Effective improvement of subtraction method for light sheet fluorescence microscopy via tangent-function subtraction coefficient

Light sheet fluorescence microscopy (LSFM) with fluorescence emission difference (FED) obtains axial resolution-enhanced images by the subtraction of signals excited by different light sheets, Gaussian light sheet and negative light sheet. Negative values are inevitable in FED LSFM with conventional subtraction coefficient, due to mismatches in the outer contours of the raw signals excited by different light sheets, causing image distortion. In this paper, we propose a method based on tangent-function subtraction coefficient to reduce image distortion. This tangent-function subtraction coefficient is introduced to minimize image distortion without lowering the axial resolution. The value of the variable subtraction coefficient is obtained from the intensity value of raw signals. We performed experiments using fluorescent beads with a diameter of 1 micron and compared the subtraction results with constant and tangent-function subtraction coefficient. The experimental results demonstrated that the proposed tangent-function subtraction coefficient has an advantage over the conventional constant coefficient. © 2018 SPIE.