Quantitative Comparison of the Sensitivity of Detection of Fluorescent and Bioluminescent Reporters in Animal Models

Abstract

Bioluminescent and fluorescent reporters are finding increased use in optical molecular imaging in small animals. In the work presented here, issues related to the sensitivity of in vivo detection are examined for standard reporters. A highsensitivity imaging system that can detect steady-state emission from both bioluminescent and fluorescent reporters is described. The instrument is absolutely calibrated so that animal images can be analyzed in physical units of radiance allowing more quantitative comparisons to be performed. Background emission from mouse tissue, called autoluminescence and autofluorescence, is measured and found to be an important limitation to detection sensitivity of reporters. Measurements of dual-labeled (bioluminescent/fluorescent) reporter systems, including PC-3M-luc/DsRed2-1 and HeLaluc/PKH26, are shown. The results indicate that although fluorescent signals are generally brighter than bioluminescent signals, the very low autoluminescent levels usually results in superior signal to background ratios for bioluminescent imaging, particularly compared with fluorescent imaging in the green to red part of the spectrum. Fluorescence detection sensitivity improves in the far-red to near-infrared, provided the animals are fed a low-chlorophyll diet to reduce autofluorescence in the intestinal region. The use of blue-shifted excitation filters is explored as a method to subtract out tissue autofluorescence and improve the sensitivity of fluorescent imaging. Mol Imaging (2004) 3, 9-23.

In vivo imaging of early stage apoptosis by measuring real-time caspase-3/7 activation

“…However, strategies that rely on fluorescent signals have drawbacks when used in vivo because of the high scattering and the high auto fluorescence rates of visible and near-infrared light in tissues and blood, thus resulting in lower signal to noise ratio [13,14]. BLI is not influenced by unspecific background signal, hence the use of bioluminescence is generally preferred over fluorescence for imaging of living organisms.…”

A dual-reporter system for specific tracing of pancreatic ß-cell lines that non-invasively measures viable in vivo islet cells

“…Current optical imaging systems have limited clinical applications, however, because of the technical problem that fluorescence does not penetrate beyond a few centimeters of tissue (Troy et al 2004;Keren et al 2008). Another limitation is that the system does not provide information about whether islet cells are actively secreting insulin in response to high glucose levels, which is the key and crucial point for evaluating the true viability and functionality of transplanted islets.…”

“…However, the primary limitations to clinical applications of fluorescent optical imaging will be the same as those faced by other methods requiring fluorescent penetration beyond a few centimeters of tissue (Keren et al 2008). The method is also complicated by background from photobleaching and autofluorescence (Troy et al 2004).…”

In vivo quantification of fluorescent molecular markers in real‐time by ratio imaging for diagnostic screening and image‐guided surgery

“…MRI, SPECT, PET, optical fluorescence tomography, hyper-spectral fluorescence imaging, and bioluminescence imaging do currently not offer such high frame rates due to the complexity of 3-Dimensional (3D) tomographic image acquisition and reconstruction [6], acquisition and unmixing of hyperspectral data cubes [7,8], or low bioluminescence photon budget [9]. By contrast, 2-Dimensional (2D) ultrasound and optical fluorescence imaging do offer high throughput imaging, whereas ultrasound typically offers B-scan images representing a section through the tissue and optical fluorescence imaging offers tissue surface images, at high resolution with relatively low technological complexity and significantly lower cost [3].…”

“…This, is a major challenge, because the in vivo fluorescence depends on many parameters other that the concentration of the fluorescent marker. Examples are variations in the tissue-to-detector geometry, autofluorescence and tissue optical properties, so that the raw fluorescence image can be subject to several artifacts that compromise accurate quantification [7,[9][10][11].…”