Scattering suppression and confocal detection in multifocal multiphoton microscopy.
We have developed a new descanned parallel (32-fold) pinhole and photomultiplier detection array for multifocal multiphoton microscopy that effectively reduces the blurring effect originating from scattered fluorescence photons in strongly scattering biological media. With this method, we achieve a fourfold improvement in photon statistics for detecting ballistic photons and an increase in spatial resolution by 21% in the lateral and 35% in the axial direction compared to single-beam non-descanned multiphoton microscopy. The new detection concept has been applied to plant leaves and pollen grains to verify the improvements in imaging quality.
Martini
Jörg
J
Bielefeld University, Department of Physics, Experimental Biophysics and Applied Nanoscience, Universitätsstrasse 25, D-33615 Bielefeld, Germany. jmartini@physik.uni-bielefeld.de
Andresen
Volker
V
Anselmetti
Dario
D
D016477:Artifacts; D007089:Image Enhancement / Q000295:instrumentation* / Q000379:methods; D007090:Image Interpretation, Computer-Assisted / Q000295:instrumentation* / Q000379:methods; D008027:Light; D018613:Microscopy, Confocal / Q000295:instrumentation* / Q000379:methods; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation* / Q000379:methods; D015203:Reproducibility of Results; D012542:Scattering, Radiation; D012680:Sensitivity and Specificity; D012815:Signal Processing, Computer-Assisted / Q000295:instrumentation*; D013382:Subtraction Technique