WiFI
Wide-Field Functional Imaging (WiFI) is a new technology development project for quantitative imaging of tissue structure and metabolism with scalable resolution and depth sectioning. WiFI combines principles of spatially modulated imaging (MI, both reflective and fluorescence) with laser speckle imaging (LSI) to simultaneously measure tissue blood flow, biochemical composition (i.e., oxy- and deoxyhemoglobin, water and lipid content), and molecular fluorescence in a single platform. Sufficient spatio-temporal resolution can be achieved to study both fast (i.e., ms timescale) and localized (i.e., tens of microns to mm) events at depths of several millimeters in thick tissues. Applications range from small animal model imaging and tomography to intra-operative guidance and endoscopy; essentially any setting where quantitative subsurface metabolic imaging is required.
Modulated Imaging
Modulated Imaging (MI) is a non-contact optical imaging technology capable of quantitative imaging, spectroscopy, and tomography of tissues over a large field-of-view (many cm) and up to a depth of ~1cm. While compatible with time-modulation methods, MI alternatively uses spatially-modulated illumination for imaging of tissue constituents. This imaging platform consists of three basic components: a light source, a spatial light modulator, and a CCD for detection (figure 1). The current instrument employs periodic illumination patterns of various spatial frequencies projected over a large area of tissue. The remitted diffuse light is detected via a CCD camera and then demodulated in order to extract absorption and scattering maps. Figure 2 demonstrates this data flow for a measurement of a human forearm.
Figure 1 Figure 2
As shown above, the spatially-varying (2D or 3D) absorption and scattering characteristics of the sample can then be reconstructed from the measured diffuse reflectance. Similarily, this concept can be extended to fluoresence. Estimation of intrinsic tissue chromophores is further enabled by quantifying absorption at many wavelengths. For example, images of tissue hemoglobin concentration (total, oxy- and deoxy-forms) and tissue oxygen saturation, lipid, water, and melanin concentration can all be quantified using appropriate red or infrared wavelengths. Finally, this technique has also demonstrated the ability for depth sectioning. Figure 3 demonstrates how higher spatial frequency (AC) is more sensitive to superficial absorbing stuctures (square) in turbid media than zero frequency (DC). Subsequently, simple image processing can be used to isolate a deeper structure (triangle).
Figure 3
The current research employing spatially structured light continues with the goal of developing novel tools for functional imaging. The research projects emphasize model development/validation in addition to in vivo studies. These in vivo studies include research on traumatic brain injuries, melanoma, wound healing, tumor angiogenesis and chemotherapy among others.
Laser Speckle Imaging
Noninvasive blood flow imaging can provide critical information on the state of biological tissue and the efficacy of approaches to treat disease. Laser Doppler flowmetry and laser Doppler imaging have been applied in numerous preclinical and clinical studies on the brain, retina, skin, and joints. A primary limitation of these methods is the need for mechanical scanning of the probe laser beam, resulting in long (on the order of min) image collection times. A method for high spatial and temporal resolution imaging of blood flow dynamics is required to provide objective evaluation of external stimuli, such as pharmacological intervention, electrical stimulation, or laser irradiation.
In 1981, Fercher and Briers proposed a laser speckle imaging (LSI) approach as an alternative to laser Doppler imaging. This method employs quantitative, spatially resolved analysis of the speckle pattern that is observed within images of laser irradiated objects. The speckle phenomenon is due to EM wave interference effects that result essentially in both spatial and temporal modulation of the imaged reflectance pattern. On the basis of this study, it was concluded that variations in speckle contrast can be used to provide directly a wide-field velocity distribution map.
With laser doppler imaging, temporal intensity fluctuations of each speckle (or a collection of speckles) is monitored at high sampling frequencies (on the order of MHz). An increase in fluctuation frequency is associated with faster blood flow. In contrast, LSI relies on acquisition and analysis of a single image captured at an exposure time that is considerably longer than a characteristic correlation time associated with the fluctuation frequency. A faster blood flow appears more blurred in the captured image than regions of slower or no flow. The degree of blurring is quantified as the local speckle contrast value (see Equation 1 below), with zero contrast representing no speckle and hence high blood flow, and unity contrast representing a fully developed speckle pattern and hence no flow.
Based on laser speckle statistics, Fercher and Briers derived the following relationship between the speckle contrast (K) and the normalized autocorrelation function of the remitted light:
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[Equation 1]
where σ is the variance, is the mean and T is the integration time of the time-averaged speckle image, and is the normalized autocorrelation function of the remitted light. For a Lorentzian velocity distribution:
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[Equation 2]
where tau_c is the correlation time. Substitution of Eq. 2 into Eq. 1 yields Eq. 3:
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[Equation 3]
For T/tau_c > 2, corresponding to K values of 0 to 0.6, tau_c can be simplified to the following algebraic expression:
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Laser speckle imaging (LSI) of blood flow dynamics. LSI permits noninvasive, wide-field imaging of blood flow dynamics, with high spatial (tens of microns) and temporal (ms scale) resolution. (Reflectance Image) With standard lamp illumination, the microvascular architecture is visible, but the degree of blood flow in each vessel is unknown. (Raw Speckle Image) With laser excitation, a grainy pattern is observed. The speckle pattern is blurred by light collected from moving red blood cells. (Speckle Contrast Image) With use of a convolution filter, we obtain a high-contrast blood flow map. The darker the pixel intensity, the higher the flow. (Speckle Flow Index Map) With assumptions made on the distribution of flow velocities, we compute a blood flow map. We use LSI as a research and clinical tool to study the microvascular response to therapy, in preclinical and clinical studies.
