Concept of trans-illumination: for fluorescence, second-harmonic, and conventional light imaging. Two-photon (red) excitation illuminates the specimen. Second harmonic (blue) or fluorescence (green) light are transmitted and collected by PMTs after separation by a dichroic mirror (DM). Two-photon light is transmitted by the DM to produce a transmitted light image. Reflected fluorescence is detected after DM (bottom right image). Top image is the combined transmitted and reflected fluorescence image with increased signal-to-noise ratio.