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23


Improved in vivo whole-animal detection limits of green fluorescent protein-expressing tumor lines by spectral fluorescence imaging

Tam, Jenny M; Upadhyay, Rabi; Pittet, Mikael J; Weissleder, Ralph; Mahmood, Umar
Green fluorescent protein (GFP) has been used for cell tracking and imaging gene expression in superficial or surgically exposed structures. However, in vivo murine imaging is often limited by several factors, including scatter and attenuation with depth and overlapping autofluorescence. The autofluorescence signals have spectral profiles that are markedly different from the GFP emission spectral profile. The use of spectral imaging allows separation and quantitation of these contributions to the total fluorescence signal seen in vivo by weighting known pure component profiles. Separation of relative GFP and autofluorescence signals is not readily possible using epifluorescent continuous-wave single excitation and emission bandpass imaging (EFI). To evaluate detection thresholds using these two methods, nude mice were subcutaneously injected with a series of GFP-expressing cells. For EFI, optimized excitation and emission bandpass filters were used. Owing to the ability to separate autofluorescence contributions from the emission signal using spectral imaging compared with the mixed contributions of GFP and autofluorescence in the emission signal recorded by the EFI system, we achieved a 300-fold improvement in the cellular detection limit. The detection limit was 3 x 10(3) cells for spectral imaging versus 1 x 10(6) cells for EFI. Despite contributions to image stacks from autofluorescence, a 100-fold dynamic range of cell number in the same image was readily visualized. Finally, spectral imaging was able to separate signal interference of red fluorescent protein from GFP images and vice versa. These findings demonstrate the utility of the approach in detecting low levels of multiple fluorescent markers for whole-animal in vivo applications.
PMID: 17711782
ISSN: 1535-3508
CID: 3983302

Real-time multichannel imaging framework for endoscopy, catheters, and fixed geometry intraoperative systems

Sheth, Rahul A; Upadhyay, Rabi; Weissleder, Ralph; Mahmood, Umar
To address the need for a clinically applicable intravital optical imaging system, we developed a new hardware and software framework. We demonstrate its utility by applying it to an endoscope-based white light and fluorescent imaging system. The capabilities include acquisition and visualization algorithms that perform registration, segmentation, and histogram-based autoexposure of two imaging channels (full-spectrum white light and near-infrared fluorescence), all in real time. Data are processed and saved as 12-bit files, matching the standards of clinical imaging. Dynamic range is further improved by the evaluation of flux as a quantitative parameter. The above features are demonstrated in a series of in vitro experiments, and the in vivo application is shown with the visualization of fluorescent-labeled vasculature of a mouse peritoneum. The approach may be applied to diverse systems, including handheld devices, fixed geometry intraoperative devices, catheter-based imaging, and multimodal systems.
PMID: 17532881
ISSN: 1535-3508
CID: 3983282

N-cadherin and keratinocyte growth factor receptor mediate the functional interplay between Ki-RASG12V and p53V143A in promoting pancreatic cell migration, invasion, and tissue architecture disruption

Deramaudt, Therese B; Takaoka, Munenori; Upadhyay, Rabi; Bowser, Mark J; Porter, Jess; Lee, Amy; Rhoades, Ben; Johnstone, Cameron N; Weissleder, Ralph; Hingorani, Sunil R; Mahmood, Umar; Rustgi, Anil K
The genetic basis of pancreatic ductal adenocarcinoma, which constitutes the most common type of pancreatic malignancy, involves the sequential activation of oncogenes and inactivation of tumor suppressor genes. Among the pivotal genetic alterations are Ki-RAS oncogene activation and p53 tumor suppressor gene inactivation. We explain that the combination of these genetic events facilitates pancreatic carcinogenesis as revealed in novel three-dimensional cell (spheroid cyst) culture and in vivo subcutaneous and orthotopic xenotransplantation models. N-cadherin, a member of the classic cadherins important in the regulation of cell-cell adhesion, is induced in the presence of Ki-RAS mutation but subsequently downregulated with the acquisition of p53 mutation as revealed by gene microarrays and corroborated by reverse transcription-PCR and Western blotting. N-cadherin modulates the capacity of pancreatic ductal cells to migrate and invade, in part via complex formation with keratinocyte growth factor receptor and neural cell adhesion molecule and in part via interaction with p120-catenin. However, modulation of these complexes by Ki-RAS and p53 leads to enhanced cell migration and invasion. This preferentially induces the downstream effector AKT over mitogen-activated protein kinase to execute changes in cellular behavior. Thus, we are able to define molecules that in part are directly affected by Ki-RAS and p53 during pancreatic ductal carcinogenesis, and this provides a platform for potential new molecularly based therapeutic interventions.
PMCID:1489079
PMID: 16705170
ISSN: 0270-7306
CID: 3983272