Pico-projector-based optical sectioning microscopy for 3D chlorophyll fluorescence imaging of mesophyll cells

A pico-projector-based optical sectioning microscope (POSM) was constructed using a pico-projector to generate structured illumination patterns. A net rate of 5.8 × 10⁶ pixel/s and sub-micron spatial resolution in three-dimensions (3D) were achieved. Based on the pico-projector's flexibility in pattern generation, the characteristics of POSM with different modulation periods and at different imaging depths were measured and discussed. With the application of different modulation periods, 3D chlorophyll fluorescence imaging of mesophyll cells was carried out in freshly plucked leaves of four species without sectioning or staining. For each leaf, an average penetration depth of 120 μm was achieved. Increasing the modulation period along with the increment of imaging depth, optical sectioning images can be obtained with a compromise between the axial resolution and signal-to-noise ratio. After ∼30 min imaging on the same area, photodamage was hardly observed. Taking the advantages of high speed and low damages of POSM, the investigation of the dynamic fluorescence responses to temperature changes was performed under three different treatment temperatures. The three embedded blue, green and red light-emitting diode light sources were applied to observe the responses of the leaves with different wavelength excitation.