A total internal reflection fluorescence imaging system, comprising: a first imaging system , wherein the first imaging system comprises N separate laser light paths, an objective lens , a first receiving device , N first light sources , a reflecting mirror , N is an integer greater than one, each laser light path of the N separate laser light paths is configured to generate light to enter the objective lens , the light passing through the objective lens illuminates a sample to excite the sample to emit fluorescence, the fluorescence passes through the objective lens and enters the first receiving device to form a first image; and a second imaging system , wherein the second imaging system is an automatic focusing system, the second imaging system comprises a light emitting source , the objective lens , and a second receiving device , the light emitting source is configured to emit light to enter the objective lens , the light passing through the objective lens illuminates the sample and is reflected, the reflected light passes through the objective lens and enters the second receiving device to form a second image; characterized in that the first imaging system further comprises N first dichroic mirrors , wherein each of the N first light sources corresponds to one of the N first dichroic mirrors , wherein the N separate laser light paths are composed by the N first light sources , the reflecting mirrors , and the N first dichroic mirrors , and wherein light generated in each of the laser light path is emitted from the first light source , the light emitted from the first light source is reflected by the reflecting mirror to enter the first dichroic mirror , the first dichroic mirror reflects the light into the objective lens . a first imaging system , wherein the first imaging system comprises N separate laser light paths, an objective lens , a first receiving device , N first light sources , a reflecting mirror , N is an integer greater than one, each laser light path of the N separate laser light paths is configured to generate light to enter the objective lens , the light passing through the objective lens illuminates a sample to excite the sample to emit fluorescence, the fluorescence passes through the objective lens and enters the first receiving device to form a first image; and a second imaging system , wherein the second imaging system is an automatic focusing system, the second imaging system comprises a light emitting source , the objective lens , and a second receiving device , the light emitting source is configured to emit light to enter the objective lens , the light passing through the objective lens illuminates the sample and is reflected, the reflected light passes through the objective lens and enters the second receiving device to form a second image; characterized in that the first imaging system further comprises N first dichroic mirrors , wherein each of the N first light sources corresponds to one of the N first dichroic mirrors , wherein the N separate laser light paths are composed by the N first light sources , the reflecting mirrors , and the N first dichroic mirrors , and wherein light generated in each of the laser light path is emitted from the first light source , the light emitted from the first light source is reflected by the reflecting mirror to enter the first dichroic mirror , the first dichroic mirror reflects the light into the objective lens . <EOS>