Development of a Multicolor Line-Focus Microscope for Rapid Acquisitions of Excitation Spectra

To conduct rapid microscope observations with the excitation spectral measurement for photosynthetic organisms, a wavelength-dispersive line-focus microscope was developed. In the developed system, fluorescence signals at multiple positions on a sample excited with different wavelengths can be detected as a two-dimensional image on the EMCCD camera at the same time. Using the developed system, one can obtain excitation spectra at every pixel over the excitation wavelength range from 635 to 695 nm, which covers the full range of the Q_y bands of both chlorophyll-a and chlorophyll-b. Recording the reference laser spectra at the same time ensures robust measurement against the moderate spectral fluctuation in the excitation laser. Using an objective lens with a numerical aperture of 0.9, the lateral and axial resolutions of 0.56 and 1.08 μm, respectively, were achieved. The theoretically limited and experimentally estimated spectral resolutions of the excitation spectral measurement were 0.86 and 1.3 nm, respectively. The validity of the system was demonstrated by measuring fluorescent beads and single cells of a model alga, Chlamydomonas reinhardtii. Intrachloroplast inhomogeneity in the relative intensity of the chlorophyll-b band could be visualized in Chlamydomonas cells. The inhomogeneity reflects the intrachloroplast variation in the local peripheral antenna size.