Stimulated emission depletion (STED) technology incorporated with fluorescence microscopy envisions biomedical samples to sub-diffraction-limit resolution. Pulsed-mode STED microscopy is attractive because milliwatt-level low average optical power can induce notable STED effect . However, it is difficult to find stable and compact optical pulse sources that are suitable for STED. The pulsed operation of a laser diode (LD) can provide the stability and compactness, but the generated optical power is not very sufficient in general . Therefore, we previously demonstrated a 1.3μm LD-based optical pulse source using an optical fiber amplifier (OFA) and second harmonic generation (SHG) to obtain high peak power (and thus high pulse energy) at 650nm band . However, when we want to generate optical pulses in yellow-orange spectral range for green fluorescent protein (GFP) and yellow fluorescent protein (YFP) labelers, we find it difficult to obtain conventional OFAs at the corresponding fundamental wavelengths for SHG. In this report, we describe a novel method to generate smooth-shaped, sub-nanosecond, 586nm optical pulse sources toward STED application.