Detection sensitivity enhancements for fluorescence imaging with multi-photon excitation microscopy

Multiphoton excitation microscopy (MPEM) offers several distinct advantages over laser scanning confocal microscopy (LSCM). One is that the resolution and the thickness of the optical section are determined by the excitation beam; a confocal detection aperture is therefore unnecessary. This feature allows the photodetector to be relocated from its usual location in the descanned emission beam within a confocal scan head so as to directly intercept the emitted fluorescent signal emerging from the microscope. The enhancement in signal gained by changing from internal to whole area (external) detection in this way has been evaluated quantitatively. In particular, longitudinal chromatic aberration has been shown to reduce the efficiency of internal detection relative to external. Also, a novel optical design is described, which allows increased collection efficiency with MPEM by capturing light emitted away from the objective lens. Together, these two enhancements typically provide an improvement in efficiency of at least 4.5 times, and even greater inside thick and scattering specimens. Such improvements are crucial for extending the longevity of living preparations by minimizing phototoxic exposure during observation. They also enable deeper imaging inside specimens where signal loss by scattering has previously limited the obtainable sectioning depth.