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| Multiplexed Imaging
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Multiplexed Imaging from Macro to Micro
Protocol Author: Dr. Richard Levenson, CRi; David T. Lynch, Ph.D., fi nal unmixed composite with pseudo-color representation that is
CRi; James R. Mansfi eld, CRi differentiated from the original RGB image. In this way, multispectral
imaging technology enables the end-user to study the activity of
Increasing sophistication in the design and interrogation of biological multiple proteins in a signaling pathway or parallel pathways at the
models, and the advent of novel fl uorescent probes have led to same time.
new demands on molecular imaging systems to deliver enhanced
sensitivity, reliable quantitation, and the ability to resolve multiple These protocols describe methods to track multiple markers at the
simultaneous signals. Sensitivity is limited, especially in the visible whole animal level by fl uorescent-based in vivo imaging in Maestro
spectral range, by the presence of ubiquitous autofl uorescence and then confi rm the analysis in vitro using Nuance 2 for imaging
signals (mostly arising from the skin and gut), which need to be resolution of multiple signaling pathway-related proteins in cancer
separated from those of targeted fl uorophores. Fluorescence-based cells in tissue sections, on a cell-by-cell basis, even if the proteins
imaging is also affected by absorbing and scattering properties are co-localized.
of tissues in the visible and to a lesser extent in the near-infrared
(NIR). However, the small size of typical animal models (usually
mice) often permit the detection of enough light arising even from
relatively deep locations to allow capture of signals with acceptable
signal-to-noise. Multispectral imaging, through its ability to separate
autofl uorescence from the label fl uorescence, can increase
sensitivity by as much as 300-fold compared to conventional
approaches, and concomitantly improves quantitative accuracy.
In the NIR region, autofl uorescence, while still signifi cant, poses
less of a problem. However, the task of disentangling signals from Figure 1. Conventional-color and Maestro-composite images showing FITC,
multiple fl uorophores remains. Multispectral imaging allows the
TRITC, and Cy3.5, as well as autofluorescence (in yellow) in live mice.
separation of fi ve or more fl uorophores, with each signal quantitated
Sample and images courtesy R. Weissleder, U. Mahmood, and J. Tam,
and visualized separately. Preclinical small-animal imaging is often
MGH, Boston.
accompanied by microscopic analysis, both before and after the in
vivo phase. This can involve tissue culture manipulations and/or
histological examination of fi xed or frozen tissue. Due to the same
advantages already discussed with respect to sensitivity, quantitation
and multiplexing, microscopy-based multispectral techniques form
an excellent complement to in vivo imaging.
Multispectral imaging is the underlying enabling technology used in
the Maestro
TM
and Nuance
TM
product lines from CRi. The software
Figure 2. Conventional-color and Nuance-unmixed composite images of
algorithms built into these products provide the end-user with the
breast carcinoma tripled-stained for ER, PR, and HER-2. Sample courtesy
ability to resolve one or more fl uorescent signals in a biological
Dr. Micheal Feldman, U Penn.
sample that are spectrally or spatially overlapping as separate
component images (spectral species) of the whole. Each of the
signals can be separated as a component image of the whole from
across the entire visible spectrum of the whole image, quantify the
contribution of each signal and store that information in memory on
the computer as an “image stack” or “image cube.” For the fi nal
display the separate component images are assigned a different
color for each spectral species and the algorithm provides the
Protocol Guide ı 2008 View entire protocol online at www.biotechniques.com/protocol ı BioTechniques ı 59
CRI Protocol2.indd 59 10/26/07 11:30:22 AM
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