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 high-sensitivity 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 HeLa-luc/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.

Noninvasive molecular imaging of small living subjects using Raman spectroscopy

“…The primary limitation to Raman imaging of larger subjects will be those also faced by other optical techniques and is limited by NIR light penetration beyond a few centimeters of tissue (17). The key advantages of the current Raman imaging strategy over fluorescence is the very high multiplexing capability and lack of confounding background signal from autofluorescence.…”

Characterization and application of single fluorescent nanodiamonds as cellular biomarkers

“…Laser-induced fluorescence is a technique widely adopted for this purpose owing to its ultrahigh sensitivity and capabilities of performing multiple-probe detection (1)(2)(3). However, in applying this technique to imaging and tracking a single molecule or particle in a biological cell, progress is often hampered by the presence of ubiquitous endogenous components such as flavins, nicotinamide adenine dinucleotides, collagens, and porphyrins that produce high fluorescence background signals (4)(5)(6). These biomolecules typically absorb light at wavelengths in the range of 300-500 nm and fluoresce at 400-550 nm (Fig.…”

Improved QD-BRET conjugates for detection and imaging

“…These properties make them appealing fluorescent probes for bioimaging applications including in situ cell/tissue labeling, live cell imaging and in vivo imaging [3]. However, in vivo imaging using quantum dots still faces challenges such as interferences from tissue autofluorescence [4] and paucity of light available at non-superficial tissue sites [5]. One approach to solve these problems is to use high quality near infrared (NIR) QDs [6], which is an area still under active development.…”