Abstract 3236: High-sensitivity, high-resolution multispectral fluorescence imaging of small (and very small) animals
Authors: Mansfield James 
Published: February 2011

Abstract
in vivo multispectral imaging (MSI) increases the sensitivity and usefulness of small animal models of development and disease, including the most popular and promising experimental systems. In mice, MSI can essentially eliminate autofluorescence that otherwise limits sensitivity, reducing detection limits by as much as 100-fold. Fluorescence imaging is a key technology for determining the where, the when and the how much of gene expression. However, the ability to image and quantitate fluorescently labeled markers in vivo has typically been limited by the bright autofluorescence of animal tissues. Small-animal imaging, which often means the imaging of mouse models, encounters autofluorescence primarily from components in skin (collagen, which fluoresces green) and food (chlorophyll, which fluoresces red). However, other small animal model systems, such as nematodes (C. elegans), zebrafish (D. rerio) embryos or adult fish, and fruit flies (D. melanogaster) pose similar problems due to the presence of autofluorescent structures that may render common labeling strategies, including the use of green fluorescent protein (GFP) labels, problematic. Various solutions have been proposed for the reduction or elimination of autofluorescence. The most prevalent is simply to use narrow bandpass emission filters in an effort to isolate the desired fluorescence signal [1]. More recently, there has been a move to use infrared-emitting labels that are excited at wavelengths that are much less likely to induce autofluorescence signals. However, autofluorescence still limits sensitivity. Fortunately, the low signal-to-background contrast problem created by autofluorescence can be substantially eliminated using MSI techniques. Moreover, MSI permits the simultaneous use of multiple labels, such as GFP and RFP, even when they spectrally overlap with themselves or autofluorescence [2].
Presented here are the results of applying an MSI strategy to the whole-animal in vivo imaging of mice, C. elegans, D. rerio and D. melanogaster at a variety of magnifications, ranging from the imaging of three whole mice simultaneously down to microscopic images of whole animals and embryos. This is accomplished using a liquid crystal tunable filter (LCTF)-based imaging system that has been adapted to either a C-mount on a fluorescence microscope or macroscope, or incorporated into a whole-animal imaging system. In each case, the sensitivity of the fluorescence measurement was greatly improved by using a MSI approach. Rapid, quantitative, imaging of GFP signals of up to a few hundred flies simultaneously in a few seconds can be easily achieved, as can high-sensitivity microscopic imaging of GFP in nematode worms. These types of imaging experiments are of value to anyone with whole-animal fluorescence-based models.
[1] Niswender KD et al, J Microsc 180, 109-116 (1995)
[2] Levenson, et al, Cytometry A. 2006 Aug; 69(8):748-58
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3236.