This paper discusses the opinion that illuminating intensity will seriously affect the effectiveness of automatic focusing techniques in the microscopic vision research field. An automatic lighting control system is developed to provide experimental conditions in illumination research. Firstly

a tool of Fast Fourier Transform (FFT) is used to analyze spectrum of a sequence defocused images and a sequence images under different lighting intensity

proving that lighting intensity conditions impact significantly on the high frequency energy of images. Then

a function that can evaluate high-frequency energy of images is proposed. Finally

some auto-focusing experiments are executed. Experimental results show that lighting intensity has a great impact on focusing curves

and higher accuracy can be achieved after illumination optimization than usual method.