Development of a 3D Confocal Laser Scanning Microscope for Applying the Saturation Pulse Method to Chlorophyll a Fluorescence

Abstract

To analyze chlorophyll fluorescence of leaf tissue three-dimensionally using the saturation pulse method, a new real-time confocal laser scanning microscope was developed. A sensitive EMCCD camera was installed in the rotating pinhole Nikpow disk scanning confocal laser microscope. The light intensity was controlled within 60–250 μM m−² s−¹in actinic light and 1,400–6,000 μM m−² s−¹.in saturation light pulse by two electromagnetic shutters with neutral density (ND) filters installed in the system. The fluctuations of actinic light and of the saturation light pulse were kept at −1.46 to +1.37% and −0.45 to +0.32% of the average intensities, respectively. The shutters open within 90 ms in the dark and 60 ms under the actinic light. To acquire rapidly sufficient focal planes for constructing the 3D image, the piezo z-scan unit was installed in the system. This scan unit made it possible to capture 64 focal planes within 1.92 s. This also enabled us to capture sufficient focal planes during illumination with one saturation light pulse. The images that were captured within 30 ms were sufficiently clear for 3D reconstruction and calculation of chlorophyll fluorescence parameters such as ϕ_PSII.