Two-Photon Excitation Microscopy

Two-photon excitation microscopy is a fluorescence imaging technique that allows imaging of living tissue up to a depth of one millimeter. The two-photon excitation microscope is a special variant of the multiphoton fluorescence microscope, employing a concept first described by Maria Goeppert-Mayer in her 1931 doctoral dissertation¹ and first observed in 1962 in cesium vapor using laser excitation by Isaac Abella².

The advantages of two-photon excitation include:

• Localized excitation
• Higher signal to noise ratio
• Better imaging depth (up to 1000 μm)
• Confined photo-damage
• Photobleaching or uncaging is possible with fine z-axis resolution

These features have made possible experiments and innovations beyond the capability of traditional confocal microscopy³.

Basic Principles

The most commonly used fluorophores have excitation spectra in the 400–500 nm range, whereas the laser used to excite the fluorophores for mulitphoton excitation lies in the ~700–1000 nm (infrared) range. If the fluorophore absorbs two infrared photons simultaneously, it will absorb enough energy to be raised into the excited state. The fluorophore will then emit a single photon with a wavelength that depends on the type of fluorophore used (typically in the visible spectrum). For one-photon excitation, the amount of light absorbed is proportional to the intensity of the incident light. This results in the emission intensity being directly proportional to the intensity of excitation. In the case of 2-photon excitation, the emission intensity depends on the average squared the incident light intensity (signal ~ <I(t)>²), which in turn decreases approximately as the square of the distance from the focus. Because of this highly nonlinear (~fourth power) behavior, only those dye molecules very near the focus of the beam are excited. This property of 2-photon excitation results in an unusual spatial profile for the excited-state population. It is essential to focus the excitation in order to obtain a high local intensity. This property of localized excitation is particularly useful in fluorescence microscopy, where it is possible to excite fluorophores at the focal point of the objective, without photobleaching the fluorophores above or below the focal plane. The benefit of localized excitation is that emission is restricted to the narrow focal region, providing sectioning ability without the use of a pinhole. This allows more flexibility in choosing the detection geometry.

In a confocal microscope, the detectors are placed beyond the confocal apertures. These apertures are necessary to eliminate out-of-focus light which would otherwise ruin the image quality. In addition, emitted light from the focal point has to be "descanned" via the scanning system before it is sent back through the confocal aperture and collected. In two-photon microscopy, the emission only comes from the focal point. Hence all emission can be collected, significantly enhancing performance. Descanning the emitted signal is therefore unnecessary and non-descanned for external detectors (see diagram below). Light emitted (blue) would be collected through the confocal aperture. Light that undergoes scattering events (red) is lost to the conventional confocal detector as it does not pass through the confocal aperture.

By designing the optics of the non-aperture detector to collect all of the emitted light, sensitivity is significantly enhanced without loss of image quality. Consequently, using the non-aperture detection approach with two-photon excitation dramatically increases collection efficiency, and is essential for maximal depth penetration into living tissue.

Although two-photon excitation microscopy does not produce images with higher optical resolution than confocal microscopy, it does allow for increased depth of penetration into thick specimens. The greater penetration depth is possible in part because of the open pinhole geometry of the two-photon microscope, the absence of out-of-focus absorption of the excitation light, and decreased scattering of the excitation light (because of its wavelength). In order to take full advantage of the depth of penetration, non-aperture detection geometries must be utilized to effect a dramatic increase in collection efficiency of scattered fluorescence photons.

References

1. Goeppert-Mayer M (1931). "Über Elementarakte mit zwei Quantensprüngen." Ann Phys 9: 273–95.
2. I. D. Abella (1962) "Optical Double-Photon Absorption in Cesium Vapor," Phys. Rev. Letters. 9, 453.
3. Denk W, Strickler J, Webb W (1990). "Two-photon laser scanning fluorescence microscopy". Science 248 (4951): 73–6.