Online Issues

Journal Vol 49 (1)

Multiplexing with Multispectral Imaging: From Mice to Microscopy

Richard M. Levenson, David T. Lynch, Hisataka Kobayashi, Joseph M. Backer, Marina V. Backer

Richard M. Levenson, MD, is Director of Research and David T. Lynch, PhD, is Senior Scientist at CRI Inc. in Woburn, Massachusetts. Hisataka Kobayashi, MD, PhD, is a staff scientist in molecular imaging at the National Institutes of Health in Bethesda, Maryland. Joseph M. Backer, PhD, is Chief Executive Officer and Marina V. Backer, PhD, is Senior Scientist with SibTech Inc. in Newington, Connecticut.

Address correspondence and reprint requests to Dr. Richard M. Levenson, Director of Research, CRI Inc., 35B Cabot Road, Woburn, MA 01801 or email rlevenson@cri-inc.com.

Abstract

Increasing sophistication in the design and application of biological models as well as the advent of novel fluorescent probes have led to new demands on molecular imaging systems to deliver enhanced sensitivity, reliable quantitation, and the ability to resolve multiple simultaneous signals. Sensitivity is limited, especially in the visible spectral range, by the presence of ubiquitous autofluorescence signals (mostly arising from the skin and gut), which need to be separated from those of targeted fluorophores. Fluorescence-based imaging is also affected by absorbing and scattering properties of tissue in both the visible and to a lesser extent the near-infrared (NIR) regions. However, the small size of typical animal models (usually mice) often permits the detection of enough light arising even from relatively deep locations to allow the capture of signals with an acceptable signal-to-noise ratio. Multispectral imaging, through its ability to separate autofluorescence from label fluorescence, can increase sensitivity as much as 300 times compared to conventional approaches, and concomitantly improve quantitative accuracy. In the NIR region, autofluorescence, while still significant, poses less of a problem. However, the task of disentangling signals from multiple fluorophores remains. Multispectral imaging allows the separation of five or more fluorophores, with each signal quantitated and visualized separately. Preclinical small animal imaging is often accompanied by microscopic analysis, both before and after the in vivo phase. This can involve tissue culture manipulations and/or histological examination of fixed or frozen tissue. Due to the same advantages in sensitivity, quantitation, and multiplexing, microscopy-based multispectral techniques form an excellent complement to in vivo imaging.

Key Words: autofluorescence; fluorescence; in vivo imaging; multiplexing; reagents