Wednesday, September 12, 2007

Two-Photon Microscopy

Our regular reader RocketRoo has recently contributed an interesting comment to the post on non-local realism of last April. A long time has past since then, and as this comment is more of a two-post than a comment, I am taking the liberty to repost it here. This is the first part:

A very interesting example of non-local realism appears in the paper entitled, "Two-Photon Microscopy: Shedding Light on the Chemistry of Vision," (Biochemistry 2007, v46, 9674-9684) . Since it is written by chemists, the going is a little tough in parts, so here are some way-points for the interested reader:


Fluorescence typically involves single photon production from a particular atomic transition in either inorganic or organic materials. TPEM relies on dual simultaneous photo-production. The key point is that, unlike ordinary fluorescence microscopy, TPEM enables 3-D imaging of living tissues and has the potential to allow noninvasive study of biochemical processes in vivo. For more details, see

The TPEM effect was predicted in 1930 by Max Born's (female) student, Maria Göppert-Mayer.

TPEM circumvents the high phototoxicity and the limited penetration depth of UV light. In addition, imaging using two-photon excitation sidesteps the need for expensive optics optimized for UV excitation and suffers less from chromatic aberration problems.

Phototoxicity and fluorophore bleaching can sometimes present a significant problem for confocal microscopy, as the intense light is shone repeatedly through the specimen. Since 1990, TPEM has revolutionized the (in vivo) study of biological structure and function by exciting fluorophores in biological specimens through the simultaneous absorption of two IR photons. This is achieved by focusing an infrared laser beam (700—1100 nm) on the specimen, so that the high concentration of photons at the focal plane substantially increases the probability of the simultaneous absorption of two photons by a molecule of the fluorophore. In TPEM, the requirement of a high infrared light intensity necessitates the use of a laser (e.g., Ti:Saph). The near-IR and red (600-700 nm) regions are considered to be the “optical window” of cells and tissues.

A variant of TPEM called Second harmonic Imaging Microscopy (SHIM). SHIM refers to the induction of a nonlinear polarization by the incident light that results in the production of photons at half the wavelength. This effect seems remarkably similar to the production of type-II entangled photons by spontaneous down-conversion. (See non-local realism discussion above).

Collagen and elastin emit enough fluorescence to provide suitable contrast for imaging. In the case of the eye, SHIM imaging has been used to investigate the organization of the collagen in the cornea and the sclera.

Credits: RocketRoo