In this work, we have established a double modulation lock-in detection technique using two semiconductor laser diodes in stimulated emission based pump-probe microscopy. By modulating the pump and probe beams at two different frequencies, f1 and f2, the signal is then recovered with the sum frequency, (f1+ f2), so as to minimize the leak-through noise due to the spontaneous emission caused by the pump beam. In this way, the DC background that is often attributed to the stimulated emission is effectively removed. Our technique has implemented in ATTO647N fluorescent dye which is applicable for many biological applications.

We have established a pump-probe microscopy with lock-in amplifier for the detection of spontaneous loss and stimulated gain. ATTO647N fluorescent is used for imaging. In order to separate the signal of interest from background, spontaneous loss is also detected, in addition to the stimulated gain. In this way, optical section images can be obtained with greatly reduced background, which is the origin of shot noise.