Validation of in vivo fluorochrome concentrations measured using fluorescence molecular tomography.

Fluorescence molecular tomography (FMT) has emerged as a means of quantitatively imaging fluorescent molecular probes in three dimensions in living systems. To assess the accuracy of FMT in vivo, translucent plastic tubes containing a turbid solution with a known concentration of Cy 5.5 fluorescent dye are constructed and implanted subcutaneously in nude mice, simulating the presence of a tumor accumulating a fluorescent molecular reporter. Comparisons between measurements of fluorescent tubes made before and after implantation demonstrate that the accuracy of FMT reported for homogeneous phantoms extends to the in vivo situation. The sensitivity of FMT to background fluorescence is tested by imaging fluorescent tubes in mice injected with Cy 5.5-labeled Annexin V. For small tube fluorochrome concentrations, the presence of background fluorescence results in increases in the reconstructed concentration. This phenomenon is counteracted by applying a simple subtraction correction to the measured fluorescence data. The effects of varying tumor photon absorption are simulated by imaging fluorescent tubes with varying ink concentrations, and are found to be minor. These findings demonstrate the in vivo quantitative accuracy of fluorescence tomography, and encourage further development of this imaging modality as well as application of FMT in molecular imaging studies using fluorescent reporters.

Quantitative analysis of chemotherapeutic effects in tumors using in vivo staining and correlative histology.

AIMS: To microscopically analyze the chemotherapeutic response of tumors using in vivo staining based on an annexinV-Cy5.5 probe and independently asses their apoptotic count using quantitative histological analysis. METHODS: Lewis Lung Carcinomas cells, that are sensitive (CS-LLC) and resistant (CR-LLC) to chemotherapy were implanted in nude mice and grown to tumours. Mice were treated with cyclophosphamide and injected with a Cy5.5-annexinV fluorescent probe. In vivo imaging was performed using Fluorescence Molecular Tomography. Subsequently tumours were excised and prepared for histology. The histological tumour sections were stained for apoptosis using a terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. A minimum of ten tissue sections were analyzed per tumour for apoptosis quantification by TUNEL staining and corresponding Cy5.5 distribution. RESULTS: We detected higher levels of apoptosis and corresponding higher levels of Cy5.5 fluorescence in the CS-LLC vs. the CR-LLC tumours. The cell count rate on CS-LLC sections over CR-LLC was found to be approximately 2 :1 where the corresponding area observed on Cy5.5 distribution measurements revealed a approximately 1.7 :1 ratio of CS-LLC over CR-LLC. These observations are consistent with the higher apoptotic index expected from the CS-LLC cell line. CONCLUSIONS: Quantitative analysis of histological slices revealed higher fluorescence and higher apoptotic count in the CS-LLC tumour images compared to the CR-LLC tumour images. These observations demonstrate that the annexinV-Cy5.5 probe sensed the chemotherapeutic effect of cyclophospamide and further confirmed in vivo FMT measurements.

Experimental determination of photon propagation in highly absorbing and scattering media.

Optical imaging and tomography in tissues can facilitate the quantitative study of several important chromophores and fluorophores. Several theoretical models have been validated for diffuse photon propagation in highly scattering and low-absorbing media that describe the optical appearance of tissues in the near-infrared (NIR) region. However, these models are not generally applicable to quantitative optical investigations in the visible because of the significantly higher tissue absorption in this spectral region compared with that in the NIR. We performed photon measurements through highly scattering and absorbing media for ratios of the absorption coefficient to the reduced scattering coefficient ranging approximately from zero to one. We examined experimentally the performance of the absorption-dependent diffusion coefficient defined by Aronson and Corngold [J. Opt. Soc. Am. A 16, 1066 (1999)] for quantitative estimations of photon propagation in the low- and high-absorption regimes. Through steady-state measurements we verified that the transmitted intensity is well described by the diffusion equation by considering a modified diffusion coefficient with a nonlinear dependence on the absorption. This study confirms that simple analytical solutions based on the diffusion approximation are suitable even for high-absorption regimes and shows that diffusion-approximation-based models are valid for quantitative measurements and tomographic imaging of tissues in the visible.

Visualization of antitumor treatment by means of fluorescence molecular tomography with an annexin V-Cy5.5 conjugate.

In vivo imaging of treatment responses at the molecular level could have a significant impact on the speed of drug discovery and ultimately lead to personalized medicine. Strong interest has been shown in developing quantitative fluorescence-based technologies with good molecular specificity and sensitivity for noninvasive 3D imaging through tissues and whole animals. We show herein that tumor response to chemotherapy can be accurately resolved by fluorescence molecular tomography (FMT) with a phosphatidylserine-sensing fluorescent probe based on modified annexins. We observed at least a 10-fold increase of fluorochrome concentration in cyclophosphamide-sensitive tumors and a 7-fold increase of resistant tumors compared with control studies. FMT is an optical imaging technique developed to overcome limitations of commonly used planar illumination methods and demonstrates higher quantification accuracy validated by histology. It is further shown that a 3-fold variation in background absorption heterogeneity may yield 100% errors in planar imaging but only 20% error in FMT, thus confirming tomographic imaging as a preferred tool for quantitative investigations of fluorescent probes in tissues. Tomographic approaches are found essential for small-animal optical imaging and are potentially well suited for clinical drug development and monitoring.