ABSTRACT
The usefulness of quantum dots for the immunofluorescent detection of surface antigens on the lymphoid cells has been studied. To optimize quantum dots detection we have upgraded fluorescent microscope that allows obtaining multiple images from different quantum dots from one section. Specimens stained with quantum dots remained stable over two weeks and practically did not bleach under mercury lamp illumination during tens of minutes. Direct conjugates of primary mouse monoclonal antibodies with quantum dots demonstrated high specificity and sufficient sensitivity in the case of double staining on the frozen sections. Because of the high stability of quantum dots' fluorescence, this method allows to analyze antigen coexpression on the lymphoid tissue sections for diagnostic purposes. The spillover of fluorescent signals from quantum dots into adjacent fluorescent channels, with maxima differing by 40 nm, did not exceed 8%, which makes the spectral compensation is practically unnecessary.
Subject(s)
Antigens, Neoplasm/immunology , Diagnostic Imaging/methods , Immunoconjugates/metabolism , Lymph Nodes/pathology , Lymphoma, Mantle-Cell , Staining and Labeling/methods , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Antibody Specificity , Antigens, Neoplasm/metabolism , Antigens, Surface/immunology , Antigens, Surface/metabolism , Biomarkers, Tumor/immunology , Biomarkers, Tumor/metabolism , Fluorescence , Frozen Sections , Humans , Immunoconjugates/immunology , Lymph Node Excision , Lymph Nodes/immunology , Lymph Nodes/metabolism , Lymphoma, Mantle-Cell/diagnosis , Lymphoma, Mantle-Cell/immunology , Lymphoma, Mantle-Cell/pathology , Mice , Microscopy, Fluorescence , Quantum DotsABSTRACT
Quantum dots (QD) nanocrystals consisting of CdSe core with ZnS shell are a novel class of fluorophores with tremendous potential in microscopy and cytometry techniques. The unique optical features of Qdots, namely, high photostability and extinction coefficient, wide absorption and narrow emission spectra, and large Stokes shift make them desirable fluorescent tags for diverse biomedical applications. Applications of this novel technology in microscopy and cytometry produce reliable multicolor specimens due to increased photostability, ability for multiplexing and narrow emission spectra of nanocrystals. QD conjugates are available on the market and could be prepared in the laboratory. This paper describes the application of QD-conjugates for immunophenotyping and FISH assessment of cells and tissues, and the requirements for microscope and flow cytometer reengineering for successful use of QD in multiplex fluorescent format. Despite the considerable progress, important methodological issues still need to be solved in terms of QD nanocrystals' size, heterogeneity, functionalization and stability of their conjugates. We discuss practical approaches and challenges that need to be addressed to make QD immunostaining a standard method in biology.
