Imaging of optically thick specimen using two-photon excitation microscopy

✍ Scribed by Gerritsen, H.C.; De Grauw, C.J.

• Publisher: John Wiley and Sons
• Year: 1999
• Tongue: English
• Weight: 124 KB
• Volume: 47
• Category: Article
• ISSN: 1059-910X
• DOI: 10.1002/(sici)1097-0029(19991101)47:3<206::aid-jemt6>3.0.co;2-h

✦ Synopsis

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.