AbstractAutofluorescence spectra and optical imaging of Platymonas subcordiformis after irradiation of diode laser were observed via laser scanning confocal microscopy (LSCM). With 488 nm Ar+ laser excitation, the horizontal and vertical dimensions of a cup‐shaped chloroplast of the irradiation group increased about 10% compared with the control group. The fluorescence spectra were similar between irradiation group and control group with a maximum fluorescence band around 682 nm, whereas the former has a higher intensity. Image of a small circular substance with stronger two‐photon autofluorescence (TPA) was obtained when using two‐photon excitation wavelength of 800 nm in single‐channel mode. Further analysis by the 800 nm excitation based on two independent‐channels mode showed an emission band of the small circular substance around 376–505 nm, which corresponded to the eyespot of P. subcordiformis. In lambda scanning mode, with two‐photon wavelength of 800 nm excitation, six fluorescence peaks that are located at 465, 520, 560, 617, 660 and 680 nm were observed; the fluorescence intensity of the irradiation group was higher than that of the control group, especially at 520, 560 and 617 nm. As a conclusion, diode laser irradiation can promote chloroplast growth of P. subcordiformis cells in the form of expanding area and the increasing content of protein, phospholipids and chlorophyll. LSCM, especially TPA imaging based on femtosecond laser excitation, provides a nondestructive, real‐time and accurate method to study changes of living algal cells under laser irradiation and other environmental factors. SCANNING 30: 443–447, 2008. © 2008 Wiley Periodicals, Inc.