Multiplane differential saturated excitation microscopy using varifocal lenses.

Saturated excitation microscopy, which collects nonlinear fluorescence signals generated by saturation, has been proposed to improve three-dimensional spatial resolution. Differential saturated excitation (dSAX) microscopy can further improve the detection efficiency of a nonlinear fluorescence signal. By comparing signals obtained at different saturation levels, high spatial resolution can be achieved in a simple and efficient manner. High-resolution multiplane microscopy is perquisite for volumetric imaging of thick samples. To the best of our knowledge, no reports of multiplane dSAX have been made. Our aim is to obtain multiplane high-resolution optically sectioned images by adapting differential saturated excitation in confocal laser scanning fluorescence microscopy. To perform multiplane dSAX microscopy, a variable focus lens is employed in a telecentric design to achieve focus tunability with constant magnification and contrast throughout the axial scanning range. Multiplane fluorescence imaging of two different types of pollen grains shows improved resolution and contrast. Our system's imaging performance is evaluated using standard targets, and the results are compared with standard confocal microscopy. Using a simple and efficient method, we demonstrate multiplane high-resolution fluorescence imaging. We anticipate that high-spatial resolution combined with high-speed focus tunability with invariant contrast and magnification will be useful in performing 3D imaging of thick biological samples.