Abstract
Advances in spectral deconvolution technologies are rapidly enabling researchers to replace or enhance traditional epifluorescence microscopes with instruments capable of detecting numerous markers simultaneously in a multiplexed fashion. While significantly expediting sample throughput and elucidating sample information, this technology is limited by the spectral width of common fluorescence reporters. Semiconductor nanocrystals (NC’s) are very bright, narrow band fluorescence emitters with great potential for multiplexed fluorescence detection, however the availability of NC’s with facile attachment chemistries to targeting molecules has been a severe limitation to the advancement of NC technology in applications such as immunocytochemistry and immunohistochemistry. Here we report the development of simple, yet novel attachment chemistries for antibodies onto NC’s and demonstrate how spectral deconvolution technology enables the multiplexed detection of 5 distinct NC-antibody conjugates with fluorescence emission wavelengths separated by as little as 20 nm.
Highlights
Immunohistochemistry (IHC), or the immunological staining and detection of cellular markers in tissue, is a mainstay technique in pathology and diagnostics research and is the current recipient of several new advances in technology; advances which may be capable of evolving IHC from its roots in limited colorimetric staining to a multiplexed fluorescence-based method capable of imaging multiple target antigens within a single tissue section
Quantum dot nanocrystal (NC) fluorescent markers in combination with multispectral imaging (MSI) technology has been a exciting union that has resulted in a respectable number of promising studies over the past few years [1,2,3,4,5,6,7,8]
A full description of the deconvolution algorithms designed and bundled with multispectral imaging software is beyond the scope of this paper, but is essentially the mathematical disentangling of different known spectral profiles weighted by their contribution to the total spectrum, i.e., If multiple wavelengths of light have combined to produce a unique intensity spectrum, those same wavelengths may be deconstructed from the total spectrum back to their component parts
Summary
Immunohistochemistry (IHC), or the immunological staining and detection of cellular markers in tissue, is a mainstay technique in pathology and diagnostics research and is the current recipient of several new advances in technology; advances which may be capable of evolving IHC from its roots in limited colorimetric staining to a multiplexed fluorescence-based method capable of imaging multiple target antigens within a single tissue section. To successfully implement the mainstream use of fluorescent NC markers into IHC across multiple disciplines such as cancer biology, immunology, and pathology will require a simple, robust procedure for producing Ab-NC conjugates coupled with standard staining techniques which can be applied in a facile yet rapid manner. We present two simplistic conjugation chemistries that enable expedient NC conjugation to monoclonal antibodies in combination with optimized staining techniques that allow a simultaneous cocktail-style protocol for multiplexed antigen detection
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