Direct observation of human microcirculation has been limited by the need to provide transillumination, fluorescent dyes for contrast enhancement, or by the size of instrumentation. Orthogonal polarization spectral imaging is a microscopy technology that allows the capture of detailed, high-contrast video images of human and animal microcirculation, similar to that observed using a research intravital microscope.
The technology is based on creating a virtual light source deep within tissue that transilluminates vessels. A metal halide arc lamp generates a high-intensity beam that is filtered to a narrowband output (40 to 70 nm) about 548 nm. Centered at an isobestic point of oxy- and deoxyhemoglobin (a wavelength at which both forms of hemoglobin absorb equally), this wavelength band provides optimal imaging of the microcirculation. This represents a compromise between using an isobestic point in the Soret region (about 420 nm), where hemoglobin absorption is maximum but the scattering pathlength is shorter, or one in the near-IR region (810 nm), where the pathlength is longer (multiple scattering is deeper in the tissue) but absorption for hemoglobin is insufficient to produce contrast for smaller vessels.
After propagating through the bandpass filter, the light is linearly polarized and reflected toward the target by a beamsplitter. An objective lens focuses the light onto a region approximately 1 mm in diameter and subsequently collects the light returning from the target. A second polarizer (analyzer) is placed on the imaging axis in front of the CCD, with its axis oriented orthogonal to the source polarization.
The polarization of light is preserved in specular reflections and for certain geometries in single scattering events. Multiple Rayleigh-scattering events in the diffuse regime are required to depolarize the source. The analyzer thus attenuates light from specular reflections and near-field scattering events. The only light returning from the subject that can pass through the analyzer results from multiple scattering events occurring relatively deep (about 10 times the single scattering length) within the tissue. This scattered, depolarized light can be thought of as a virtual source that effectively back-illuminates any absorbing material in the foreground. The returning light forms an image of the illuminated region on a CCD video camera. This is linked to photon migration studies. The use of crossed polarizers gates photons by their migration path. Photons that have a longer diffusion path are more likely to pass through the analyzer.
In initial tests, we determined that light scattered in the region between the vessel and the objective actually does affect image contrast and photometric measurements. The birefringent nature of human or animal tissue rotates the axis of linearly polarized light, so even light from specular reflections and coherent regime scattering events is rotated to some degree from the original orientation of the source polarization. The analyzer cannot thus fully attenuate these components, which leads to diminished image contrast with a component that varies as the orientation of the source relative to the fast axis of the tissue. The measured vessel optical density depends on local image contrast and therefore varies with the relative orientation of the instrument.
To address this concern, we developed illuminator optics that work in conjunction with the objective to image the source. The source illumination is imaged onto the object plane in the tissue such that the pattern lies outside of the imaging system field of view. Any component of the source directed back into the objective enters at a high angle. By design, this is higher than the acceptance angle for the objective and the light is eliminated. This additional filtering step eliminates most of the coherent-regime scattering components and the orientation dependence in image contrast.
Professor Jean-Louis Vincent, chief of Intensive Care Medicine, Erasmus University Hospital (Brussels, Belgium), has used such a system to monitor the microcirculation in critical care patients. In the future, the system is likely to guide therapy at bedside, limiting complications. oe
Christopher Cook is a scientific consultant at Rheologics Inc., Exton, PA.