Maestro™ FAQs
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Why is Maestro so highly sensitive for in vivo fluorescence imaging?
Maestro’s sensitivity is a result of its ability to effectively remove autofluorescence from the image, and its advanced algorithms that ensure accurate unmixing. The limiting factor for sensitivity for in vivo fluorescence detection in any wavelength regime is the interference from the ubiquitous tissue autofluorescence. Since autofluorescence emits at the same wavelengths as fluorophores of interest, it is necessary to separate, or unmix, the two, and in so doing, increase sensitivity.
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What is multispectral imaging?
Multispectral imaging is an advanced imaging technique which acquires images where each pixel contains a spectrum of the captured light at that location, rather than the typical 3 data points (red, green and blue) found in an RGB image. Multispectral imaging allows 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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Which fluorophores can I use in Maestro?
Maestro’s equipped with the EX filter were designed to be able to image any fluorophore emitting above 450 nm
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How quickly can I acquire images?
The acquisition of multispectral images in Maestro typically takes about 2 to 5 seconds. Using DyCE (Dynamic Contrast Enhancement) technology, monochrome images can be acquired as fast as 20 frames per second.
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What is the best spatial resolution Maestro can image?
Maestro can image from up to three whole mice down to an image with 25 microns/pixel spatial resolution. If higher resolution is required, a Nuance™ multispectral imaging system mounted on a macroscope or microscope can be used, to give resolutions down to the conventional optical resolution limit.
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What advantages does fluorescence have over bioluminescence?
In vivo fluorescence measurements have several advantages over in vivo bioluminescence measurements. First, it is possible to do many more types of experiments with fluorescence. Every bioluminescence experiment has an analogous fluorescence experiment (using fluorescent proteins), butthe converse is not necessarily true. Second, it is difficult to validate in vivo bioluminescence results with ex vivo imaging, as bioluminescence is only created in living cells. Fluorescent proteins, on the other hand, can be easily imaged ex vivo to give high-resolution microscopic images of what was generating the in vivo signals (made more powerful still with the combination of Maestro and Nuance in order to maintain continuity of reported results). Lastly, the use of ‘activatable’ fluorescent reagents, which can turn on in the presence of particular enzymes or conditions, has no analog in bioluminescence. Fluorescence is a more broadly based, versatile imaging modality.
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Is your system automated?
Yes, the Maestro 2 system is fully automated and computer-controlled.
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How many cells or how much fluorophore can I see?
This is a very difficult question to answer, as it very much depends on the brightness of the cells and their spatial distribution (concentrated or dispersed). The brighter the cells/fluorophores and the more concentrated they are, the easier they are to detect. The smallest number of cells that have been imaged using a Maestro is 3000. [1] These were not particularly brightly labeled, as the detection limit for a monochrome system was 1,000,000 of these cells, which is considered fairly high. It is likely that with brighter cells, Maestro would yield a detection limit lower than 3000 cells.
[1] Tam JM, Upadhyay R, Pittet MJ, Weissleder R, Mahmood U. “Improved in vivo whole-animal detection limits of green fluorescent protein-expressing tumor lines by spectral fluorescence imaging.” Mol Imaging. 2007 Oct-Dec;6(4):269-76.
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Where can I see a system?
You can see the Maestro system at some of these events, or you can contact CRi to set up a demonstration.
