FIGURE 5.

Comparison of the depth-of-focus of 1P and 2P illumination. Fluorescent microspheres of 1 μm (Molecular Probes Orange FluoSpheres) were dried on a coverslip and immobilized in a mounting medium provided with an MP PS-Speck Microscope Point Source Kit (Molecular Probes). A center of a microsphere was scanned in the Z direction in 0.1 μm steps. (A) 1P scan. Pinhole diameter 35 μm. λ_ex = 568 nm, LP 590 nm emission filter. The bright peak at right is the light reflected by a coverslip. The microsphere profile is at left. HWHM of the profile = 2.2 μm. (B) 2P scan. No pinhole. λ_ex = 820 nm, no emission filters, HWHM = 1.2 μm.