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Imaging of optically thick specimen using two-photon excitation microscopy
1Debye Institute, Utrecht University, 3508 TA Utrecht, The Netherlands. H.C.Gerritsen@phys.uu.nl
Microscopy Research and Technique
|November 2, 1999
Summary
Two-photon excitation microscopy offers superior depth penetration for imaging compared to confocal microscopy. A water immersion objective preserves resolution and fluorescence intensity at greater depths, unlike oil immersion objectives.
Area of Science:
- Biomedical Optics
- Microscopy Techniques
Background:
- Confocal microscopy is a standard for high-resolution imaging.
- Two-photon excitation microscopy offers potential for deeper tissue imaging.
Purpose of the Study:
- To compare the in-depth imaging capabilities of two-photon excitation microscopy and confocal microscopy.
- To evaluate the impact of objective type on two-photon excitation microscopy performance at depth.
Main Methods:
- Dental biofilm imaging was performed using confocal and two-photon excitation microscopy.
- Point spread functions (PSFs) were measured at various depths using fluorescent beads.
- Experiments utilized both high numerical aperture oil and water immersion objectives.
Main Results:
- Confocal microscopy imaged to 40 microm; two-photon excitation microscopy imaged beyond 100 microm.
- Oil immersion objectives caused rapid resolution and intensity degradation due to spherical aberrations.
- Water immersion objectives maintained axial and lateral resolution and fluorescence intensity with depth.
Conclusions:
- Two-photon excitation microscopy significantly outperforms confocal microscopy in imaging depth.
- Water immersion objectives are crucial for maintaining two-photon excitation microscopy performance in deep-tissue imaging.
- The choice of objective lens critically impacts deep-tissue imaging quality.