Nuance™ FAQs
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What is multispectral imaging?
Multispectral imaging is an advanced imaging technique which acquires images where each pixel contains a spectrum (intensity as a function of wavelength) of the collected light at that location, rather than the typical 3 data points (red, green and blue) found in an RGB image. Multispectral imaging allows much more quantitative and sensitive measurement of subtle differences in spectral emissions, which in turn is what provides the increased sensitivity from unmixing.
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For which applications is Nuance best suited?
Nuance has been developed for multispectral imaging applications in microscopy in the life sciences. In particular, Nuance is well-suited to the imaging of tissue sections in both brightfield (chromogenic) and fluorescence modes. For fluorescence imaging of tissues, Nuance is able to eliminate tissue autofluorescence, greatly increasing sensitivity, as well as to eliminate bleed-through of overlapping spectral signatures. In brightfield chromogenic applications, Nuance can image multiple markers even when co-localized in the same cellular compartment.
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What advantages does Nuance have over confocal multispectral systems?
Nuance has two main advantages over confocal multispectral systems. First, it is able to do brightfield imaging of chromogenic molecular markers – confocal systems are restricted to fluorescence-based applications. It can also act as an excellent color camera. Secondly, in fluorescence microscopy, in samples which are autofluorescent (which is most tissue sections, particularly paraffin-embedded, formalin-fixed sections) Nuance is superior to most laser-scanning multispectral confocal systems at separating tissue autofluorescence from signals of interest. This is partly because of Nuance’s light-efficient CCD-based imaging system, which is excellent for shot-noise limited samples (ie, those with autofluorescence) and partly because of Nuance’s proprietary algorithms for determining the pure spectral signatures of fluorophores from tissue sections with autofluorescence.
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Which fluorophores or chromogens can I use with Nuance?
Nuance was designed to be able to image any fluorophore or any chromogen with optical activity within its broad spectral range. Which fluorophore you can use depends more on which filter cubes you have in your microscope, but any chromogen can be imaged using Nuance.
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How many colors can I use?
The answer to this depends far more on one’s ability to put markers onto a tissue section than on Nuance. Nuance is capable of imaging 8 or 10 markers simultaneously, more if the markers are not co-localized. But since this is more than can typically be deployed in tissue sections with current labeling methodologies, the Nuance is not the limiting factor – staining methodology is.
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What protocols exist for multi-label chromogenic imaging?
There has been a great deal published on multicolor chromogenic staining. In addition, there are now commercially available kits for dual color IHC staining.
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What microscopes are compatible with Nuance?
Nuance is compatible with any inverted or upright microscope with a C-mount camera mount attachment.
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Is the Nuance system compatible with any other imaging software programs?
Nuance is compatible with both MetaMorph® (Molecular Devices) and IPLab (Scanalytics, BD Biosciences), allowing for hardware usage and scripting (contact CRi for details). Nuance images and spectral datasets can also be saved as TIFF files and viewed in any program that reads this file format.
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Where can I see a system?
You can see the Nuance system at some of these events, or you can contact CRi to set up a demonstration.
