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Published on: April 22, 2014
Time Gated Luminescence Imaging of Immunolabeled Human Tissues
Ting Chen1, Rui Hong2, Darren Magda3
1Department of Chemistry, University of Illinois at Chicago , 845 West Taylor Street, Chicago, Illinois 60607, United States.
Insights
Time-gated luminescence microscopy with Lumi4-Tb offers enhanced sensitivity and multiplexing for tissue imaging. This method overcomes limitations of conventional immunofluorescence, improving disease diagnosis and proteomic profiling.
Area of Science:
- Biomedical Imaging
- Analytical Chemistry
- Pathology
Background:
- Multiplexed immunofluorescence is crucial for tissue proteomic analysis and disease diagnosis.
- Conventional methods using organic dyes face limitations in sensitivity, multiplexing capacity, and compatibility with standard staining protocols like hematoxylin and eosin (H&E).
- Tissue autofluorescence can reduce the sensitivity of traditional immunofluorescence techniques.
Purpose of the Study:
- To evaluate the advantages of time-gated luminescence microscopy (TGLM) using a terbium(III) complex (Lumi4-Tb) for multiplexed immunofluorescence imaging.
- To compare TGLM with conventional organic dye-based immunofluorescence in terms of sensitivity, multiplexing potential, and protocol compatibility.
- To demonstrate the utility of TGLM for visualizing low-abundance tissue markers and its compatibility with archived or clinical tissue specimens.
Main Methods:
- Utilized time-gated luminescence microscopy with Lumi4-Tb, an emissive terbium(III) complex.
- Employed temporal separation of long-lifetime Tb(III) emission and short-lifetime organic dye signals for multiplexing.
- Investigated compatibility with tyramide signal amplification and hematoxylin and eosin (H&E) staining.
- Visualized low-abundance markers like Bcl-6 and MSH-6.
Main Results:
- TGLM significantly increased the signal-to-noise ratio compared to conventional steady-state detection, enhancing visualization of low-abundance markers.
- Temporal separation enabled a second dimension for multiplexing and detection of Tb(III)-to-dye Förster resonance energy transfer (FRET).
- Lumi4-Tb demonstrated compatibility with tyramide signal amplification and H&E staining, unlike conventional organic dyes.
Conclusions:
- Time-gated luminescence microscopy with Tb(III) labels provides a highly sensitive and robust method for multiplexed immunofluorescence.
- This technique overcomes key limitations of conventional immunofluorescence, offering improved performance for tissue imaging.
- TGLM is suitable for analyzing archived and clinical tissue specimens, advancing proteomic profiling and disease diagnostics.
Abstract:
Multiplexed immunofluorescence imaging of formalin-fixed, paraffin-embedded tissues is a powerful tool for investigating proteomic profiles and diagnosing disease. However, conventional immunofluorescence with organic dyes is limited in the number of colors that can be simultaneously visualized, is made less sensitive by tissue autofluorescence background, and is usually incompatible with commonly used hematoxylin and eosin staining. Herein, we demonstrate the comparative advantages of using time-gated luminescence microscopy in combination with an emissive Tb(III) complex, Lumi4-Tb, for tissue imaging in terms of sensitivity, multiplexing potential, and compatibility with common immunohistochemistry protocols. We show that time-gated detection of millisecond-scale Tb(III) emission increases signal-to-noise ratio relative to conventional steady-state detection of organic dye fluorescence and permits visualization of low-abundance tissue markers such as Bcl-6 or MSH-6. In addition, temporal separation of long- and short-lifetime (∼nanosecond) signals adds a second dimension for multiplexing and also permits detection of intermolecular Tb(III)-to-dye Förster resonance energy transfer. Furthermore, we demonstrate that the Lumi4-Tb complex is compatible with tyramide signal amplification and, unlike conventional organic dyes, can be reliably used on tissue stained with hematoxylin and eosin. Our results indicate that time-gated luminescence microscopy using Tb(III) labels can provide a sensitive and robust method to perform multiplexed immunofluorescence on archived or clinical tissue specimens.
