Customer Success Stories
Nuance: Fluorescence Microscopy
Issue: How to image multiple (up to 6) mRNA targets simultaneously in the same tissue section?
Solution: Utilize the Nuance™ multispectral imaging system to unmix multiple fluorophores from the strong autofluorescence found in paraffin-embedded sections.
Gene and protein mapping using microarray analysis has been used to identify many useful gene or protein signatures for predicting outcome of cancers. However, little of this to date has been translated into clinically effective diagnostic tools because these microarrays require high-quality, fresh-frozen tissue samples. Utilizing more commonly available formalin-fixed, paraffin-embedded tissues (FFPE) would help to facilitate their utility. Moreover, the imaging of these in situ hybridization (ISH) or antibody distributions in tissues can provide architectural and morophologic information which is not present in the homogenized microarray analyses.
Drs. Richard Byers (University of Manchester) and Massimo Loda (Dana Farber Cancer Institute) et als. implemented a methodology which combined ISH labeling of multiple mRNA targets in samples with acute leukemia and follicular lymphoma using quantum dots (qdot). The Nuance system was used to separate the qdot signals from each other and from the strong, ubiquitous autofluorescence signal usually found in FFPE samples. In this manner clear, strong ISH signals could be seen, even when co-localized with each other or with autofluorescence.
Without the use of a Nuance system for imaging, it is unlikely that this project would have been successful. Not only does Nuance enable the imaging of multiple fluorophores even when co-localized, it dramatically increases the sensitivity of measurement of fluorescent signals which are mixed with autofluorescence, enabling what would otherwise be weak signals to be confidently quantitated.
References:
- Richard J. Byers, Dolores Di Vizio, Fionnuala O'Connell, Eleni Tholouli, Richard M. Levenson, Kirk Gossage, David Twomey, Yu Yang, Elisa Benedettini, Joshua Rose, Keith L. Ligon, Stephen P. Finn, Todd R. Golub and Massimo Loda "Semiautomated Multiplexed Quantum Dot-Based in Situ Hybridization and Spectral Deconvolution" J Mol Diagn. 2007; 9: 20-29
- Eleni Tholouli, Judith A. Hoyland, Dolores Di Vizio, Fionnuala O'Connell, Sarah A. MacDermott, David Twomey, Richard Levenson, John A. Liu Yin, Todd R. Golub, Massimo Loda, Richard Byers, "Imaging of multiple mRNA targets using quantum dot based in situ hybridization and spectral deconvolution in clinical biopsies" Biochemical and Biophysical Research Communications 348 (2006) 628-636.
Nuance: Chromogenic Microscopy
Issue: How to visualize multiple co-localized IHC chromogens in the same tissue section?
Solution: By using the Nuance™ multispectral imaging system to unmix the chromogens, they have been able to image up to 4 co-localized IHC markers in a single tissue section.
The use of multiple chromogenic markers in immunohistochemistry has a long history, with dual labeling techniques going back to the mid-1980's. However, until recently, these dual staining techniques needed to focus on color combinations where the co-localized color was distinguishable by the human eye (or an RGB camera). Unfortunately, even for these higher-contrast staining methods, the co-localized color is distinguishable only when the amounts of color in a single compartment/pixel are very similar - ie, when there are more-or-less equal amounts of color deposited. If one color exceeds the other (say, in an 80:20, 90:10 or worse ratio), the co-localizations can no longer be visualized and only the stronger chromogen will be able to be seen.
Dr. Chris van der Loos of the Academic Medical Center in Amsterdam has been working on developing double, triple and even quadruple staining methods for brightfield IHC for many years, and is one of the leading authorities on this. But until he purchased his Nuance system he was limited in whether his antigens were co-localized or not. If they were co-localized, it was very difficult even to do two colors well, and even then that was without using a counterstain (such as hematoxylin).
Now that he has a spectral imaging system, Dr. van der Loos has been able to completely free himself from which color combinations he needs to use and can focus on making the best IHC stainings possible without worry about which colors are where, because the Nuance multispectral imaging system is capable of quantitatively separating 4 or 5 colors from each other even when they are spatially co-localized. The results are revolutionary. According to Dr. van der Loos "Every time when I have to opportunity to talk about the Nuance system people get totally amazed by what you show them."
Reference:
- Chris van der Loos "Multiple immunoenzyme staining: methods and visualizations for the observation with spectral imaging" Journal of Immunochemistry & Cytochemistry Volume 56(4): 313-328, 2008.
