Limitations on the number of biomolecules that can be quantified in biological samples, especially at the single-cell level, impede advances in accurate molecular diagnosis and understanding of normal cell physiology and disease pathogenesis. Spectral overlap of commonly available fluorophores is the largest culprit hindering the multiplexing capacity of conventional fluorescence microscopy approaches. While methods have been developed to try and overcome these limitations, issues still remain regarding consistent quantification, specimen degradation and increased background.
Researchers at Arizona State University have developed a method for detecting multiple targets in biological samples using probes labeled with cleavable fluorophores. In this method, probes labeled with cleavable fluorophores are utilized to detect their molecular targets in biological samples immobilized on a solid surface. After fluorescence imaging and data storage, the cleavable fluorophores coupled to probes are efficiently removed. Upon continuous cycles of target recognition, fluorescence imaging, and fluorescence signal erasing, this approach has the potential to quantify over 100 different molecular targets at the optical resolution in biological samples. This approach enhances detection sensitivity while reducing imaging time by 1-2 orders of magnitude.
This multiplexed molecular profiling technology should have wide applications in research, molecular diagnoses and targeted therapies.
• Molecular diagnostics & prognostics
• Targeted therapies (monitoring the effect of treatments)
• Basic biological studies (systems biology, cell heterogeneity, signaling pathway analysis, and more)
• Fluorescent tags for different biology probes in:
o Protein quantification
o DNA or RNA in situ hybridization & metabolic analyses
Benefits and Advantages
• 88 times more sensitive than direct immunofluorescence
• 35 times more sensitive than indirect immunofluorescence
• Can detect 50-100 different proteins/nucleic acids expressed at different levels in the same tissue
• Can detect low-abundant proteins or short RNAs
• Can be used with formalin fixed tissues
• Protein antigenicity not affected by fluorophore cleavage
• Reduced imaging and overall assay time
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