09 Oct 2007
Two-color two-photon absorption has been used to image blood vessels with micrometer resolution, both in vitro and in vivo.
A US team has developed a technique to image hemoglobin in red blood cells with micrometer resolution, but without the need to inject external contrast agents or dyes into the blood. The method might also differentiate between oxy- and deoxyhemoglobin, which would be a crucial step in cancer studies. (Optics Letters, 32, 2641).
The technique employs two-color two-photon absorption, which offers the same performance and resolution as two-photon fluorescence but does not require dyes to be injected into the blood stream. Two collinear synchronized ultrashort laser pulse trains at different wavelengths function as pump and probe, and are coupled into a laser scanning microscope for imaging purposes.
"With different pump-probe wavelength combinations we could potentially image oxygenation on the same micrometer level, exploiting oxygen's effect on the molecule's excited-state dynamics," Dan Fu of Princeton University told optics.org. "This is very important, because we could watch blood vessel growth non-invasively and monitor tissue oxygenation state during the development of a tumor."
Fu says that the pump-probe spectroscopy technique has been around for many decades, but that nobody has ever thought about using it in tissue microscopy before.
One limiting factor in the team's early images was a reduction in spatial intensity caused by scattering of the probe photons at 650 nm. The signal-to-noise ratio was improved by increasing the probe wavelength to 775 nm and pumping at 650 nm, which enabled observations of complex 3D blood vessel structures.
The technique's depth penetration is limited to approximately 70 µm, also due to high scattering, but the team believes this could be improved with longer pump and probe wavelengths, higher powers, and a microscope objective lens with a higher numerical aperture.
"Our laser sources are standard off-the-shelf products using an ultrafast laser oscillator and optical parametric oscillator," Fu explained. "The system setup is a little more complex than standard two-photon fluorescence because we are using two lasers, but in principle we could use one broadband laser and derive the two color beams we need. We've already done initial studies on that."
The team is also refining its ability to image oxygenation levels in blood vessels, and is planning to integrate second-harmonic generation detection with two-photon fluorescence detection in their system. "With these techniques in place, we can start to study the early stages of angiogenesis in tumor development," commented Fu.