Two-photon excitation microscopy

This contrasts with confocal microscopy, where the spatial resolution is produced by the interaction of excitation focus and the confined detection with a pinhole.

Two-photon excitation can be a superior alternative to confocal microscopy due to its deeper tissue penetration, efficient light detection, and reduced photobleaching.

These techniques use focused laser beams scanned in a raster pattern to generate images, and both have an optical sectioning effect.

The longer wavelength, lower energy (typically infrared) excitation lasers of multiphoton microscopes are well-suited to use in imaging live cells as they cause less damage than the short-wavelength lasers typically used for single-photon excitation, so living tissues may be observed for longer periods with fewer toxic effects.

Effectively, excitation is restricted to the tiny focal volume (~1 femtoliter), resulting in a high degree of rejection of out-of-focus objects.

Two-photon microscopy was pioneered and patented by Winfried Denk and James Strickler in the lab of Watt W. Webb at Cornell University in 1990.

The Ti-sapphire laser normally used has a pulse width of approximately 100 femtoseconds (fs) and a repetition rate of about 80 MHz, allowing the high photon density and flux required for two-photon absorption, and is tunable across a wide range of wavelengths.

Higher-order photodamage becomes a problem and bleaching scales with the square of the laser power, whereas it is linear for single-photon (confocal).

For very thin objects such as isolated cells, single-photon (confocal) microscopes can produce images with higher optical resolution due to their shorter excitation wavelengths.

In scattering tissue, on the other hand, the superior optical sectioning and light detection capabilities of the two-photon microscope result in better performance.

Two-photon microscopy has been involved in numerous fields including: physiology, neurobiology, embryology and tissue engineering.

[27] Currently, two-photon microscopy is widely used to image the live firing of neurons in model organisms including fruit flies (Drosophila melanogaster), rats, songbirds, primates, ferrets, mice (Mus musculus), zebrafish.

[34] In general, all commonly used fluorescent proteins (CFP, GFP, YFP, RFP) and dyes can be excited in two-photon mode.

[citation needed] Several green, red and NIR emitting dyes (probes and reactive labels) with extremely high 2-photon absorption cross-sections have been reported.

Two-photon excitation microscopy of mouse intestine . Red: actin . Green: cell nuclei . Blue: mucus of goblet cells . Obtained at 780 nm using a Ti-sapphire laser .
Two-photon fluorescence image (green) of a cross section of rhizome colored with lily of the valley . The excitement is at 840 nm, and the red and blue colors represent other channels of multiphoton techniques which have been superimposed.
Schematic representation of the energy levels (Jabłoński diagrams) of the fluorescence process, example of a fluorescent dye that emits light at 460 nm. One (purple, 1PEF), two (light red, 2PEF) or three (dark red, 3PEF) photons are absorbed to emit a photon of fluorescence (turquoise).
Optical response from a point source. From left to right: calculated intensity distributions xy (top) and rz (bottom), with logarithmic scale, for a point source imaged by means of a wide field (a), 2PEF (b) and confocal microscopy (c). The 2PEF and confocal forms have a better signal-to-noise ratio than the wide field. The 2PEF distribution is larger due to the fact that a wavelength twice as long as in the case of a wide or confocal field is responsible for the intensity distribution. These intensity distributions are also known as point spread functions. Optical conditions: the excitation wavelengths are 488 nm and 900 nm respectively for 1PEF and 2PEF; the emission wavelength is 520 nm; the numerical aperture is 1.3 with an oil immersion objective .
A diagram of a two-photon microscope.
Diagram of in vivo brain function imaging. Shows the general schematic for imaging, along with neuronal and vascular images. Imaging was performed using various fluorescent dyes.