A line-scanning semi-confocal multi-photon fluorescence microscope with a simultaneous broadband spectral acquisition and its application to the study of the thylakoid membrane of a cyanobacterium Anabaena PCC7120.
We describe the construction and characterization of a laser-line-scanning microscope capable of detection of broad fluorescence spectra with a resolution of 1 nm. A near-infrared femtosecond pulse train at 800 nm was illuminated on a line (one lateral axis, denoted as X axis) in a specimen by a resonant scanning mirror oscillating at 7.9 kHz, and total multi-photon-induced fluorescence from the linear region was focused on the slit of an imaging polychromator. An electron-multiplying CCD camera was used to resolve fluorescence of different colours at different horizontal pixels and fluorescence of different spatial positions in a specimen at different vertical pixels. Scanning on the other two axes (Y and Z) was achieved by a closed-loop controlled sample scanning stage and a piezo-driven objective actuator. The full widths at half maximum of the point-spread function of the system were estimated to be 0.39-0.40, 0.33 and 0.56-0.59 mum for the X (lateral axis along the line-scan), Y (the other lateral axis) and Z axes (the axial direction), respectively, at fluorescence wavelengths between 644 and 690 nm. A biological application of this microscope was demonstrated in a study of the sub-cellular fluorescence spectra of thylakoid membranes in a cyanobacterium, Anabaena PCC7120. It was found that the fluorescence intensity ratio between chlorophyll molecules mainly of photosystem II and phycobilin molecules of phycobilisome (chlorophyll/phycobilin), in the thylakoid membranes, became lower as one probed deeper inside the cells. This was attributable not to position dependence of re-absorption or scattering effects, but to an intrinsic change in the local physiological state of the thylakoid membrane, with the help of a transmission spectral measurement of sub-cellular domains. The efficiency of the new line-scanning spectromicroscope was estimated in comparison with our own point-by-point scanning spectromicroscope. Under typical conditions of observing cyanobacterial cells, the total exposure time became shorter by about 50 times for a constant excitation density. The improvement factor was proportional to the length of the line-scanned region, as expected.
Kumazaki
Shigeichi
S
Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan. kumazaki@kuchem.kyoto-u.ac.jp
Hasegawa
Makoto
M
Ghoneim
Mohammad
M
Shimizu
Yugo
Y
Okamoto
Kenji
K
Nishiyama
Masayoshi
M
Oh-Oka
Hirozo
H
Terazima
Masahide
M
D017033:Anabaena / Q000648:ultrastructure*; D002734:Chlorophyll / Q000378:metabolism; D019044:Image Cytometry / Q000379:methods*; D018613:Microscopy, Confocal / Q000295:instrumentation*; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D017785:Photons; D052980:Phycobilins / Q000378:metabolism; D020524:Thylakoids / Q000502:physiology / Q000648:ultrastructure*
D052980:Phycobilins; D002734:Chlorophyll