Three-photon excitation in fluorescence microscopy

Abstract

We show experiments proving the feasibility of scanning fluorescence microscopy by three-photon excitation. Three-photon excitation fluorescence axial images are shown of polystyrene beads stained with the fluorophore 2,5-bis(4-biphenyl)oxazole (BBO). Three-photon excitation is performed at an excitation wavelength of 900 nm and with pulses of 130 fs duration provided by a mode-locked titanium-sapphire laser. Fluorescence is collected between 350 and 450 nm. The fluorescence image signal features a third-order dependence on the excitation power, also providing intrinsic 3-D imaging. The resolution of a three-photon excitation microscope is increased over that of a comparable two-photon excitation microscope.

Far‐field fluorescence microscopy with three‐dimensional resolution in the 100‐nm range

“…Unfortunately, these microscopes require detrimental specimen preparation to obtain a three-dimensional (3D) image. While confocal (Wilson, 1990) and multiphoton microscopes (Denk et al, 1990;Hell et al, 1996;Wokosin et al, 1996;Schrader et al, 1997) can image transparent specimens in 3D, these microscopes could not significantly change the resolution issue. The resolution of confocal and multiphoton microscopes does not exceed l/3 and l in the lateral and axial directions, respectively, with l denoting the wavelength of light.…”

3D microscopy of transparent objects using third‐harmonic generation

“…A number of nonlinear optical techniques, including twophoton absorption (Webb, 1990;Denk et al, 1990;Piston & Webb, 1991), three-photon absorption Hell et al, 1996), multifrequency excitation (Hell & Wichmann, 1994) and surface second harmonic generation (Hellwarth & Christensen, 1974;Campagnola et al, 1997) have been introduced to (laser scanning) microscopy over the past few years. Made possible by the continuing advances in ultrashort pulse laser technology, these techniques have opened new fields in microscopical research.…”

From microscopy to nanoscopy via visible light

“…The two-photon microscope always provides clear imaging at 10 times the depth of other microscopes (Figure 6). A higher order of excitation was experimentally realized by Hell et al 55 in 1996, which further resulted in the invention of three-photon microscope.…”