocusing of the coherent light, the problems of non-invasively refocusing and locating of linearly-excited fluorescent beads inside the scattering medium have not been thoroughly explored. In this paper, we linearly excited the fluorescent beads inside a scattering medium by using our homemade optical con-focal system, collected the fluorescence scattering light as the optimized target, and established a theoretical model of target contrast enhancement, which is consistent with the experimental data. By improving both the cost function and variation rate within the genetic algorithm, we could refocus the fluorescence scattering field while improving the contrast enhancement factor to 12.8 dB. Then, the positions of the fluorescent beads are reconstructed by subpixel accuracy centroid localization algorithm, and the corresponding error is no more than 4.2 μm with several fluorescent beads within the field of view. Finally, the main factors such as the number of fluorescent beads, the thickness of the scattering medium, the modulating parameter, the experimental noise and the system long-term stability are analyzed and discussed in detail. This study proves the feasibility of reconstructing fluorescent labeled cells inside biological tissues, which provides certain reference value for deep imaging of biological tissues.