Two-Photon Microscopy

Two-photon microscopy uses non-linear absorption of two photons to induce fluorescence that is confined to a very small region. A laser beam is scanned laterally across the sample to generate 2D fluorescence images from an extremely thin optical section within the sample. This optical section can be varied in depth, building up a stack of images to produce a 3D rendering the sample. OBEL was part of the consortium successfull in securing the Centre for Microscopy and Microanalysis’s state-of-the-art million-dollar two-photon microscope, commissioned in 2004.

Two-photon excitation

Two-photon excitation (2PE) is based on the concept that a molecule (or fluorophore) can simultaneously absorb two low-energy photons in the same quantum event (Figure 1). The resulting fluorescence emission is a photon of higher energy. The probability of two photons being simultaneously absorbed is extremely low, and two-photon excitation will only occur where the density of photons is sufficiently high. Thus, fluorescence is emitted only at the focal point and out-of-focus blur is eliminated, enabling two-photon microscopy to image the sample at greater depths. This is also due to the greater penetration depth of the longer wavelength excitation light. The lower energy of the laser source results in less photodamage to the specimen. This is in contrast to conventional Laser Scanning Confocal Microscopy (LSCM), in which fluorescence emission results from one-photon excitation (1PE), and occurs for all fluorophores along the beam-path (Figure 2).

Optical clearing

Ongoing research at OBEL and close collaboration with the European Laboratory for Nonlinear Spectroscopy in Florence, Italy and Centre for Microscopy and Microanalysis at UWA is aimed at improving the imaging depth and contrast in living specimens, through the addition of so-called clearing and contrast agents. The hyperosmotic agents glycerol, propylene glycol and glucose have enabled an improvement in imaging depth and contrast in ex vivo human skin (Figure 3A), and their non-toxic nature may lead on to the enhancement of in-vivo deep-tissue imaging.

We are using two-photon microscopy and optical clearing to study the nature of skeletal muscle (Figure 3B) and muscle damage due to pathological conditions, in a collaborative effort with the Skeletal Muscle Research Group and Centre for Microscopy and Microanalysis, both at UWA.

Figure 3. (A) Image stacks for a skin tissue sample immersed in PBS (upper) and after immersion for 7 minutes in glycerol (lower). The optical clearing agent (OCA) glycerol has improved the penetration depth of the two-photon microscopy1. (B) Two-photon microscopy 2D and 3D images of freshly sectioned mouse skeletal muscle.

References

1. R. Cicchi, F. S. Pavone, D. Massi, D. D. Sampson, Contrast and depth enhancement in two-photon microscopy of human skin ex vivo by use of optical clearing agents, Opt. Express, vol. 13, pp. 2337-2344, 2005.

Figure 1.
Energy-level (Jablonski) diagrams for one-photon (1PE) and two-photon (2PE) excitation.
Figure 2.
Schematic of fluorescence emission in one-photon excitation Laser Scanning Confocal Microscopy (LSCM) versus Two-photon microscopy (TPM). LSCM (blue): fluorescence (green) all along beam path. TPM (red): fluorescence (green) only at focal point.
Adapted from Nature Methods (2005) 2, pp. 932-940