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.     

In vivo mice lung tumor follow-up with fluorescence diffuse optical tomography

We present in vivo experiments conducted with a new fluorescence diffuse optical tomographic (fDOT) system on cancerous mice bearing mammary murine tumors. We first briefly present this new system that has been developed and its associated reconstruction method. Its main specificity is its ability to reconstruct the fluorescence yield even in heterogeneous and highly attenuating body regions such as lungs and to enable mouse inspection without immersion in optical index matching liquid (Intralipid and ink). Some phantom experiments validate the performance of this new system for heterogeneous media inspection. Its use for a mice study is then related. It consists in the follow-up of the lungs at different stages of tumor development after injection of RAFT-(cRGD)4-Alexa700. As expected, the reconstructed fluorescence increases along with the tumor stage. These results validate the use of our system for biological studies of small animals.     

In vivo fusion of circulating fluorescent cells with dystrophin-deficient myofibers results in extensive sarcoplasmic fluorescence expression but limited dystrophin sarcolemmal expression

To investigate the therapeutic potential of bone marrow transplantation in Duchenne muscular dystrophy, green fluorescent protein-positive (GFP+) bone marrow cells were transplanted into irradiated wild-type and dystrophin-deficient mdx mice. Tibialis anterior muscles showed fivefold to sixfold more GFP+ mononucleated cells and threefold to fourfold more GFP+ myofibers in mdx than in wild-type mice. In contrast, dystrophin expression in mdx mice remained within the level of nontransplanted mdx mice, and co-expression with GFP was rare. Longitudinal sections of 5000 myofibers showed 160 GFP+ fibers, including 9 that co-expressed dystrophin. GFP was always visualized as full-length sarcoplasmic fluorescence that exceeded the span of sample length (up to 1500 microm), whereas dystrophin expression was restricted to 11 to 28% of this length. Dystrophin expression span was much shorter in GFP+ fibers (116 +/- 46 microm) than in revertant fibers (654 +/- 409 microm). These data suggest that soluble GFP diffuses far from the fusion site with a pre-existing dystrophin(-) myofiber whereas dystrophin remains mainly expressed close to the site of fusion. Because restoration of dystrophin in whole muscle fiber length is required to expect functional improvement and clinical benefits for Duchenne muscular dystrophy, future applications of cell therapies to neuromuscular disorders could be more appropriately envisaged for replacement of defective soluble sarcoplasmic proteins.     

Portable two-color in vivo flow cytometer for real-time detection of fluorescently-labeled circulating cells

The recent introduction of the in vivo flow cytometer for real-time, noninvasive detection and quantification of cells circulating in the vasculature of small animals has provided a powerful tool for tracking the roles of different types of cells in disease progression. We describe a portable version of the device, which provides the capability to: a) excite and detect fluorescence at two distinct colors simultaneously, and b) perform data analysis in real time. These advances improve significantly the utility of the instrument and provide a means of increasing detection specificity. As examples, we present the depletion kinetics of circulating green fluorescent protein (GFP)-labeled breast cancer cells in the vasculature of mice, and the specific detection of circulating hematopoietic stem cells labeled in vivo with two antibodies.     

Ultrafiltration of circulating blood in vivo

Summary A method of continuous ultrafiltration of circulating blood in the intact rat is described. Thus, the serum concentrations of substances can be continuously followed, whose measurement in whole blood is made impossible by the corpuscular and macromolecular elements. As an example, conductivity curves in portal venous blood of the conscious rat after intraduodenal hydration are presented.     

New fluorescence imaging probe with high spatial resolution for in vivo applications

A new fiberized fluorescence imaging probe is presented. This device can potentially be used for a wide range of biological or medical applications. By exploiting the chromatic aberrations of gradient index lenses, the excitation blue or near-UV excitation light is focused on the sample surface, while the red fluorescence signal is efficiently launched back to collecting fibers. The excitation fiber is single mode at the working wavelength so that a resolution of 5 microm is obtained over a scanning area of several square millimeters. Experimental fluorescence images are presented. They concern either self-fabricated fluorescent microsamples or views of leaves that constitute an example of biological tissues analysis. The probe can also be adapted for spectroscopic investigations.     

A virtual source pattern method for fluorescence tomography with structured light

In order to reduce both acquisition and reconstruction times, illumination and detection in fluorescence diffuse optical tomography (FDOT) have recently evolved from a point-based to a pattern-based approach. The use of structured illumination, offering the ability to project any pattern of light onto the object, associated with the compression of the acquired fluorescence images has paved the way for a new generation of fast reconstruction algorithms for FDOT. However, the choice of the most appropriate set of source patterns is still an open problem. Here, the use of typical source patterns is investigated on experimental data. Reconstructions of similar qualities are obtained for the different types of source patterns. We found that the performances of structured illumination are limited by the required positivity of the source patterns. To alleviate this problem, we introduce a novel method, namely the virtual source pattern method, which allows for considering any kind of patterns, e.g., with negative and complex intensities. This new method provides a significant increase of the contrast of the reconstruction and also a reduction of the reconstruction error, especially when virtual wavelet source patterns are considered.     

Clastogenic effect of fenfluramine in mice bone marrow cells in vivo

Fenfluramine, an amphetamine derivative used in the treatment of obesity, has been evaluated in vivo in the bone marrow cells of Swiss albino mice using two cytogenetic endpoints for assessing its genotoxic and clastogenic potentials. Concentrations of 0.75, 1.5, 3.0, and 5.0 mg/kg b.w. were administered orally for the study of sister chromatid exchange frequencies and chromosome aberrations (CA). SCE frequencies showed a positive dose response; 1.5 mg/kg being the minimum effective concentration. Fen caused a prolongation of cell cycle at all concentrations. Except for the minimum therapeutic dose (0.75 mg), all other doses (1.5, 3.0, and 5.0 mg) showed a significant increase in the percentage of damaged cells over that of the vehicle control. The degree of clastogenicity was directly proportional to the dosage used and inversely related with the duration of treatment. A gradual reduction of the clastogenic potential was observed after 12 and 24 hr of exposure, indicating that the maximum effect occurs at the middle or late synthetic phase of the cell cycle. This study, probably the first detailed screening of the drug for its genotoxicity, shows that Fen is moderately clastogenic and a DNA damaging agent in vivo.     

Silver nanostructure sensing platform for maximum-contrast fluorescence cell imaging

We present herein a silver nanostructure-assisted sensing platform which consists of a combined structure of Ag nanowire (NW) and nanodot (ND) array. Highly enhanced fluorescence from fluorophore is attributed to a strongly coupled optical near-field interaction between proximately located Ag NW and NDs. We obtained enhanced fluorescence intensity with up to 140 folds, as contrasted from background intensity, reaching a theoretical maximum value. On the other hand, fluorescence lifetime was greatly reduced to 0.27 ns (from 2.17 ns for the same fluorophores without nanostructure). This novel platform can be a promising utility for optical imaging and labeling of biological systems with a great sensitivity.     

T-prolymphocytic leukemia with circulating carrot-like cells

A case of T-prolymphocytic leukemia leading to rapid demise of a 67-year-old man is reported. He presented with multiple skin lesions and splenomegaly. A unique feature was that a proportion of circulating leukemic cells assumed bizarre shapes, resembling carrots. The leukemic cells expressed the T-cell markers T11, T8 and Dako-T2, and the natural killer cell markers NKH1, Leu7 and Leu 11b.     

Solid-phase Clq-binding fluorescence immunoassay for detection of circulating immune complexes.

A fluorescence immunoassay for detection of immune complexes bound to solid-phase C1q was developed. The method was standardized by using human aggregated immunoglobulin G (IgG) to simulate immune complexes. A linear relationship existed between the concentrations of the aggregated IgG standards and the resulting fluorescent intensity. The method was found to be reproducible and capable of detecting as little as 10 micrograms of aggregated IgG per ml of heat-inactivated human serum. Antigen-antibody complexes prepared in vitro were detectable from equivalence to moderate antigen excess. Endogenous serum C1q inhibited the binding of aggregated IgG to solid-phase C1q. Pretreatment of test sera with EDTA was ineffective in eliminating this competitive effect. Heating the sera at 56 degrees C alleviated, but did not abolish, interference of endogenous C1q. Elevated levels of immune complexes were detectable in sera fro seven of nine patients wit systemic lupus erythematosus, provided the samples were heat inactivated before testing. Heparin and DNA were also found to interfere with the detection of aggregated IgG added to human serum. Assay values were falsely decreased due to competitive inhibition by these anions. Lipopolysaccharides from a variety of bacterial preparations produced no detectable interference. A comparative study was conducted on samples that had previously been tested by fluid-phase C1q-binding radioimmunoassay. The two methods were concordant in assigning normal or elevated levels of immune complexes in 70% of the samples tested. This solid-phase fluorescence immunoassay is proposed as a possible alternative to radioimmunoassay for the detection of circulating immune complexes.     

Neutrophils do not affect clonal growth of granulocyte progenitor cells cultured in vivo in diffuse chambers

The effect of polymorphonuclear leukocytes on the growth of granulocytic progenitors in the diffusion chamber culture (CFU-dG) has been studied. It has been shown that peripheral blood neutrophils do not affect the myeloid colony formation in this culture system. The inhibition of CFU-dG growth, observed in the presence of peripheral blood leukocytes was dependent on prostaglandins release from monocytes.     

Lifetime-based photoacoustic oxygen sensing in vivo

The determination of oxygen levels in blood and other tissues in vivo is critical for ensuring proper body functioning, for monitoring the status of many diseases, such as cancer, and for predicting the efficacy of therapy. Here we demonstrate, for the first time, a lifetime-based photoacoustic technique for the measurement of oxygen in vivo, using an oxygen sensitive dye, enabling real time quantification of blood oxygenation. The results from the main artery in the rat tail indicated that the lifetime of the dye, quantified by the photoacoustic technique, showed a linear relationship with the blood oxygenation levels in the targeted artery.     

Application of infrared light for in vivo neural stimulation

A novel method for damage-free, artifact-free stimulation of neural tissue using pulsed, low-energy infrared laser light is presented. Optical stimulation elicits compound nerve and muscle potentials similar to responses obtained with conventional electrical neural stimulation in a rat sciatic nerve model. Stimulation and damage thresholds were determined as a function of wavelength using a tunable free electron laser source (lambda = 2 to 10 microm) and a solid state holmium:YAG laser (lambda = 2.12 microm). Threshold radiant exposure required for stimulation varies with wavelength from 0.312 Jcm2 (lambda = 3 microm) to 1.22 Jcm2 (lambda = 2.1 microm). Histological analysis indicates no discernable thermal damage with suprathreshold stimulation. The largest damage/stimulation threshold ratios (>6) were at wavelengths corresponding to valleys in the IR spectrum of soft tissue absorption (4 and 2.1 microm). Furthermore, optical stimulation can be used to generate a spatially selective response in small fascicles of the sciatic nerve that has significant advantages (e.g., noncontact, spatial resolution, lack of stimulation artifact) over conventional electrical methods in diagnostic and therapeutic procedures in neuroscience, neurology, and neurosurgery.     

Aggregation-enhanced fluorescence in PEGylated phospholipid nanomicelles for in vivo imaging

We report polymeric nanomicelles doped with organic fluorophores (StCN, (Z)-2,3-bis[4-(N-4-(diphenylamino)styryl)phenyl]-acrylonitrile), which have the property of aggregation-enhanced fluorescence. The fluorescent nanomicelles have two unique features: (1) They give much brighter fluorescence emission than mono-fluorophores. (2) The nanomicelles with amphiphilic copolymers [e.g., phospholipids-PEG (polyethylene glycol)] make the encapsulated fluorophores more stable in various bio-environments and easy for further conjugation with bio-molecules. After chemical and optical characterization, these fluorescent nanomicelles are utilized as efficient optical probes for in vivo sentinel lymph node (SLN) mapping of mice. The StCN-encapsulated nanomicelles, as well as their bioconjugates with arginine-glycine-aspartic acid (RGD) peptides, are used to target subcutaneously xenografted tumors in mice, and in vivo fluorescence images demonstrate the potential to use PEGylated phospholipid nanomicelles with aggregation-enhanced fluorescence as bright nanoprobes for in vivo diagnosis of tumors.