Cleavable Tyramide Imaging for Multiplexed In Situ Tissue Profiling
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods for in situ analysis of proteins and nucleic acids in biological samples are limited in their ability to detect a small number of molecules and often miss low-copy transcripts, lacking sensitivity and multiplexing capabilities.
Innovation Solution
A cleavable detectably-labeled tyramide (CLT) is used, comprising a detectable marker tethered via a chemically cleavable linker, allowing for multiplexed in situ analysis by sequential staining, imaging, and signal removal through agents like PTA and TCEP, enabling detection of multiple biomolecules in intact tissues with single-molecule sensitivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional immunohistochemistry and immunofluorescence methods are used, then the detection process is simple and straightforward, but only a handful of proteins can be detected in one tissue sample, limiting multiplexing capability
Solution Approach 1:
The detection process is segmented into multiple sequential cycles, where each cycle detects one or more biomolecules using HRP-conjugated targeting agents and cleavable tyramide. After each cycle, the detectable label is removed and HRP activity is deactivated, allowing the same tissue sample to be reused for detecting additional biomolecules in subsequent cycles with different targeting agents
Solution Approach 2:
The detectable label attached to the tyramide is temporarily discarded after imaging by treating with PTA and TCEP to remove the fluorophore while preserving the HRP-conjugated targeting agents on the tissue. This allows recovery and reuse of the tissue sample for additional detection cycles, enabling multiplexed analysis of numerous biomolecules from a single sample
2Measurement precision
If existing in situ analysis methods are used, then the methodology is established and reliable, but the sensitivity is insufficient to detect low-copy transcripts and proteins
Solution Approach 1:
The invention changes the chemical parameters of the detection system by using cleavable tyramide with high HRP catalytic efficiency instead of conventional fluorophore-conjugated antibodies. This allows signal amplification through the HRP-mediated oxidation of tyramide, generating multiple fluorescent tyramide derivatives per HRP molecule, thereby significantly enhancing detection sensitivity for low-copy transcripts and proteins while maintaining reliability through the established HRP detection chemistry
3Adaptability or versatility
If multiple biomolecules are detected in sequential cycles, then comprehensive molecular profiling is achieved, but the time required for analysis increases significantly
Solution Approach 1:
The detection process is designed as a continuous cyclic workflow where HRP-conjugated targeting agents bind to biomolecules, cleavable tyramide is added for signal generation, imaging is performed, then the detectable label is removed and HRP activity is deactivated in preparation for the next cycle. This continuous cyclic process minimizes idle time between detection events and enables efficient multiplexed analysis of numerous biomolecules from a single tissue sample
Solution Approach 2:
HRP-conjugated targeting agents are applied and allowed to bind to the tissue sample before the cleavable tyramide detection reagents are added. This preliminary binding step ensures that the detection system is pre-positioned and ready for rapid signal generation when the tyramide is introduced, reducing the overall time required for each detection cycle and enabling faster comprehensive profiling
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The method enables comprehensive, high-throughput, and sensitive detection of numerous proteins and nucleic acids in intact tissues, maintaining protein antigenicity and allowing for accurate quantification and correlation analysis across multiple cycles.
Implementation Method 1
wherein R is a detectable marker. Exemplary detectable markers include, without limitation, fluorophores
Implementation Method 2
comprising a detectable marker tethered via a chemically cleavable linker
Data Source
AI summary
Provided herein are methods for multiplexed in situ analysis of biomolecules in a tissue. In particular, provided herein are methods for multiplexed single-cell in situ protein and nucleic acid profiling in fixed or fresh tissues, that allows the investigation of the different cell compositions and their spatial organizations in intact tissues through consecutive cycles of probe hybridization, fluorescence imaging, and signal removal.