Maximum likelihood tomographic reconstruction of extremely sparse solutions in diffuse fluorescence flow cytometry.

We apply reparameterization and the maximum likelihood method to a specific fluorescence-mediated tomography problem where the solution is known a priori to be extremely sparse (i.e., all image values are zero except for one). Our algorithm performs significantly better than a standard image reconstruction method, particularly for deep-seated targets, and achieves close to 150 µm accuracy in a 3 mm diameter cross-sectional area with only 12 measurements. Moreover, results do not depend on the selection of a regularization parameter or other ad hoc values, and since reconstructions can be computed very quickly, the algorithm is also suitable for real-time implementation.

Tomographic sensing and localization of fluorescently labeled circulating cells in mice in vivo.

Sensing and enumeration of specific types of circulating cells in small animals is an important problem in many areas of biomedical research. Microscopy-based fluorescence in vivo flow cytometry methods have been developed previously, but these are typically limited to sampling of very small blood volumes, so that very rare circulating cells may escape detection. Recently, we described the development of a 'diffuse fluorescence flow cytometer' (DFFC) that allows sampling of much larger blood vessels and therefore circulating blood volumes in the hindlimb, forelimb or tail of a mouse. In this work, we extend this concept by developing and validating a method to tomographically localize circulating fluorescently labeled cells in the cross section of a tissue simulating optical flow phantom and mouse limb. This was achieved using two modulated light sources and an array of six fiber-coupled detectors that allowed rapid, high-sensitivity acquisition of full tomographic data sets at 10 Hz. These were reconstructed into two-dimensional cross-sectional images using Monte Carlo models of light propagation and the randomized algebraic reconstruction technique. We were able to obtain continuous images of moving cells in the sample cross section with 0.5 mm accuracy or better. We first demonstrated this concept in limb-mimicking optical flow photons with up to four flow channels, and then in the tails of mice with fluorescently labeled multiple myeloma cells. This approach increases the overall diagnostic utility of our DFFC instrument.

Instrument for fluorescence sensing of circulating cells with diffuse light in mice in vivo.

Accurate quantification of circulating cell populations in mice is important in many areas of preclinical biomedical research. Normally, this is done either by extraction and analysis of small blood samples or, more recently, by using microscopy-based in vivo fluorescence flow cytometry. We describe a new technological approach to this problem using detection of diffuse fluorescent light from relatively large blood vessels in vivo. The diffuse fluorescence flow cytometer (DFFC) uses a laser to illuminate a mouse limb and an array of optical fibers coupled to a high-sensitivity photomultiplier tube array operating in photon counting mode to detect weak fluorescence signals from cells. We first demonstrate that the DFFC instrument is capable of detecting fluorescent microspheres and Vybrant-DiD-labeled cells in a custom-made optical flow phantom with similar size, optical properties, linear flow rates, and autofluorescence as a mouse limb. We also present preliminary data demonstrating that the DFFC is capable of detecting circulating cells in nude mice in vivo. In principle, this device would allow interrogation of the whole blood volume of a mouse in minutes, with sensitivity improvement by several orders of magnitude compared to current approaches.

Validation of a device for fluorescence sensing of rare circulating cells with diffusive light in an optical flow phantom model.

Detection and quantification of rare circulating cells in biological tissues is an important problem and has many applications in biomedical research. Current methods normally involve extraction of blood samples and counting of cells ex vivo, or the use of microscopy-based fluorescence in vivo flow cytometry. The goal of this work is to develop an instrument for non-invasively enumerating very rare circulating cells in small animals with diffuse light with several orders of magnitude sensitivity improvement versus current approaches. In this work, we describe the design of our system and show that single, fluorescent microspheres can be detected in limb-mimicking optical flow phantoms with varying optical properties chosen to simulate in vivo conditions. Further, we demonstrate single cell counting capabilities using fluorescently (Vybrant-DiD) labeled Jurkat and Multiple Myeloma cells. Ongoing work includes in vivo testing and characterization of our system in mice.