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Multifocal multiphoton microscopy (MMM) enhances imaging speed by parallelization. It is not well understood why the imaging depth of MMM is significantly shorter than conventional single-focus multiphoton microscopy (SMM). In this report, we show that the need for spatially resolved detectors in MMM results in a system that is more sensitive to the scattering of emission photons with reduced imaging depth. For imaging depths down to twice the scattering mean free path length of emission photons (2xl (s) (em)), the emission point spread function (PSF(em)) is found to consist of a narrow, diffraction limited distribution from ballistic emission photons and a broad, relatively low amplitude distribution from scattered photons. Since the scattered photon distribution is approximately 100 times wider than that of the unscattered photons at 2xl (s) (em), image contrast and depth are degraded without compromising resolution. To overcome the imaging depth limitation of MMM, we present a new design that replaces CCD cameras with multi-anode photomultiplier tubes (MAPMTs) allowing more efficient collection of scattered emission photons. We demonstrate that MAPMT-based MMM has imaging depth comparable to SMM with equivalent sensitivity by imaging tissue phantoms, ex vivo human skin specimens based on endogenous fluorophores, and green fluorescent protein (GFP) expressing neurons in mouse brain slices.

This article reviews our research activities in the area of optical mammography and relates them to the historical developments and the current state and trends in the field. The guiding threads for this article are the roles played in optical mammography by spatial and spectral information. The first feature, spatial information, is limited by the diffusive nature of light propagation but can take advantage of the exceptionally high optical contrast featured by blood vessels and blood-rich areas in the breast. We describe a method to correct for edge effects, a spatial second-derivative algorithm, and a two-dimensional phased-array approach that enhance the image contrast, the spatial resolution, and the depth discrimination in optical mammograms. The second feature, spectral information, is the most powerful and unique capability of optical mammography, and allows for functional measurements associated with hemoglobin concentration and oxygenation, water concentration, lipids content, and the wavelength dependence of tissue scattering. We present oxygenation-index images obtained from multi-wavelength optical data that point to the diagnostic potential of oxygenation information in optical mammography. The optimization of the spatial and spectral information in optical mammography has the potential to create a role for this imaging modality in the detection and monitoring of breast cancer.

We have previously reported a comparison between edge-corrected near-infrared optical mammograms and those that have undergone a further image-processing step based on a spatial second derivative. In this work, we go a step further by combining the second-derivative images from four wavelengths (690, 750, 788, and 856 nm) to obtain oxygenation-index images. While the spatial second derivative improves contrast and allows for visibility of fine structures in the images, thereby improving the sensitivity to tumor detection, additional information is needed to avoid false-positive results. The oxygenation-index images are introduced to address this issue. Oxygenation information may help discriminate benign from malignant breast lesions, thereby effectively complementing single-wavelength optical mammograms that display optically dense regions within the breast with high sensitivity.

We present an image-processing method that enhances the detection of regions of higher absorbance in optical mammograms. At the heart of this method lies a second-derivative operator that is commonly employed in edge-detection algorithms. The resulting images possess a high contrast, an automatic display scale, and a greater sensitivity to smaller departures from the local background absorbance. Moreover, the images are free of artifacts near the breast edge. This second-derivative method enhances the display of structural information in optical mammograms and may be used to robustly select areas of interest to be further analyzed spectrally to determine the oxygenation level of breast lesions.