Imaging of optically thick specimen using two-photon excitation microscopy.
The in-depth imaging properties of two-photon excitation microscopy were investigated and compared with those of confocal microscopy. Confocal imaging enabled the recording of images from dental biofilm down to a depth of 40 microm, while two-photon excitation images could be recorded at depths greater than 100 microm. Two-photon excitation point spread functions (PSFs) were recorded at depths ranging from 0 to 90 microm depth using 220-nm diameter fluorescent beads immersed in water. PSFs were measured using both a high numerical aperture oil immersion objective and a water immersion objective. The experiments carried out using the oil immersion objective showed a rapid degradation of both the axial and lateral resolution due to spherical aberrations. In addition, the detected fluorescence intensity rapidly decreased as a function of depth. The experiments carried out using the water immersion objective showed no significant degradation of both the axial and lateral resolution and the fluorescence intensity.
Gerritsen
H C
HC
Debye Institute, Utrecht University, 3508 TA Utrecht, The Netherlands. H.C.Gerritsen@phys.uu.nl
De Grauw
C J
CJ
D001419:Bacteria; D018441:Biofilms; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D009055:Mouth / Q000382:microbiology; D017785:Photons