A novel imaging method which can acquire highcontrast image of collagen and elastin,twochannel sequential detection imaging based on twophoton excited autofluorescence and second harmonic generation of biological tissues,is presented by using a Zeiss LSM 510 META laser scanning microscopy system. And this method is successfully used to obtain highcontrast image of collagen and elastin in the stromal layer of esophageal tissue at 84μm and 150μm depths. The experimental findings show that the use of two independent channels obviously increases imaging contrast of collagen and elastin. Both twophoton excited autofluorescence and second harmonic signals of tissue samples are collected in a backscattering geometry. This makes twochannel sequential detection imaging technique have a clinic application potential for pathological diagnosis.